Porto de Galinhas, Pernambuco, Brazil, May 13 th to 17th , 2013 Enotel
Abstract Book
Porto de Galinhas, Pernambuco, Brazil, May 13th to 17th, 2013 Enotel www.worldleish5.org
Abstract Book
Brazil – Pernambuco – Ipojuca
Fifth World Congress on Leishmaniasis
WorldLeish5 13th to 17th May 2013 Porto de Galinhas, Pernambuco, Brazil The World Congress on Leishmaniasis is the most important meeting on a disease of high priority in the World Health Organization’s list of Tropical Neglected Diseases. The leishmaniases are going to be around for a very, very long time because almost all of them have an animal reservoir, which excludes the possibility of elimination. It is therefore essential to define guidelines targeting the control. WL5 gathers leishmaniacs from all around the world. Unlike other meetings it does not focus on one academic or public health field but on one group of diseases – the leishmaniasis. Research on Leishmania parasites and the diseases they cause has increased over the last decade. Indeed, they have proven to be exciting models for studying parasite-host and vector-host interactions, immunological mechanisms, molecular biology, genetics, epidemiology and other related areas. The principal aims of WorldLeish are to foster interaction between leishmaniacs from around the world and to provide a unique forum for discussing the state-of-the-art of leishmaniasis research, including the perspectives related to the control and prevention. Besides this it allows participants to share their most recent findings and to meet and interact with some of the world’s leading experts. The first World Congress on Leishmaniasis (WL1) was held in Istanbul, Turkey, in 1997 and was a great success. WL2 was held in Crete, Greece, in 2001, WL3 took place in Sicily, Italy in 2005 and WL4 took place in 2009 in Lucknow, India. The meeting’s success and popularity continued to increase as did the number of participants. However, they all took place in the Old World which made it difficult for younger American leishmaniacs to attend. Now, after three meetings in Europe and one in Asia WorldLeish is finally taking place in an American country. Brazil represents one of most important leishmaniasis endemic areas, with the occurrence of many varieties of parasites which cause different clinical forms, including cutaneous, mucocutaneous and visceral leishmaniasis. Brazil has one of the world’s biggest critical masses of scientists and public health authorities working on leishmaniasis. This is due to the ever increasing importance of the disease and the levels of funding from different federal and state governmental bodies.
WL5 has broken all previous records. A total of 1,426 abstracts have been received which exceeded by far the number that was expected. Although a pleasant surprise, this created certain logistical problems. The challenge was how to accommodate so many high quality contributions as oral presentations since the number of sessions was limited. So, some had to be changed to posters. To help in analyzing these abstracts the Organizing Committee counted on both the International Scientific Committee and the Brazilian Scientific Committee, composed of 52 Brazilian leishmaniacs. The final timetable has 5 simultaneous daily sessions with 1 satellite symposium, which to date is the most extensive WL program ever. Our actual Program introduces, besides 43 oral sessions and 14 Satellite Symposia, 22 special sessions designated as Hot Spots. The subjects for the Hot Spots are those that need greater focus or are considered to be controversial according to a survey performed on Leish-L. Abstracts in these specific areas were selected for presentation in the relevant Hot Spot. A total of 962 posters are presented in 3 evening sessions and specially selected posters will be located in 6 different areas, that are being called “Thinking Boxes”, to serve as discussion canters. The number of participants has broken all records. Approximately 1,200 individuals from all walks of science and public health will be attending. We are grateful to the enthusiasm of all who have guaranteed WL5´s success, especially the younger ones from Latin America for whom this is possibly a once in a lifetime opportunity to attend a WL meeting. We want to acknowledge our deepest gratitude to the organizations that have generously supported WL5 financially: Health Surveillance Secretary/Ministry of Health/Brazil, Oswaldo Cruz Foundation, Bill & Melinda Gates Foundation, Belgian Development Cooperation, NIH, PAHO/WHO, CNPq, CAPES, DECIT, FACEPE, and the Sponsorship of BAYER, SANOFI, ZOETIS/PFIZER, HERTAPE CALIER, PALADIN, BIOMANGUINHOS and MSD. Without their funding WL5 would probably not have taken place. Due to committee’s dedication together with the generosity of funding agencies WL5 has been able to award registration waivers that include help with accommodation to 37 applicants selected from 15 countries were funds are scarce and where the disease is a significant public health issue. We have put together a scientific program that has something for everybody and hopefully will open new horizons for both young and old leishmaniacs. We also wish that all of you enjoy the tranquility of Porto de Galinhas’s warm and clear waters, together with its excellent variety of local and international cuisine. These settings will create the perfect atmosphere for making new friends and reuniting with old ones. Have a wonderful time!
The Organizing Committee
- GENOMICS, TRANSCRIPTOMICS, PROTEOMICS, METABOLOMICS [756] P 152 - AN INSIGHT INTO THE PROTEOME OF CRITHIDIA FASCICULATA APPLIED TO THE SEARCH OF DISTINCTIVE FEATURES OF LEISHMANIA SPECIES INVOLVED IN CUTANEOUS AND VISCERAL LEISHMANIASIS. ALONSO, A.; ALCOLEA, P.J.; GARCIA, F.; LARRAGA, V. CENTRO DE INVESTIGACIONES BIOLOGICAS, CSIC, MADRID, SPAIN.
Keyword:crithidia fasciculata; gene expression; proteome
Abstract: The life cycle of the trypanosomatid Crithidia fasciculata is monogenetic as the unique hosts of these parasites are different species of mosquitoes. Choanomastigotes differentiate into nonmotile round amastigotes attached to the gut epithelium and are released to the environment. As they do not infect mammals, the comparison of these parasites with other evolutionary close genera developing digenetic life cycles grouped within the family Trypanosomatidae as Leishmania is of outstanding interest. We aimed to compare the proteome of C. fasciculata with the proteomes of L. infantum and L. amazonensis, which are etiological agents of visceral and cutaneous leishmaniasis. For this purpose, first we prepared total protein extracts of logarithmic and stationary phase promastigotes of L. infantum and L. amazonensis and choanomastigotes of C. fasciculata. Thereafter, we performed a 2DE analysis and the subsequent protein identification by MALDI-TOF/TOF. As a result, important differences in proteins involved in catabolic processes, gene expression regulation, signalling, redox homeostasis and protein modification have been found between the motile stages of Leishmania spp. and C. fasciculata. First of all, broader and more stringent gene expression control likely takes place in L. infantum and L. amazonensis at the beginning of differentiation processes rather than in C. fasciculata. Regarding the catabolic processes, the motile stage of C. fasciculata up-regulates key enzymes involved in the glycolitic pathway at early stages of differentiation unlike in L. infantum and L. amazonensis. The utilization of this pathway is mantained throughout growth of Leishmania spp. and its regulation is more complex provided the digenetic life cycle. We have observed the alternation of sugar, amino acid and lipid catabolic pathways depending on the environment in the Leishmania spp. studied. The arginase is over-expressed in mature L. amazonensis promastigotes as a pre-adaptation to survival in mammalian host phagocytes. The CACK and LACK orthologues are over-expressed at early stages of differentiation in C. fasciculata and L. amazonensis unlike in L. infantum, where the abundance of this protein is constant in the motile stage. For this reason, this protein may have an important role in visceral leishmaniasis. In fact, we observed promising results using this antigen in a recombinant vaccine against canine leishmaniasis in dogs experimentally infected. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [759] P 153 - INVOLVEMENT OF CD4+T CELLS PRODUCING GRANZYME B AND IFN- IN THE IMMUNE RESPONSE AGAINST EXCRETED/SECRETED PROTEINS OF LEISHMANIA MAJOR BOUSSOFFARA, T.; NAOUAR, I.; BEN AHMED, M.; BELHAJ HMIDA, N.; SALAH, A.B.; HASSOUNA, N.B. PASTEUR INSTITUTE OF TUNIS, TUNIS, TUNISIA.
Keyword:excreted secreted proteins; cd4+tcells; granzyme b
Abstract: The potential immunogenicity of Leishmania major excreted/secreted (LmES) proteins was evaluated in humans using peripheral blood mononuclear cells (PBMCs) from individuals with history of zoonotic cutaneous leishmaniasis and from appropriate healthy controls. Our results show that PBMCs from all immune individuals proliferate and produce high levels of IFN- and granzyme B (GrB) when stimulated with LmES. Interestingly, significant high levels of IFNand GrB were detected in supernatants of CD8-depleted peripheral cells stimulated by LmES suggesting that such mediators are mainly produced by CD4+ T cells. In attempt to identify the sub-population of CD4+T cells involved in IFN- and GrB production, CD4+CD25+ and CD4+CD25- T cell subsets were purified from the peripheral blood of a representative set of immune individuals. Our results showed that both populations produced IFN- and GrB when stimulated with LmES. Further experiments showed that CD4+CD25+CD127dim/- regulatory T cells were not involved in such production. Collectively, our results emphasize the involvement of potentially cytotoxic CD4+ T cells in the immune response against excreted/secreted proteins from L. major.
- GENETICS, EVOLUTION AND TAXONOMY [761] P 154 - GENOTYPIC CHARACTERIZATION OF LEISHMANIA DONOVANI FIELD POPULATION OF BIHAR, INDIA TIWARY, P.; SINGH, N.; LAL, S.; KUMAR, D.; SINGH, B.; RAI, M.; SUNDAR, S. BANARAS HINDU UNIVERSITY, VARANASI, INDIA.
Keyword:hsp 70; pcr-rflp; leishmania donovani
Abstract: The 70 kDa heat-shock protein (HSP70) is conserved across prokaryotes and eukaryotes, and the protein as well as its encoding gene have been applied in phylogenetic studies of different parasites. In spite of the frequent use of New World Leishmania species identification on the basis of restriction fragment length polymorphisms (RFLP) in the hsp70 gene, it was never sequenced extensively for studying evolutionary relationships. The present study applies PCR-RFLP of heat shock 70 gene and RAPD for studying the polymorphism among 630 strains of Leishmania donovani from endemic regions of Bihar, India collected since 2004. PCR-RFLP revealed the presence of both Leptomonas sp. and Leishmania donovani. Therefore, we conclude that there was a mixed infection of Leptomonas sp. and L. donovani in this isolate. While RAPD revealed intra specific variations in case of L. donovani. Further work regarding creation of phylogenetic tree based on RAPD is going on.
- CELLULAR AND MOLECULAR BIOLOGY [762] P 155 - THE ROLE OF ER STRESS AND TOLL LIKE RECEPTORS IN PKR ACTIVATION DURING LEISHMANIA AMAZONENSIS INFECTION DIAS TEIXEIRA, K.L.1; DA COSTA, A.P.1; SILVA, A.M.2; REAL, F.3; LOPES, U.G.1 1.INSTITUTO DE BIOFĂ?SICA CARLOS CHAGAS FILHO - UFRJ, RIO DE JANEIRO, RJ, BRAZIL; 2.DEPARTAMENTO DE MORFOLOGIA - UFMG, MINAS GERAIS, MG, BRAZIL; 3.DEPARTAMENTO DE MICROBIOLOGIA IMUNOLOGIA E PARASITOLOGIA - UNIFESP, SAO PAULO, SP, BRAZIL.
Keyword:pkr; er stress; leishmania amazonensis
Abstract: The protein kinase R (PKR) is a critical component of the innate immune response against viral infection and modulates the expression of several immune mediators. Latent PKR is activated by binding to one of its activators, dsRNA or the protein PACT. We have previously shown that Leishmania amazonensis was able to activate and increase the PKR expression in infected macrophages. PKR activation promoted parasite intracellular proliferation in an IL-10dependent fashion. The signaling pathways triggered by toll like receptors induce the production of innate immune mediators, which in turn may rely on PKR activation. Moreover, TLRactivated XBP1 was required for optimal and sustained production of pro-inflammatory cytokines in macrophages. Activation of IRE1 by ER stress acted in synergy with TLR activation for cytokine production through activation of XBP-1. We previously demonstrated in peritoneal macrophages from TLR2 or TLR4-KO B6 mice, a dramatic reduction of parasite infection, similar to the observed with PKR-KO macrophages. Our data suggested that toll-like receptors pathways do support L. amazonensis infection. Additionally, western blotting assays showed that PKR phosphorylation was reduced in infected macrophages from TLR2-KO or TLR4-KO mice, suggesting that PKR phosphorylation seems to be partially dependent on TLR engagement. We also observed that the expression of the suppressor cytokine IL-10 was reduced in infected macrophages from TLR2 and TLR4-KO, suggesting a role for IL-10 in the infection. We also showed that the L. amazonensis infection was able to induce a cytosolic mRNAsplicing event that generates a mature (spliced) XBP1 protein with a potent transactivation domain, indicating a possible role of ER stress in this process. Accordingly, we showed an increase in the parasite burden when macrophages were treated with tunicamycin, a potent inducer of ER stress. Moreover, we observed in immunofluorescence assays an increase in PACT levels in macrophages infected with L. amazonensis. Tunicamycin is able to induce the activation of PKR and PACT leading to ER stress, our working hypothesis is that ER stress is induced during L. amazonensis infection, leading to PKR activation via PACT. Our data corroborate this notion and we also propose that PKR-dependent intracellular proliferation of L. amazonensis may depend on TLR2 and TLR4 engagement together with the induction of ER stress components. Supported by FAPERJ and CNPq. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [764] P 156 - PHAGOCYTOSIS OF PROMASTIGOTES OF LEISHMANIA AMAZONENSIS BY B-1 CELLS GERALDO, M.M.1; BARBOSA, F.M.C.1; GONZAGA, W.F.2; VIVANCO, B.C.2; LOPES, J.D.2; XANDER, P.1 1.UNIVERSIDADE FEDERAL DE Sﾃグ PAULO CAMPUS DIADEMA, DIADEMA, SP, BRAZIL; 2.UNIVERSIDADE FEDERAL DE Sﾃグ PAULO, Sﾃグ PAULO, SP, BRAZIL.
Keyword:l. amazonensis; phagocytosis; b-1 cells
Abstract: Leishmaniasis are caused by Leishmania parasites which mainly infect macrophages. The promastigotes stage of Leishmania are internalized by phagocytic cells and followed are transformed to the obligate intracellular amastigote stage. B-1 cells are a subpopulation of B cells resident in the peritoneal and pleural cavities in mice. These cells are able to differentiate in vitro into mononuclear phagocyte-like cells with phagocytic properties. B-1 cells phagocytose by several receptors such as mannose receptor (MR), Fc gamma receptors (Fc Rs) and third complement receptor (CR3). Leishmania binds to the same receptors in macrophage cells. Nevertheless, the role of B-1 cells in phagocytosis of Leishmania has not yet been clarified. This study aimed to investigate the phagocytosis of L. amazonensis by mononuclear phagocytes
derived from B-1 lymphocytes in vitro and in vivo. In this report we demonstrated that phagocytes derived from B-1 cells were able to internalize in vitro promastigotes of L. amazonensis. The internalized promastigotes became into amastigotes. Our results showed that phagocytic index was higher in phagocytes derived from B-1 (2885 ± 264.46), compared to peritoneal macrophages (1236.33 ± 355.92) and bone-marrow-derived macrophages (421.5 ± 6 36; p <0.001). In addition, L. amazonensis stimulated TNF-alpha production by B-1 cells. TNFalpha amounts were higher in B-1 cells that phagocytosed L. amazonensis, as compared with B1 control (cells non-stimulated with parasites). The in vivo phagocytic ability of B-1 cells was also evaluated. A flow cytometric method was performed to evaluate the peritoneal B-1 cells-L. amazonensis interaction. Parasites were labeled with stable intra-cytoplasmic fluorocrome, 5,6carboxyfluorescein diacetate succinimidyl ester (CFSE) and subsequently inoculated in peritoneal cavities of mice. After 8, 16 or 24 h peritoneal cells were collected and labeled with anti-CD19 and anti-CD23. We found that many CD19+ CD23- cells were positive for FITC, which we attributed to phagocytosis of the parasite by B-1 cells. This is the first report that B-1 cells can internalize Leishmania species. - GENOMICS, TRANSCRIPTOMICS, PROTEOMICS, METABOLOMICS [766] P 157 - IS AXENIC CULTURE SO GOOD MODEL FOR LEISHMANIA PROMASTIGOTES? ALCOLEA ALCOLEA, P.J.1; ALONSO, A.1; MOLINA, R.2; JIMENEZ, M.I.2; GOMEZ, M.3; POSTIGO, M.3; LARRAGA, V.1
1.CENTRO DE INVESTIGACIONES BIOLÓGICAS, CSIC, MADRID, SPAIN; 2.CENTRO NACIONAL DE MICROBIOLOGÍA, VIROLOGÍA E INMUNOLOGÍA SANITARIAS, ISCIII, MADRID, SPAIN; 3.CENTRO DE ASTROBIOLOGÍA, INTA-CSIC, MADRID, SPAIN.
Keyword:leishmania infantum; phlebotomus perniciosus; gene expression profiling
Abstract: Axenic cultures of Leishmania spp. promastigotes were developed in the 1960 and 70 decades to reproduce in vitro the conditions inside the gut of the sand fly. As a difference with axenic culture of amastigotes, the promastigote culture model is routinely used because it is considered to be stable and reproducible, keeping the original features. It has been described that infectivity and virulence become attenuated after several passages in culture, thus requiring further passages through laboratory animals. We called into question whether the culture conditions accurately reproduce the environment inside the phlebotomine sand fly gut. So then, we compare the variable transcriptome of promastigotes in stationary phase of axenic culture with promastigotes isolated from the digestive tract anterior to the stomodeal valve of Phlebotomus perniciosus (Lin-Pper). For this purpose, a group of about 150 sand flies were fed with a suspension of differentiated U937 cells infected with L. infantum in defibrinated and heat inactivated rabbit blood and maintained at 27-28 ºC. Promastigotes were obtained from the dissected guts, after 7 days to recover Lin-Pper, and from axenic cultures. RNA was immediately isolated and mRNA successfully amplified. These samples were reversely transcribed, labeled and compared by whole genome L. infantum custom DNA microarray hybridization analysis. As a result, we observed higher rate of variable transcripts in Lin-Pper than in cultured promastigotes, as well as qualitatively different variable transcripts in each condition with several exceptions like the over-expression of the amino acid transporter LinJ.31.1850. The up-regulation of genes involved in sugar anabolic processes in Lin-Pper and the down-regulation of genes related with branched chain amino acid catabolism points to different nutritional requirements of the parasite in each of the environments indirectly compared in the last stages of differentiation prior to infection of the mammalian host. Important differences in the expression of genes involved in gene expression regulation transport, proteolysis and signaling pathways have been also observed, suggesting alternative differentiation processes in each environment. Differences in the glycocalix are also presumably appear as the GIPL-galactofuranose transferase gene is over-expressed in Lin-Pper. Taken
together, these data support that the axenic culture model does not reproduce the conditions of the sand fly gut as accurately as it has been considered so far. - CLINICAL LEISHMANIASIS [772] P 158 - LINPHOMA EXPLAINING NON-RESPONSE IN MUCOSAL LEISHMANIASIS HUEB, M.; GONÇALVES, A.M.; FERREIRA SOARES, M.C.; DE OLIVEIRA, T.F.; SOUSA NASCIMENTO LEITE, K.S.; RIBEIRO, L.C. UFMT, CUIABA, MT, BRAZIL.
Keyword:ulcer; mucocutaneous; treatment failure
Abstract: M.I.M.S., 57 ANOS, COM QUEIXA DE OBSTRUÇÃO NASAL HÁ UM ANO E CINCO MESES, COM EPIDEMIOLOGIA POSITIVA PARA LTA, SEM HISTÓRICO DE LESÃO CUTÂNEA PRÉVIA, LESÃO INFILTRATIVA COM PONTOS HEMÁTICOS EM REGIÃO SEPTAL ANTERO-SUPERIOR A ESQUERDA. APRESENTOU IRM NÃO REATOR E BIÓPSIA DA LESÃO (03/03/11) COM INFILTRADO INFLAMATÓRIO RICO EM MACRÓFAGOS, LINFÓCITOS E PLASMÓCITOS, COM PROLIFERAÇÃO DE TECIDO DE GRANULAÇÃO. APÓS MEDIDAS LOCAIS, apresentou piora clínica, sendo SOLICITADO NOVO IRM, NÃO REATOR, e REALIZADO NOVA BIÓPSIA LOCAL COM os mesmos achados anteriores. Iniciado GLUCANTIME 17,3 MG/KG/DIA POR 30 DIAS. APÓS ESQUEMA COM SBV, APRESENTAVA PIORA DA LESÃO ASSOCIADO A CELULITE DE FACE. Internada para tratamento, sendo realizado drenagem de abscesso que obstruía seio paranasal, antibioticoterapia e anfotericina b lipossomal (DA: 1G). No retorno, após alta, havia lesão hipertrófica em toda cavidade nasal e. TEVE EPISÓDIOS RECORRENTES DE INFECÇÃO BACTERIANA SECUNDÁRIA, TRATADAS. REALIZADA NOVA BIÓPSIA DE MUCOSA, SENDO DESCARTADA NEOPLASIA,sugestiva de LEISHMANIOSE SEM DESCARTAR VASCULITE. APÓS NOVA PIORA CLÍNICA, FOI REINICIADA ANFOTERICINA B LIPOSSOMAL (DA:2,1G. INSTITUIu-SE MEDICAÇÕES ANTIINFLAMATÓRIAS SEM RESPOSTA. REALIZADO INVESTIGAÇÃO PARA IMUNODEFICIÊNCIA, SEM DADOS POSITIVOS. REINICIADO ESQUEMA DE ANFOTERICINA B LIPOSSOMAL (DA:1,45G). REINTERNOU EM com lesão infiltrativa de lábio superior, cruenta e sanguinolenta, extendendo para vestíbulo nasal e mucosa jugal, SEM ATIVIDADE, preservando restante de cavidade oral. Em acompanhamento EVIDENCIOU-SE desnutrição grave, anemia de doença crônica E DISCRETA ESPLENOMEGALIA (15,3 CM). Diante da piora clinica da lesão, da febre, do TRATAMENTO PARA LTA E das pesquisas negativas para fungos, bactérias e vasculite, optou-se por TRATAMENTO empíricO para micobacteriose. Após revisão de laminaS em serviço de Brasília, e imunohistoquímica em são Paulo, não HOUVE CONCLUSÃO QUANTO LTA ou vasculite, NEM EVIDÊNCIAS DE BK OU FUNGOS. DEVIDO A ESTAGNAÇÃO da LESÃO, instituiU-se tratamento para vasculite com metotrexate e colchicina, SEM RESPOSTA FAVORÁVEL. Paciente evoluiU progressivamente com hiporexia, adinamia, hipoatividade,DESCONFORTO RESPIRATORIO, além de piora da lesão e extensão de acometimento (palato mole e nasal). Por sugestão de patologista e cirurgião oncológico, foi feita ressecção da lesão para análise. Anatomopatológico sugestivo de neoplasia MALIGNA INFILTRATIVA DE CÉLULAS PREDOMINANTEMENTE ARREDONDADAS. Em 05/12/12, IMUNOHISTOQUÍMICA: MONOCLONALIDADE PARA LINFÓCITOS T favorecendo O DIAGNÓSTICO DE LINFOMA EXTRANODAL DE CÁLULAS T/NK, DO TIPO NASAL, COM ÍNDICE DE PROLIFERAÇÃO CELULAR DE 60%. Realizado ESTADIAMENTO E PROPOSTO INÍCIO DE PROTOCOLO CHOP + MADIT, tendo realizado 2 CICLOS ( 07/12 E 28/12). Evoluiu com melhora clinica, estando em suporte intensivo sob os cuidados da hematologia. Atualmente está afebril, em VM POR TRAQUEOSTOMIA, com IRA e regressão de massa tumoral.
- VACCINE DEVELOPMENT AND TRIALS [776] P 159 - LEISHMANIA EXCRETED-SECRETED ANTIGENS AS AMAZING SOURCE OF VACCINE CANDIDATES. BRAS-GONCALVES, R.1; PETITDIDIER, E.1; PAGNIEZ, J.1; HOLZMULLER, P.2; PAPIEROK, G.3; LEMESRE, J.1 1.IRD, MONTPELLIER, FRANCE; 2.CIRAD, MONTPELLIER, FRANCE; 3.BVT, LA SEYNE SUR MER, FRANCE.
Keyword:vaccine; excreted-secreted antigens; leishmaniasis
Abstract: Both Leishmania promastigote and amastigote forms synthesize complex glycoconjugates which are either displayed on their surface and that have been shown to play important roles in parasite virulence both in the sandfly and the mammalian host, or are excreted-secreted. Due to their location or because they are released into the host environment, these excreted-secreted antigens (ESA) are the first to establish a close contact with the immune system of the host modulating its functions. Previous studies have demonstrated that antigens secreted by microorganisms can quickly bind to class-I and -II molecules of major histocompatibility complex priming CD4+ and CD8+ T lymphocytes. In contrast, somatic antigens, encapsulated by cellular membranes and protected from the antigen processing machinery of eukaryotic cells, are only processed after pathogen destruction, leading to a delay in the T cell stimulation. Antigens released from promastigote forms have been identified as potent modulators of the immune system, having a crucial role in promoting the establishment and maintenance of the infection by interfering in the activation of effector mechanisms, such as the microbicidal activity of macrophages and cytokine production. Moreover, interesting studies using released antigens from Leishmania sp. as a model for the development of affordable vaccines have been conducted. The above considerations support the hypothesis that antigens released by Leishmania species may have potential to induce host protection and be used for vaccine development against leishmaniasis. The present work exposes our study strategy of Leishmania ESA, their production and their tremendous potential either as vaccine candidate of first generation and as a rich pool of other vaccine generations. From the successfully development of a completely defined serum-free medium that readily supports the continuous in vitro cultivation of promastigotes of most Leishmania species, we have been access to antigens naturally excreted-secreted by parasites, thus opening up a great opportunity to easily study them. The biological potential of these ESA is showed by our studies using of these naturally excreted-secreted antigens as a vaccine candidate of first generation, inducing a long-lasting and strong protective effect against canine visceral leishmaniasis, and as a rich source of major immunogenic antigens, conducting to new prospects to gain access to new vaccine generations for human use. - CELLULAR AND MOLECULAR BIOLOGY [788] P 160 - NATURAL KILLER CELLS AS INDICATOR OF HUMAN TEGUMENTARY LEISHMANIASIS PROGNOSIS GOMES, A.H.S.; BRASIL, R.; KANAMURA, C.T.; PEREIRA, J.C.; IGLEZIAS, S.D.; GAVA, R.; CHIOCCOLA, V.L.P. INSTITUTO ADOLFO LUTZ, Sﾃグ PAULO, SP, BRAZIL.
Keyword:leishmaniasis; natural killer ; immunohistochemistry
Abstract: American tegumentary leishmaniasis (ATL) is an important public health disease with high morbidity and constant expansion into new geographic areas. Experimental studies of innate
response in visceral and cutaneous leishmaniasis have been demonstrated the association of cytokine production as element that can influence the disease development. Stimulated natural killer cells (NK) develop two main mechanisms including the parasite recognition and cytokine production. In addition, in human infection, the cytokine production is one the most important event in parasite evolution as is involved in the lesions development, cure evolution or worsening relapsing form. This study was aimed to demonstrate the presence of NK cells in skin lesions of patients, in the initial phase of ATL caused by L. (V.) braziliensis. We studied 19 biopsies of skin lesions collected from individuals, residents in Sorocaba, a southeastern region of São Paulo State, Brazil with recently infected with cutaneous leishmaniasis. The etiologic diagnosis was confirmed by, parasitological and histopathological methods; immunohistochemistry and polymerase chain reaction (PCR). Morphological and immunological characteristics were evaluated by HE staining and immunohistochemistry using an anti-Leishmania spp polyclonal antibody. The NK cell identification was performed by immuno-staining-tissue (CD57) in tissue sections included in paraffin. NK cells were shown in superficial, middle and deep dermis in lesions of all studied patients . However, a higher NK concentration was shown in superficial dermis with a greater amastigote concentration. These findings indicate a good prognostic in destroying amastigotes, especially early stages of the disease, but also indicate the need to investigate other cells as macrophages and neutrophils that also induce cytokine production. Support: FAPESP:2011/13939-8 - CELLULAR AND MOLECULAR BIOLOGY [793] P 161 - INTRACELLULAR TRAFFIC OF LEISHMANIA AMAZONENSIS DURING PARASITOPHOROUS VACUOLE FORMATION. QUINTAL, A.P.N.; TEIXEIRA, T.L.; TEIXEIRA, S.C.; DA SILVA, C.V. UNIVERSIDADE FEDERAL DE UBERLÂNDIA, UBERLÂNDIA, MG, BRAZIL.
Keyword:phagolysosome; inhibitors; endocytic vesicules
Abstract: Engulfment of Leishmania promastigotes by macrophages is initiated by the recognition of surface receptor molecules by the parasite and the host cell. During the intracellular traffic the parasitophorous vacuole is formed during invasion, undergo fusion with endocytic vesicules such as lysosomes to form the phagolysosome. The aim of our study were to determinate the activity during the phagolysosome formation by using different inhibitors of metabolic pathways. Invasion and kinetics assays were proceeded: 2x105 BALB/c peritoneal macrophages cells were invaded with L. amazonensis previously treated with the following inhibitors: Ly 294002, MEK 1, MEK ½, mTOR (Rapamycin), NRAS (Farnesyl), AKT (Calbiochen) and ERK/MAPk (Iodotubercidin). Kinetics were proceeded during 30 min, 1, 3, 6, 12, 24, 48 and 72 hours pos-invasion. Anti-lamp and anti-leishmania and respective conjugated antibodies were used for immunofluorescence and visualized in confocal microscopy. Our preliminary results showed dependence on the activity of AKT, that involves growth factor binding to a receptor tyrosine kinase and activation of PI-3 Kinase, which phosphorylates the membrane bound PIP2 to generate PIP3. Financial support: FAPEMIG/CAPES. - VACCINE DEVELOPMENT AND TRIALS [801] P 162 - THE ROLE OF TLR9 ON IMMUNITY AGAINST LEISHMANIA AMAZONENSIS INFECTION PRATTI, J.E.S.; BRAGA, D.S.F.S.; BERGMANN, B.R.; GUEDES, H.L.M. UFRJ - IBCCF, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:leishmania amazonensis; tlr9; laag
Abstract: TLR9 is involved in immunity against several diseases and it has been used as target to induce immune response against vaccines, mainly in nasal mucosa vaccines where it has a great expression. Intranasal LaAg vaccine induces protection against L. amazonensis infection, the etiologic agent of diffuse cuttaneous Leishmaniasis. Genomic DNA of L. amazonensis has capacity to activate TLR9. LaAg vaccine is composed by the whole antigens of L. amazonensis promastigotes, including DNA. In this work, we investigate the role of TLR9 on L. amazonensis infection and in the efficacy of LaAg vaccine by intranasal route. C57BL6 mice (WT) and C57BL6 deficient in TLR9 (TLR9 -/-) were infected with 5 x 105 promastigotes in footpad. The lesion development was evaluated by paquimeter and the parasite load by limited-dilution. WT and TLR 9 -/-, male and female, displayed the same profile of lesion development. However, TLR9 -/- showed an increase in lesion size after day 45 of infection. In WT, the resolution of the lesion started after day 45 and in deficient mice after day 75. In the chronic phase, parasite load of TLR9 -/- was higher than WT, suggesting the importance of TLR9 to parasite control. Besides, WT vaccinated with LaAg by intranasal route showed resistance since the beginning of infection (day 32 after infection) observed by smaller lesion. Immunization of TLR9 -/- did not induce protection in the lesion development, however, accelerated the process of lesion control, which was not observed in the no-vaccinated TLR9 -/- mice. Parasite load after lesion resolution in chronic phase demonstrated the reduction of parasite load on vaccinated mice (WT and TLR9-/-) compared to controls no-vaccinated. These results indicate the role of TLR9 on immunity against L. amazonensis. - CELLULAR AND MOLECULAR BIOLOGY [810] P 163 - INFLUENCE OF DIFFERENT CULTURE MEDIA IN LEISHMANIA INFANTUM GROWTH PATTERNS DE SOUSA, C.B.1; CORTES, S.2; FARINHA, A.3; CARVALHO, A.S.1; DIONÍSIO, L.4; CAMPINO, L.5
1.CCMAR/FACULDADE DE CIÊNCIAS E TECNOLOGIA (FCT)/UNIVERSIDADE DO ALGARVE (UALG), FARO, PORTUGAL; 2.U. PARASITOL. MÉDICA/CMDT/INSTITUTO HIGIENE MEDICINA TROPICAL(IHMT)-UNIVERSIDADE NOVA LISBOA(UNL), LISBOA, PORTUGAL; 3.U. PARASITOLOGIA MÉDICA/IHMT-UNL. FACULDADE DE CIÊNCIAS/UNIVERSIDADE DE LISBOA, LISBOA, PORTUGAL; 4.FCT/UALG. ICAAM/UNIVERSIDADE ÉVORA, FARO, PORTUGAL; 5.U. PARASITOL. MÉDICA/IHMTUNL. DEP.CIÊNCIAS BIOMÉD. E MEDICINA-UALG, LISBOA, PORTUGAL.
Keyword:in vitro; culture media; dynamics
Abstract: Protozoan parasites of the genus Leishmania undergo several developmental transitions during their life cycle, from extracellular promastigote forms in the vector to intracellular amastigote forms in vertebrate host. In laboratory conditions, it is possible to achieve promastigote growth outside the sandfly using several established culture media.To carry out in vitro cultivation of Leishmania species, liquid culture media that enables the production of higher numbers of promastigotes are needed. The main objective of the present study was to evaluate the effect on promastigote growth patterns of Leishmania infantum cultivated in different media used for in vitro studies. Morphologic analysis and percentage of parasite metacyclic forms was also assessed in each culture medium. The in vitro growth patterns of L. infantum MHOM/PT/88/IMT151, was studied in five supplemented media: Dulbecco's Modified Eagle Medium (DMEM), Grace's Insect Medium (Grace), Basic LIT Medium (LIT), RPMI-1640 Medium (RPMI) and Schneider's Insect Medium (Schneider). Promastigote cultures were maintained in the different media, supplemented with 20% fetal calf serum, at 25°+1°C. Microscopic observation of parasites was performed over 17 days.
Results have shown higher parasites densities in Schneider's medium. However, this medium, together with LIT's, have evidenced a more pronounced and earlier reduction of cell density. RPMI and DMEM presented lower parasite densities with kinetics between them. In all media, a higher percentage of metacyclic forms was observed after logarithmic phase, i.e., when a decrease in parasites density occurred. Our results suggested that medium formulation influence the kinetics and parasite morphology development. These results may be relevant for studies involving Leishmania parasites dynamics in different in vitro conditions. Funding from Portuguese Foundation for Science and Technology (FCT) research projects PTDC/CVT/ 112371/2009 and PTDC/MAR/103957/2008. C. Bruno de Sousa C. and S. Cortes hold PhD (SFRH/BD/78062/2011) and post-doctoral (SFRH/BPD/44450/2008) grants, respectively, from FCT. C. Bruno de Sousa and S. Cortes contributed equally for this work. scortes@ihmt.unl.pt, carolbrunos@yahoo.com - CLINICAL LEISHMANIASIS [811] P 164 - ORAL LESIONS OF MUCOSAL AMERICAN TEGUMENTARY LEISHMANIASIS: HOW TO MANAGE A DIFFICULT DIAGNOSTIC? PRELIMINARY INFORMATION. SILVA-COSTA, D.C.1; VALETE-ROSALINO, C.M.1; CONCEIÇÃO-SILVA, F.; SHCUBACH, A.O.1; MARTINS, A.C.C.1; QUINTELLA, L.P.1; MOREIRA, J.S.1; PALMEIRO, M.R.1; INÁCIO, R.R.2; SAHEKI, M.N.1; TORRACA, T.S.S.1; KRUMBIEGEL, G.B.2; TERCEIRO, B.R.F.1 1.IPEC-FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 2.IOC-FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:oral cavity; differential diagnosis; american tegumentary leishmaniasis
Abstract: INTRODUCTION: Leishmaniasis is a parasitic disease caused by protozoa of the genus Leishmania which is present in 88 countries, including Brazil. It priority affects the populations living in poverty with problems to access the treatment. Two basic clinical forms are recognized: visceral leishmaniasis (VL) or kala-azar and tegumentary leishmaniasis (TL) which is characterized by cutaneous and/or mucosal lesions. Mucosal Leishmaniasis (ML) may occur in both nasal and oral mucosa. However, despite the impressive tissue destruction, little is known about the oral involvement. Oral lesions are characterized by an intense inflammatory reaction associated a tissue damage and it is routinely confounding with lesions of others diseases (i.e. Wegener’s granulomatosis, Sarcoidosis, Tuberculosis, paracoccidioidosemycosis, periodontal disease, traumatic ulcerations or cancers). The difficulty to have a correct diagnosis delay the specific treatment what increases the risk of sequels. METHODS: In order to produce better tools to improve the rapid and efficient diagnostic of ML our group is studding ML patients followed at the VigiLeish (IPEC-Fiocruz). The clinical presentation, lesion localization, macroscopy and microscopy findings, laboratorial results, parasite detection and response to the correct treatment are compiled and compared. PRELIMINARY INFORMATION: the clinical presentation of ML can be altered by the presence of poor tooth conservation, denture or associated infections. However, even in these cases a good anamneses including epidemiological data can indicate ML as an hypothesis. Despite the parasite detection be a gold stander, association of other findings such as positive Montenegro Skin Test and specific serology can help the diagnosis when Leishmania parasites are not detected by the routine procedures and PCR is not available. Thereby, increase the knowledge about the evolution, clinical aspects, pathology, morphology, immunological characteristics, endemic areas and have professionals trained and prepared to identify this suspicions would permit an early diagnosis and effective treatment.
- VACCINE DEVELOPMENT AND TRIALS [818] P 165 - LEISHMANIA (L.) INFANTUM VISCERALIZATION-RELATED GENES FOR THE DEVELOPMENT OF RECOMBINANT AND LIVE VACCINES CANAVACI, A.M.C.1; MACHADO, L.F.M.1; SERUFO, A.V.1; DORO, D.H.2; NASCIMENTO, F.C.1; ANTONELLI, L.R.3; GAZZINELLI, R.T.4; TEIXEIRA, S.M.R.4; MARTINS, V.P.5; FERNANDES, A.P.1 1.DEPARTAMENTO DE ANÁLISES CLÍNICAS E TOXICOLÓGICAS – FACULDADE DE FARMÁCIA – UFMG, BELO HORIZONTE, MG, BRAZIL; 2.DEPARTAMENTO DE GENÉTICA – INSTITUTO DE CIÊNCIAS BIOLÓGICAS – UFMG, BELO HORIZONTE, MG, BRAZIL; 3.LABORATÓRIO DE IMUNOPATOLOGIA – CENTRO DE PESQUISAS RENÉ RACHOU – FIOCRUZ, BELO HORIZONTE, MG, BRAZIL; 4.DEPARTAMENTO DE BIOQUÍMICA E IMUNOLOGIA – INSTITUTO DE CIÊNCIAS BIOLÓGICAS – UFMG, BELO HORIZONTE, MG, BRAZIL; 5.DEPARTAMENTO DE BIOLOGIA CELULAR – INSTITUTO DE CIÊNCIAS BIOLÓGICAS – UNB, BRASÍLIA, DF, BRAZIL.
Keyword:visceral leishmaniasis; recombinant vaccine; l. tarentolae
Abstract: Several strategies of vaccination have been tested against leishmaniasis, including the use of avirulent organisms. Leishmania tarentolae can differentiate to amastigotes, but is unable to persist in macrophages. The A2 gene, first described in L. donovani, is amastigote-specific, has potent CD8 epitopes and it plays an important role in virulence and visceralization of Leishmania species. Recent studies have shown that L. tarentolae expressing A2 protein had an increased infectivity and survival in in vitro and in vivo experiments. The recombinant parasite was able to protect BALB/c mice against challenge with L. (L.) infantum by inducing increased production of IFN- before and after challenge. Novel genes of L. donovani have been associated to visceralization after transfection into L. major, and may also be alternative candidates to improve leishmaniasis vaccines. However, studies assessing their importance as virulence genes and/or in the generation of protection have not been performed. In order to test that, we describe cloning, production, analysis and purification of recombinant proteins related to visceralization process. Initially the protein sequences of the putative antigens were submitted to in silico analysis for T cell epitope prediction that showed epitopes with high scores for TCD8 cells. Peptides were synthesized and used in proliferation assays which confirmed the antigens immugenicity. The coding regions of Li40, Cyclo and Fc genes were amplified by PCR from genomic DNA of L. (L.) infantum. The PCR product was ligated into pGEM, and then transformed into XL1-blue competent bacteria. After selection on LB-Amp with Xgal/IPTG, colonies were evaluated by PCR. The insert was removed from pGEM by restriction digestion and religated in the expression vector pLEXSYneo2 (parasite expression) or pET15b (bacteria expression). Cloning was confirmed by PCR as well as sequencing of inserts. The expression of the recombinant proteins was confirmed by Western blot assays and the proteins were purified by affinity chromatography. Therefore, we have produced recombinant proteins that have predicted epitopes for TCD8 cells, requirements to be further explored for the development of a multi-component vaccine against Visceral Leishmaniasis. In vivo protection assays, combining A2 and the other proteins, are currently being performed as well as the generation of L. tarentolae recombinant parasites. Financial Support: INCTV; CNPq; FAPEMIG. - CELLULAR AND MOLECULAR BIOLOGY [819] P 166 - LEISHMANIA INFANTUM CHAGASI HAS A PAF- ACETYLHYDROLASE POSSIBLY INVOLVED IN VIRULENCE. COSTA-FERREIRA, V.S.1; ENTRINGER, P.F.2; SILVA-CARDOSO, L.3; ATELLA, G.C.3; SOTO, M.4; BARRAL, A.1 1.SCHOOL OF MEDICINE, FEDERAL UNIVERSITY OF BAHIA – UFBA, SALVADOR, BA, BRAZIL; 2.GONÇALO MONIZ RESEARCH CENTER- CPQGM, FIOCRUZ-BA., SALVADOR, BA, BRAZIL; 3.MEDICAL BIOCHEMISTRY INSTITUTE, FEDERAL UNIVERSITY OF RIO DE JANEIRO., RIO DE JANEIRO, RJ, BRAZIL; 4.CENTER OF MOLECULAR BIOLOGY, SEVERO OCHOA, UNIVERSIDAD AUTONOMA DE MADRID., MADRID, SPAIN.
Keyword:l. infantum chagasi; paf acetylhydrolase; lipid mediators
Abstract: Leishmania parasites developed evasion mechanisms, such as the production of molecules able to modulate the host immune response. Lipid mediators, as platelet activating factor (PAF), are molecules derived from phospholipids that play an important role in the inflammatory response. PAF has chemotactic ability, is able to induce activation of macrophages and neutrophils, and the generation of NO, cytokines and chemokynes. PAF bioactivity is abrogated by PAF acetylhydrolase, the enzyme that degrades it. The involvement of lipid mediators, such as eicosanoids and PAF, has been identified in the immunopathogenesis of leishmaniasis. PAF contributed to the infection by L. amazonensis control. However, information is limited to this specie and it is unclear how the parasite is able to subvert these effects and succeed the infection. The aim of this study is to evaluate the involvement of the PAF acetylhydrolase in the modulation of lipid mediators activities and their role in virulence. A putative phospholipase A2 with high similarity with PAF acetylhydrolases of mammalians was identified, in silico, from L. infantum genome. The sequence is localized in the position “LinJ.35.3070”, encoding a protein with 453 amino acids and predicted weight of 49kDa. The gene was cloned from L. infantum chagasi genome (BA262 strain) and recombinantly expressed in the Leishmania tarentolae platform as a 69kDa protein. The phospholipase A2 activity of recombinant protein was assayed and it was only able to hydrolyze short length substrates, independently of calcium. This profile is typical of PAF-acetylhydrolase. The effect of PAF on infection of monocytes obtained from the human peripheral blood mononuclear cells (PBMC) of healthy volunteers, by L. i. chagasi was evaluated. PAF 10-6 M was able to inhibit 50% of the monocyte infection. These results together suggest that PAF may function as a host response against infection, however this response would be suppressed by the PAF acetylhydrolase expressed by the parasite. Other experiments are in course to better understand the role of PAF and the PAF acetylhydrolase on L. i. chagasi pathogenesis. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [820] P 167 - THE TH17/IL-23 AXIS IN DIFFERENT CLINICAL FORMS OF AMERICAN CUTANEOUS LEISHMANIASIS DUE L.(L.) AMAZONENSIS AND L.(V.) BRAZILIENSIS MENEZES, J.P.B.1; DE MORAIS, I.B.1; GAMA, M.E.1; CAMPOS, M.B.2; LAURENTI, M.D.3; CORBETT, C.E.P.1; SILVEIRA, F.T.1; GOMES, C.M.C.3
1.FACULDADE DE MEDICINA DA UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL; 2.UNIVERSIDADE FEDERAL DO MARANHÃO, SÃO LUIZ, MA, BRAZIL; 3.INSTITUTO EVANDRO CHAGAS, BELÉM, PA, BRAZIL.
Keyword:th17/il-23; l.(l.) amazonensis ; l.(v.) braziliensis
Abstract: Recent studies point to the presence of Th17 cells playing a role in the immune response against parasite infections. Th17 cells are characterized by the production of IL-17, which constitutes a pro-inflammatory cytokine secreted primarily by activated T CD4+ and CD8+ cells. The production of IL-17 by mononuclear cells is favored by the presence of cytokines such as TGF, IL-1 , IL-23 and IL-6. IL-23 is member of the IL-12 cyrokine family produced by dendritic cells and macrophages. IL-23 is indispensable for Th17 effector functions in immune disorders and maintenance of Th17 cells. This study aimed to evaluate the profile of cells expressing IL17 and IL-23 in skin lesions of patients presenting different clinical forms of American cutaneous leishmaniasis (ACL) caused by L.(V.) braziliensis and L.(L.) amazonensis, addressing a better understanding on the role of Th17/IL-23 axis in the immunopathogenesis of ACL in Brazil. Thirty-one patients were examined, 6 from anergic diffuse cutaneous leishmaniasis (ADCL); 6 from borderline disseminated cutaneous leishmaniasis (BDCL), both caused by L.(L.) amazonensis infection (DTH–); 19 from localized cutaneous leishmaniasis (LCL), 13 caused by L.(L.) amazonensis and 6 caused by L. (V.) braziliensis. Paraffin-embedded biopsies were submitted to immunohistochemistry using as primary antibody anti-IL-17 (SC-7927) 1:250
diluition and anti-IL-23 (SC-271279), 1:200 dilution. For amplification and visualization of the reaction Novolink max polymer was used. The immunostained cells were counted in 10 fields (400x) in section by using an image analysis system (Zeiss). The comparison of IL-17+ cells density in the clinical-immmunological spectrum of ACL showed a progressive increase in IL17+ expression from the central LCL (DTH+) caused by L. (V.) braziliensis to the polar forms, ADCL and BDCL (DTH–) caused by L. (L.) amazonensis, as follows: ADCL DHT[506]>BDCL DHT-[384]>LCL/La[290]>LCL/Lb[289]. The density of IL-23+ cells showed a similar profile in the spetrum of disease, ADCL DTH-[946]>BDCL DTH-[817]>LCL/La[528] Supported by: LIM50/HC FMUSP; FAPESP (06/56319-1) - CELLULAR AND MOLECULAR BIOLOGY [821] P 168 - THE GAMMA-SECRETASE COMPLEX IN LEISHMANIA DEGROSSOLI, A.1; TEIXEIRA-PINTO, L.A.L.2; DE SIMONE, S.G.1 1.FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 2.UNIVERSIDADE FEDERAL FLUMINENSE, NITERÓI, RJ, BRAZIL.
Keyword:presenilin; gamma-secretase complex; pen-2
Abstract: Previous works in our laboratory led to the characterization of several proteinases from Leishmania parasite. One of these proteinases was identified as an aspartyl proteinase that has homology to the human presenilin, the enzyme of the gamma-secretase complex. In humans, the gamma-secretase complex cleaves type I transmembrane proteins and contains four proteins: presenilin, PEN-2, nicastrin and APH-1. Although the gamma-secretase complex is very well characterized in humans, its presence and function are not established in Leishmania. In this work we have identified the gamma-secretase complex proteins in L. amazonensis and L. infantum cells, as wells as described the molecular and structural characterization of the Leishmania presenilin. Using human sequences, we have investigated the presence of gammasecretase complex proteins in the Leishmania genome database by homology approaches. The expression of the complex proteins in Leishmania was verified by reverse transcriptase polymerase chain reaction (RT-PCR). The presenilin sequences from L. amazonensis and L. infantum were aligned using Clustal W. Transmembrane (TM) topology of the presenilin was investigated by bioinformatics approaches and hydropathy plots. Among the four proteins of the complex, we have identified only PS and PEN-2 in the Leishmania genome, with 31% and 35% of homology to each human protein, respectively. The nicastrin and APH-1 sequences were not identified in the Leishmania genome. The two forms of Leishmania, amastigote and promastigote, expressed the PS and the PEN-2 proteins, as verified by RT-PCR. The PS protein of L. amazonensis presented high degree of sequence identity with PS from L. infantum (94.32%). Its TM topology studies demonstrated that Leishmania PS contains eight hydrophobic regions that can function as TM domains. In this work we have demonstrated that genome of L. amazonensis and L. infantum has the sequence of two proteins of gamma-secretase complex, PS and PEN-2, and these proteins are expressed, suggesting the presence of the complex in Leishmania. We have also showed that the PS sequence is conserved between two different species of Leishmania. The PS protein of Leishmania presented TM domains indicating that it is a membrane integral protein, as well as the human presenilin. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [831] P 169 - TOLL-LIKE RECEPTORS 2, 4 AND 9 EXPRESSION IN SKIN AND MUCOSAL LESIONS OF AMERICAN CUTANEOUS LEISHAMANIASIS CAUSED BY LEISHMANIA (V.) BRAZILIENSIS AND L. (L.) AMAZONENSIS IN AMAZONIAN BRAZIL CAMPOS, M.B.1; MENEZES, J.1; GOMES, C.M.C.2; LAURENTI, M.D.2; CORBETT, C.E.P.2; LIMA, L.D.R.1; RAMOS, P.K.S.1; SILVEIRA, F.T.1 1.EVANDRO CHAGAS, BELEM, PA, BRAZIL; 2.UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL.
Keyword:toll-like receptors 2, 4 and 9; american cutaneous leishmaniasis; immunohistochemistry
Abstract: Background: Toll-like receptors (TLRs) seem to play a crucial role for promoting the activation of the innate and adaptive immune responses. This work investigated the expression of TLRs 2, 4 and 9 in the clinical spectrum of American cutaneous leishmaniasis (ACL) caused by L. (V.) braziliensis/L.b and L. (L.) amazonensis/L.a: localized cutaneous leishmaniasis (LCL), borderline disseminated cutaneous leishmaniasis (BDCL), mucosal leishmaniasis (ML) and anergic diffuse cutaneous leishamniasis (ADCL). Methods: Twenty ACL patients were examined: 5 cases of ADCL and 4 of BDCL and LCL by L. (L.) amazonensis; and 4 cases of LCL and 3 of ML by L. (V.) braziliensis. Paraffinembedded biopsies were submitted to immunohistochemistry using as primary antibody antiTLRs 2 (code and dilution), 4 (code and dilution) and 9 (code and dilution). The immunostained cells were counted in 10 fields (400x) in section by using an image analysis system (Zeiss). Data were stored in Biostat 5.0 program and analyzed by Kruskal-wallis test (P< 0.05). Results: TLR2 expression in LCL/L.b (419 mm2) was higher than those in ML/L.b (279 mm2), ADCL/L.a (274 mm2), BDCL/L.a (210 mm2) and LCL/L.a (160 mm2). TLR4 expression in LCL/ L.a (325 mm2) was higher than those in ADCL/L.a (249 mm2), BDCL/L.a (170 mm2) and ML/L.b (268 mm2), with exception in LCL/L.b (324 mm2). TLR9 expression in LCL/L.b (363 mm2) was also higher than those in DBCL/L.a (218 mm2), ADCL/L.a (202 mm2), ML/L.b (129 mm2), with exception in LCL/L.a (343 mm2). Conclusion: These results suggest the existence of a higher TLRs 2, 4 and 9 expression in LCL by L. (V.) braziliensis, which shows a clear T-cell hypersensitivity compared to that in LCL by L. (L.) amazonensis.
- CLINICAL AND EXPERIMENTAL IMMUNOLOGY [834] P 170 - ROLE OF GROUP V PHOSPHOLIPASE A2 IN L. MAJOR INFECTION ZAMITH-MIRANDA, D.; BRITO, R.S.; DIAZ, B.L. INSTITUTO DE BIOFĂ?SICA CARLOS CHAGAS FILHO - UFRJ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:phospholipase a2; th2; l. major
Abstract: L. major infection control is dependent on a Th1 response, while a Th2 response is associated with progressive lesions as seen in infected Balb/c mice. Innate immune cells, such as macrophages, neutrophils and dendritic cells, also play important roles in L. major infection in Balb/c mice by harboring the parasite and/or contributing to Th2 differentiation. Group V Phospholipase A2 (PLA2GV) is expressed in Th2 lymphocytes and in innate immune cells and was demonstrated to play a role in fungal phagocytosis by macrophages and antigen processing by dendritic cells leading to an allergic response. Thus, we investigated the hypothesis that PLA2GV participates in L. major infection by promoting parasite entry into macrophages and generating a Th2 immune response. Infection with 2 x 106 L. major promastigotes into the hind paw of PLA2GV deficient mice progressed indistinctly from lesions in wild type Balb/c throughout the first 3 weeks. Lesions in wild type mice continued to progress after 4 weeks while lesions in PLA2GV deficient mice plateaued. After 6-8 weeks of infection, PLA2GV
showed significantly smaller lesions and lower levels of IL-4 in footpad macerates, however there was no significant difference in parasite load. 72 h-activation by anti-CD3 of 3 weeksinfection lymph node cells led to reduced production of IL-4 in PLA2GV deficient cells and no difference in production of IFN-gamma, IL-10 or IL-17. There were also no differences in IL-6, IL-10 or IL-12 production by bone marrow-derived dendritic cells infected in vitro with L. major promastigotes from either genotype. Peritoneal macrophages or neutrophils from PLA2GV deficient or wild type animals infected with L. major in vitro showed no difference in cell activation. Our results suggest that PLA2GV may play a role in L. major infection by contributing to mount the Th2 response associated with susceptibility to infection. We found no indication that the role of PLA2GV in adaptive immune response in L. major infection also extends to innate cells. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [835] P 171 - PERSISTENT INFECTION BY LEISHMANIA BRAZILIENSIS MEDIATED BY CYTOKINES AND VIANA, S.M.; BRILHANTE, M.A.; GUERRA, P.V.; FIGUEIREDO, W.M.E.; CHAVES, C.S.; TEIXEIRA, M.J. UNIVERSIDADE FEDERAL DO CEARÁ, FORTALEZA, CE, BRAZIL.
Keyword:persistent infection; leishmania braziliensis; balb/c mice
Abstract: Leishmania braziliensis is the main causative agent of American cutaneous leishmaniasis in Brazil. Altough L. braziliensis infection is self-limited it is distinguished by its latency and chronicity and can persist in mice and patients even after spontaneous clinical cure or treatment. Little is known about the persistence feature in L. braziliensis’ infection in humans and mice. Thereby, the aim of this study was to charactherize the parasite’s persistence in the murine model. The parasite load, lesion thickness and pattern of citokynes and chemokines involved in persistence were evaluated through BALB/c mice infection with a L. braziliensis strain and observation for 90 days after infection. The results showed that parasites gradually disappear in the footpad, while the parasite load was sustained in the draining lymph node until 90 days postinfection (p.i). In the footpad, TNF- expression was higher at 30 days p.i, followed by the decrease in parasite load and lesion thickness in the site. IL-10 and TGF- were more expressed at first and decreased after 30 days of infection. A higher concentration of TNF- and IFN- was observed at 15 and 30 days post-infection in the draining lymph node, while IL-4 was significantly increased at 15 days post-infection, and IL-10 and TGF- were the predominant citokynes after 90 days of infection. CCL2, CCL3, CXCL1 e CXCL10 were expressed in the footpad, with a peak at 30 days p.i. and reduction at 60 days. In the draining lymph node, CCL2 and CXCL10 presented a low expression at first, increasing to a peak at 45 days p.i., while CCL3 was more expressed at 30 days p.i., the same period of the maximum lesion thickness. These data suggest that the continued production/expression of IL-10, TGF- , CCL2 and CXCL10 in the draining lymph node leads to a continuous recruitment of host cells and Th1 effective cells. This could contribute to parasite persistence and low parasite load maintenance in the lymph node, avoiding a future recurrence of the disease. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [836] P 172 - ROLE OF B-1 CELLS IN EXPERIMENTAL LEISHMANIA AMAZONENSIS INFECTION GONZAGA, W.F.K.M.1; GERALDO, M.M.2; VIVANCO, B.C.1; LOPES, J.D.1; XANDER, P.2 1.UNIVERSIDADE FEDERAL DE SÃO PAULO, SÃO PAULO, SP, BRAZIL; 2.UNIVERSIDADE FEDERAL DE SÃO PAULO CAMPUS DIADEMA, DIADEMA, SP, BRAZIL.
Keyword:l. amazonensis; b-1 cells; il-10
Abstract: Leishmaniasis belong to a group of diseases caused by protozoan parasites from Leishmania genus. The clinical forms depend both on the Leishmania species and the host response. L. amazonensis can lead to various clinical manifestations of leishmaniasis: localized cutaneous, diffuse cutaneous, and more rarely a severe form of visceral leishmaniasis. Experimental evidence suggests that B cells and/or antibodies are potentially involved in the development of infection caused by L. amazonensis. B-1 cells are a subtype of B lymphocytes whose role in the physiology of the immune system as well as in the pathogenesis of various diseases is still poorly understood. These cells have ability to engulf pathogens, to migrate to inflammatory focus and to modulate immune response in several experimental models, such as paracoccidioidomycosis and murine melanoma. In addition, B-1 cells produce large amounts of IL-10 cytokine, which plays a key role in immunosuppression in several diseases, such as leishmaniasis. However, the importance of these cells in leishmaniasis has not been clarified. The objective of this study was investigate the role of B-1 cells in experimental infection of L. amazonensis. Preliminary results demonstrated that Balb/c mice subcutaneously infected at the right hind-foot with 1x107 promastigotes of L. amazonensis for 70 days showed significant reduction in peritoneal B-1 cells population (28,46% Âą 5,43), as compared to non-infected group (45,30% Âą 5,05; p<0,01). These preliminary data suggest that B-1 cells population can be modulated in response to L. amazonensis infection. Further studies have been conducted to better understand the biological significance of these findings. The role of B-1 cells in the development of cutaneous leishmaniasis is also under investigation in our lab. - CELLULAR AND MOLECULAR BIOLOGY [838] P 173 - MINI-CIRCLE DIVERSITY IN LEISHMANIA CHAGASI GUSHI, L.T.; ALONSO, D.P.; RIBOLLA, P.E.M. DEPARTAMENTO DE PARASITOLOGIA - IBB - UNESP, BOTUCATU, SP, BRAZIL.
Keyword:mini-circles; kinetoplast; leishmania chagasi
Abstract: American Visceral Leishmaniasis (AVL) is a neglected tropical disease in expansion in Brazil. Leishmania chagasi, a protozoan belonging to the order Kinetoplastida named after the discovery of its most peculiar organelle, the kinetoplast, is the causative agent of the disease. Leishmania kinetoplast has an organized DNA network (kDNA) containing maxicircles, responsible for respiratory functions and minicircles, involved in the production of guide RNAs, which play a role in the RNA editing of maxicircles. The minicircles are divided into an approximately 120 bp conserved region and an approximately 600 bp variable region. The focus of this work is the sequence analysis of the minicircles variable region in order to understand its distribution on different life stages of Leishmania chagasi. Samples were collected from dogs and symptomatic patients by blood marrow aspiration and, in some cases, from primary cultures. After DNA extraction kDNA was PCR amplified, producing a fragment of 720 bp, and cloned for sequencing. The 182 obtained sequences were compared with the ones deposited at the GenBank. A phenetic tree was built to compare all individual sequences and also Principal Component Analysis (PCA) was undertaken. When analyzed in more detail sequences proved to be conserved, but some SNPs (Single Nucleotide Polymorphisms) could be found; these SNPs were used as criteria to separate classes. Based on these data, sequences were categorized in 53 classes. Some classes were observed only in amastigote samples. There was an interesting finding while comparing sequences with the ones available in the GenBank. Despite limited coverage, similarity to chromosome 27 of the Leishmania donovani was observed in all sequences, which leads us to question whether minicircles could have a post transcriptional editing role not only in maxicircles but also in nuclear DNA.
- CLINICAL AND EXPERIMENTAL IMMUNOLOGY [848] P 174 - EFFECTS OF CXCL10/GAMMA INTERFERON-INDUCIBLE PROTEIN 10 TREATMENT IN BALB/C MICE INFECTED WITH LEISHMANIA INFANTUM CHAGASI FIGUEIREDO, W.M.E.; VIANA, S.M.; GUERRA, P.V.; COÊLHO, Z.C.B.; CHAVES, C.S.; POMPEU, M.M.L.; TEIXEIRA, M.J. UNIVERSIDADE FEDERAL DO CEARÁ, FORTALEZA, CE, BRAZIL.
Keyword:l. infantum chagasi; cxcl10; citokynes
Abstract: Visceral leishmaniasis caused by Leishmania infantum chagasi is characterized by the loss of the ability of host to generate an effective immune response. In this study it was investigated the role of CXCL10 chemokine in controlling L. infantum chagasi infection in vivo. Groups of BALB/c mice were treated or not with recombinant CXCL10 chemokine (5 g/kg) with 1, 3 and 7 days of infection and after 1, 7 and 23 days of treatment, some parameters were evaluated: parasite load, levels of IFN-g, IL-4, TGF- and IL-10, and the histopathological alterations in the liver. After 23 days of treatment, CXCL10 induced in the spleen a significant reduction on the number of parasites as compared to control group (p=0,0272). In the liver, parasite load decreased in treated group between the 7th and 23th day post treatment (p<0,05). IFN- was induced most significantly in treated group than in controls, and reached its maximum production (100pg/mL) on day 23 after treatment, correlating with the reduction in parasite burden in target organs (p<0,05). IL-4 was produced in low doses, in both groups, although treated animals had shown higher levels than control group. Regarding to anti-inflammatory cytokines, after the 23th day of treatment, IL-10 levels in treated animals were smaller than in control group (p=0,0029). Production of TGF- after 7 days of treatment was 2 times lower in treated group when compared to control (p=0,0092), and after the 23th day of treatment, this cytokine remained with lower levels than those observed in control (p=0,0069). In the histopathological analysis of the liver after the 1st day of treatment, were found in both groups more immature granulomas (IG) than non-granulomatous infiltrate (NG), and some few mature granulomas (MG) were only observed in treated group. After 7 days of treatment, the amount of NG infiltrates began to decrease, and GI were still the most frequent in both groups, besides a slight increase of GM was observed in treated group. At the light of the found results, it is possible to suggest an important leishmanicidal role to CXCL10 in BALB/c mice infected by L. infantum chagasi, which seems to be mediated by a significant IFN-g production, and suppression of immunoregulatory cytokines, IL-10 and TGF- , opening the hypothesis that this would be associated to a decrease in the frequency of regulatory T cells induced by CXCL10 in these animals. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [849] P 175 - B-1 CELLS INCREASE SUSCEPTIBILITY TO MURINE VISCERAL LEISHMANIASIS XAVIER, V.1; VIVANCO, B.C.1; GONZAGA, W.F.K.M.1; BARBIERI, C.L.1; LOPES, J.D.1; XANDER, P.2
1.UNIVERSIDADE FEDERAL DE SÃO PAULO, SÃO PAULO, SP, BRAZIL; 2.UNIVERSIDADE FEDERAL DE SÃO PAULO CAMPUS DIADEMA, DIADEMA, SP, BRAZIL.
Keyword:l. (l.) chagasi; b-1 cells; il-10
Abstract: The imune response to leishmaniasis is complex and the result of infection depends both on the genetic composition of different species of Leishmania and the host immunity. Clinical and
experimental evidences suggest that activation of B cells leads to exacerbation of visceral leishmaniasis and contributes to susceptibility of cutaneous leishmaniasis. B-1 cells are a subtype of B lymphocytes whose biological role in immunity is not completely elucidated. In this study we investigated the role of B-1 cells in the pathogenesis of visceral leishmaniasis. Experiments were performed with BALB/c, BALB/Xid or BALB/Xid mice that receive adoptively peritoneal B-1 lymphocytes. These groups were infected or uninfected with Leishmania (Leishmania) chagasi. Our results showed that BALB/Xid mice, a mouse strain deprived of B-1 cells, infected for 45 days with L. (L.) chagasi had a significant reduction in parasite load in the spleen (0.63 × 107 ± 0.4 parasites/mg tissue) when compared to control animals BALB/c (1.87 × 107 ± 0.6 parasites/mg of tissue) or BALB/Xid that received adoptive transfer of B-1 cells (1.44 × 107 ± 0.5 parasites/mg tissue). These data were also confirmed by qPCR. Flow cytometry analysis demonstrated changes in peritoneal B-1 cell population. Infected BALB/c mice showed a significant increase in the percentage of peritoneal B-1 cells (70.0% ± 1.50) compared to control (63.3% ± 1.88). On the other hand, BALB/Xid that received adoptive transfer of B-1 cells and were infected with L. (L.) chagasi showed a significant decrease in the percentage of peritoneal B-1 cells (6.14 ± 3.01%) when compared to uninfected group (25.48 ± 7.60%). The cytokines production was evaluated in supernatants from spleen homogenates of uninfected and infected mice. No differences were detected between BALB/c uninfected and infected group. However, BALB/Xid that received adoptive transfer of B-1 cells and were infected with parasite had significant higher levels of IL-10 and IFN- when compared with unifected mice. However, Xid sera samples had lower amounts of IL-10 when compared to BALB/c or Xid that received adoptive transfer of B-1 cells. Our results suggest a possible role of B-1 cells in susceptibility to L. (L.) chagasi. - CELLULAR AND MOLECULAR BIOLOGY [850] P 176 - INFLUENCE OF AFAP1L1, SEPTIN 4 AND 14 IN INVASION AND INTRACELLULAR MULTIPLICATION OF LEISHMANIA AMAZONENSIS NETO, C.P.; SILVA, F.; MACHADO, F.C.; TEIXEIRA, T.L.; ARAÚJO, K.C.; QUINTAL, A.P.; ANDRADE, P.S.; SILVA, C.V. UNIVERSIDADE FEDERAL DE UBERLÂNDIA, UBERLÂNDIA, MG, BRAZIL.
Keyword:leishmania amazonensis; afap1l1; invasion
Abstract: Leishmania amazonensis is the agent responsible for the cutaneous form of leishmaniases. In the vertebrate host, such as humans, L. amazonensis keeps its cycle inside leukocytes, mostly, macrophages. Engulfment of Leishmania promastigotes by macrophages is conducted actively by the cytoskeleton of the host cell. During the intracellular traffic, the cytoskeleton leads several endocytic vesicules forming the phagolysosome which allows replication for the amastigotes forms. Here, we wanted to verifiy the importance of several host cytoskeleton molecules during crucial points of invasion and intracellular multiplication using knockdown macrophages for AFAP1L1 (Actin filament-associated protein 1 like 1), Septin 4 and 14. These cells lineages were already obtained by transduction of wild-type macrophages with lentiviral particles (Santa Cruz Biotechnology, Inc.) carrying a small harpin RNA capable of interfering in the expression of those molecules. Now, invasion (2 hours) and multiplication (24, 48 and 72) assays are being performed using these new lineages. For final analysis, 100 of total cells will be counted for invasion score and 100 infected cells for multiplication score. Financial support: FAPEMIG/UFU/CNPq.
- CLINICAL AND EXPERIMENTAL IMMUNOLOGY [852] P 177 - DIFFERENTIATED IMMUNE RESPONSE IN THE LIVER AND SPLEEN OF DOGS WITH CANINE LEISHMANIASIS. MOREIRA, P.R.R.; BANDARRA, M.B.; MUNARI, D.P.; ROZZA, D.B.; VASCONCELOS, R.O. UNESP - UNIVERSIDADE ESTADUAL PAULISTA, JABOTICABAL, SP, BRAZIL.
Keyword:canine leishmaniasis; immune response; cytokines
Abstract: The evaluation of the immune response in different compartments during a chronic infection is an useful strategy for the scientific study of the immune response in parasitic diseases. The aim of this study was to analyze the liver and spleen of dogs with canine leishmaniasis (CL), considering the parasite load, the density of natural killer cells (NK), apoptotic cells and cells immunostaining for cytokines TGF- and TNF- . These results were compared with the different stages of the disease by immunohistochemistry. We used 71 dogs naturally infected from an endemic area for CL, ranked in the symptomatic group (S), asymptomatic (A) and oligosymptomatic (O). The antibodies used were CD56 (Biocare), cleaved caspase3 (Cell Signaling), TGF- and TNF- (Santa Cruz Biotechnology) and for the parasite load was used positive hyperimmune serum for CL. The spleen was the organ with the highest parasite load, higher rate of apoptosis of lymphocytes and higher intensity of diffuse granulomatous inflammation in dogs with severe disease (group S). The liver was resistant to the parasite multiplication, due to lower parasite load, the cellular profile of granulomas, rich in lymphocytes and NK cells participate in the control of the parasite. NK cells in the spleen did not have a cytotoxic role in parasitized macrophages. The cytokines TGF- and TNF- had a low expression in both the spleen and the liver. However the group S was predominant TGFimmunostaining cells (Th2). In the asymptomatic group predominated TNF- (Th1). We conclude that there was a distinct pattern of immune response against the parasite in the liver and spleen, characterized by cellular and cytokine profile in each organ, principally in the group of symptomatic dogs. Financial support: Fapesp (Fundação de Amparo à Pesquisa do Estado de São Paulo, processo n° 2009/15736-7). - CELLULAR AND MOLECULAR BIOLOGY [858] P 178 - CHARACTERIZATION OF PUTATIVE HEMOLYSINS TYPE III GENES IN LEISHMANIA MAJOR MIRANDA, N.; GRYMBERG, P.; DIAS, S.; FRANCO, G.R.; HORTA, M.F.M. UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL; ; ; ; .
Keyword:leishmania; leishporin; caracterization
Abstract: Previous works from our group had shown that Leishmania amazonensis promastigotes have a molecule that lyses erythrocytes and damages macrophages, which we named leishporin. This cytolysin has already been characterized in a number of biochemical aspects. However, its molecular identity is still unknown. Since the genome of the Leishmania major is now completely sequenced, we have performed a data mining effort to identify putative cytolysin genes in this species, in attempt to identify a gene that would possibly code leishporin . We found four sequences containing the Pfam domain PF03006, named hemolysins type III-related. It is also worth noting that these sequences showed a significant homology to other characterized pore-forming-proteins. Real time PCR was performed to investigate the relative quantities of these four genes in L.major promastigotes and amastigotes. Our results demonstrated a higher expression of LmjF36.5520 mRNA in both pro and amastigotes, while the expression levels of the LmjF36.5500 and LmjF06.0750 mRNAs did not differ much.
LmjF36.5510 mRNA was not detected in the experiment, suggesting that this gene is poorly expressed. In order to determine the subcellular localization of these proteins, polyclonal antibodies for three of them (except for LmjF36.0750, which is still being cloned) were produced in BALB/c mice and used in confocal immunofluorescence microscopy. Apparently, the proteins encoded by the genes LmjF36.5520 and LmjF36.5500 are localized at a lysossome compartiment, termed multivesicular tubule (MVT) that extend from near the flagelar pocket to the posterior end of the cell. Confirming the qRT PCR results, we could not detect fluorescence for the protein encoded by the gene Lmjf36.5510. We are now performing co-localization with an antibody against the lysossome to confirm these results. Stable transfected parasites, overexpressing the hemolysins type III genes will be generated in order to verify the hemolytic and the pore-forming activity of the proteins encoded by these genes. In addition, orthologues of the genes presented in L.guianensis will be silenced by interference RNA in order to evaluate changes in hemolytic phenotype. Supported by CAPES and CNPq.
- CLINICAL LEISHMANIASIS [864] P 179 - A STUDY ABOUT THE CLASSIFICATION AND LOCATION OF THE LESIONS OF PATIENTS WITH AMERICAN CUTANEOUS LEISHMANIASIS ATTENDED AT THE CENTER OF TROPICAL MEDICINE OF RONDONIA, WESTERN AMAZON, BRAZIL FERNANDES, L.S.1; JUNIOR, C.F.S.2; BAIÃO, A.1; AZEVEDO, F.S.1; CANTANHÊDE, L.M.3 1.FEDERAL UNIVERSITY OF RONDONIA, PORTO VELHO, RO, BRAZIL; 2.FEDERAL UNIVERSITY OF RONDONIA AND CENTER OF TROPICAL MEDICINE OF RONDONIA, PORTO VELHO, RO, BRAZIL; 3.OSWALDO CRUZ FOUNDATION, PORTO VELHO, RO, BRAZIL.
Keyword:lesions; american cutaneous leishmaniasis; rondonia
Abstract: The American Cutaneous Leishmaniasis (ACL) is a parasitic infectious disease characteristic of tropical regions of the world and is transmitted through a mosquito vector of the genus Lutzomyia. The disease may present a wide variety of clinical forms and ulcerated lesions on various parts of the body, single or multiple form. The immunopathology and species of Leishmania spp. can be decisive factors in the manifestation of various clinical forms of ACL in several endemic regions. The objective of this study was to observe the location of the lesions and classify them according to known clinical forms of the disease manifestation by the Ministry of Health of Brazil. It was observed 58 patients diagnosed with ACL treated at the Center of Tropical Medicine of Rondonia (CEMETRON) between June 2011 and December 2012, being an observational descriptive case series. During the period the patients were seen at the ambulatory care of dermatology at CEMETRON and had their injuries recorded and photographed by digital camera for later analysis of the locations and classifications more prevalent. Of 58 patients with ACL, 35 (60.34%) had Localized Cutaneous Leishmaniasis form (LCL), 7 (12.06%) with Multiple Localized Cutaneous Leishmaniasis form (MLCL), 10 (17.24%) with Mucocutaneous Leishmaniasis form (MCL), 4 (6.89%) with Disseminated Cutaneous Leishmaniasis form (DCL) and 2 (3.44%) with Borderline Disseminated Cutaneous Leishmaniasis (BDCL). About the location of the injury in the patients, 16 (27.58%) had lesions only in the left lower limb, 12 (20.68%) only in the right lower limb, 7 (12.06%) in the nasal region, 4 ( 6.89%) only in the right upper limb, 3 (5.17%) only in the left upper limb, 7 (12.06%) in other body regions and 9 (15.51%) in two or more different regions. After analyzing the results it is possible to ascertain that the most common form of ACL found in patients treated at CEMETRON during that period is the cutaneous form, with 82.73% of cases, being the localized form (LCL) the most prevalent. It can also be concluded that the lower limbs are the regions most affected by injuries (48.27%), probably because it is the area most exposed
to the contact of the vectors that transmit the disease. Therefore, the using of pants, closed shoes and long-sleeved shirts in endemic areas can minimize infection risks by ACL. - CELLULAR AND MOLECULAR BIOLOGY [865] P 180 - 4E3AG AMASTIGOTE ANTIGEN OF LEISHMANIA (VIANNIA) PANAMENSIS IS A MARKER FOR METACYCLOGENESIS AND FOUND IN EXOSOMES CASTILHO, T.M.1; MOON, K.2; LAMBERTZ, U.2; REINER, N.E.2; FOSTER, L.J.2; MCMAHONPRATT, D.1
1.YALE UNIVERSITY, NEW HAVEN, UNITED STATES; 2.THE UNIVERSITY OF BRITISH COLUMBIA, VANCOUVER, CANADA.
Keyword:leishmania(viannia); metacyclogenesis; exosomes
Abstract: The mechanisms underlying the pathogenesis of infections caused by Leishmania (Viannia) subgenus organisms are not fully understood, yet it is dependent on both host and parasite factors. It is well-documented that mechanisms of pathogenesis and consequent immune responses vary amongst the Leishmania species. Therefore, identification of molecules selectively expressed by L. (Viannia) metacyclic promastigotes or amastigotes could provide particular insights concerning pathogenesis. BALB/c mice infected with late stationary phase promastigotes (LSPP) of L.(V.)panamensis present chronic lesions. Interestingly, amastigote antigens of L.(Viannia) organisms are also expressed in a small subpopulation of LSPP. Among these, 4E3Ag is detected on the surface and cytoplasm within axenic amastigotes and LSPP. Metacyclogenesis in L.(Viannia) organisms has not been extensively studied; however in previous work with L.(V.)panamensis promastigotes, increased infectivity was associated with a reduction of parasite size. CGS15943 (9-Chloro-2-(2-furanyl)-[1,2,4]triazolo[1,5-c]quinazolin5-amine), an adenosine transporter antagonist, prevents cell growth and induces metacyclogenesis in L.(Leishmania) organisms. In L.(V.)panamensis, CGS15943 not only ablates promastigote growth but also induces 4E3Ag expression to the higher levels found in axenic amastigotes. Preliminary data indicate that CGS-treated promastigotes generate chronic lesions in BALB/c mice. These data validate the 4E3Ag as a metacyclic marker for L.(V.)panamensis. Further, the presence of the 4E3Ag on the L.(V.)panamensis surface and in the flagellar pocket area suggested a connection with secreted membrane vesicles found in these locations in the case of L.(Leishmania). Indeed, 4E3Ag is found within the exosomes of axenic L. (V.) panamensis amastigotes. The cargo of exosomes from both life cycle stages is being characterized by quantitative mass spectrometry. Preliminary analysis indicates at least 12 proteins that have their secretion up-regulated in exosomes of amastigotes and at least 16 are selectively found in promastigote exosomes. In summary, the amastigote 4E3Ag has been found to be a marker for L.(V.)panamensis metacyclogenesis; 4E3Ag is also present in amastigote exosomes, which contain stage-specific cargo. This work identifies genes potentially involved in unique aspects of the L.(V.)panamensis-host pathogenesis, which will be of interest for further studies. - CELLULAR AND MOLECULAR BIOLOGY [869] P 181 - MOLECULAR MODELING OF THE INTERACTION BETWEEN THE ENDONGENOUS CYSTEINE PROTEASE INHIBITOR OF LEISHMANIA INFANTUM AND LYSOSOMAL CYSTEINE PROTEASES B AND H COSTA, G.F.; MENDES, T.A.O.; MOURA, A.L.; BARTHOLOMEU, D.C.; FUJIWARA, R.T. UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL.
Keyword:cystein ; protease; leishmania
Abstract: The Leishmania genus includes several species of protozoa parasites that cause diseases to human. These parasites infect phagocytic cells surviving within vacuoles parasitophorous (VP) that fuse to lysosomes. These organelles have different components related to degradation of intracellular and exogenous proteins such as parasite macromolecules and among these components are cysteine protease as cathepsins B, H and L. However, those events do not result in parasite elimination because it probably have mechanisms to survive in this enviroment. Studies have demonstrated the existence of a cysteine protease inhibitor (ICP) encoded in the genome of L. mexicana and this protein was also observed in bacterias and other trypanosomatids. Previous works showed this endogenous ICO of L. mexicana may bind reversibly with human cathepsins B. ICP also occurs in trypanosomes and bacteria. The ICP structure of L. mexicana (LmICP) was elucidated and it suggested that this molecule plays a more prominent role in parasite-host interaction than in parasite enzymes control. We identify and evaluate the binding mode of ICP of L infantum (LiICP) with two cysteine proteases lysosomes specific named cathepsin B and H. LiICP has 73% of identity with previously identified ortholog LmICP and ICP structure of L. infantum was modeled based on the structure of protein L. mexicana using the program MODELLER 9.9. The stereochemical quality of the generated model was evaluated using the platform Procheck. The binding mode of LiICP with lysosomal cysteine proteases has been obtained by the program HADDOCK 2.1 with fixed structure for protease and ICP flexible. LiICP has distinct regions that bind differently in the catalytic-site of each cathepsins. For cathepsin B, activity inhibitory aminoacids of LiICP are present in discontinuous motifs concentrated in the amino and carboxyl-terminal regions, while cathepsin H inhibitory amino acids are in central regions of protein. These amino acids are also highly conserved among ICPs orthologs of L. mexicana, L. braziliensis and L. major suggesting that inhibit different types of lysosomal cysteine proteases and this may be a mechanism conserved among the different species of the genus Leishmania that may contribute to parasite survival within a parasitophorous vacuole fused with lysosome.
- CLINICAL AND EXPERIMENTAL IMMUNOLOGY [871] P 182 - DIFFERENT METHODS FOR OBTAINING RECOMBINANT CANINE INTERLEUKIN-7 AND INTERLEUKIN -15 WITH BIOLOGICAL ACTIVITY IN VITRO PAIXĂƒO, L.S.; PINHEIRO, C.G.M.; DE OLIVEIRA, B.M.N.; DE CARVALHO, L.C.P.; OLIVEIRA, G.G.S. CENTRO DE PESQUISAS GONÇALO MONIZ, SALVADOR, BA, BRAZIL.
Keyword:canine leishmaniasis; imunotherapy; cytokines
Abstract: Most dogs with visceral leishmaniasis (VL) subjected to conventional chemotherapy relapses after discontinuation of treatment, possibly due incapability of maintaining specific cellular immune responses. Many studies have demonstrated the essential role of IL-7 and IL-15 in the homeostasis of T-cells. The IL-7 is a growth and anti-apoptotic factor of T lymphocytes, essential for maintaining naive, mature and especially memory CD4+ T cells. IL-15 presents a wide spectrum of biological activities. It is crucial for the development, proliferation, survival and differentiation of multiple cells types of innate immunity as well as of adaptive immunity, and plays an essential role in the maintenance of CD8+ memory T cells. These cytokines are described in the literature as being capable of promoting long term cellular immune response. In this study, canine IL-7 and IL-15 was expressed, different protocols for refolding them were
tested and the biological activity in vitro was assessed by cell proliferative assay. Canine IL-7 and IL-15 were expressed in E. coli BL21(DE3)pLysS strain, and purified by Sepharose-Ni affinity chromatography. Canine IL-7 was refolded by dilution method whilst canine IL-15 was refolded by dilution and dialysis methods. For evaluation of biological activity of the produced cytokines, a sample of IL-15 obtained in both refolding methods was used to stimulate CTLL-2 cells while a sample of IL-7 of the single method was used to stimulate lymphoblasts from a healthy adult dog. As a result, although both protocols used for IL-15 refolding have been effective, yielding around 90-95% of refolded protein, they stimulate weakly CTLL-2 cells to proliferate. Nevertheless, despite the protocol used for canine IL-7 has yielded only 6% of refolded protein, this cytokine was able to stimulate lymphoblasts proliferation. In order to improve the obtainment of refolded canine IL-7 and IL-15, at this time, a baculovirus insect cell system, an eukaryotic protein production method, is being used. Furthermore, experiments to evaluate canine IL-7 and IL-15 biological activities and toxicity assays in vivo are in progress aiming at using these cytokines in the immunotherapy against canine visceral leishmaniasis. - CELLULAR AND MOLECULAR BIOLOGY [872] P 183 - HIV-1 AND LEISHMANIA CO-INFECTION: THE ROLE OF VIRAL PROTEIN TAT MEDIATED BY PROTEIN KINASE R (PKR) SIGNALING IN IMUNE RESPONSE VIVARINI, A.C.1; GRIMMER, G.H.1; PEREIRA, R.M.S.1; SOUZA, V.B.2; SARAIVA, E.M.3; BOUHABIB, D.C.2; LOPES, U.G.1 1.IBCCF-UFRJ, RIO DE JANEIRO, RJ, BRAZIL; 2.FIOCRUZ-RJ, RIO DE JANEIRO, RJ, BRAZIL; 3.IMPPG-UFRJ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:leishmania; pkr; tat-hiv1
Abstract: Protozoan parasites appear as human immunodeficiency virus (HIV)-1 co-pathogens, resulting in a mutual enhancement of viral and parasite replication, and facilitating the clinical progression of both diseases. The molecular mechanisms by which HIV-1 induces upmodulation of protozoan replication are unknown. In this work, we investigated the role of HIV1 transcriptional transactivator (Tat) protein in the enhancement of Leishmania amazonensis replication in macrophages cells in a Protein Kinase R (PKR)-dependent manner. Tat is a regulatory protein of HIV-1 responsible for viral transcription and replication. It also modulates the expression of several host genes through the activation of the transcription factor NF- B through PKR signaling. Previous data demonstrated that the treatment of human macrophages with recombinant Tat favors L. amazonensis infection, and that this protozoa parasite is able to increase PKR expression and activities. To investigate whether Tat modulates the infection of Leishmania through PKR, we pursued in this work analyze the signaling pathway between Tat and PKR that converge to L. amazonensis replication. Using PKR dominant-negative cells and the treatment with 2-aminopurine (2AP), a PKR pharmacological inhibitor, versus wild-type cells, we verify that recombinant Tat promotes increased of Leishmania in murine and human macrophages, that is reversed in the absence of PKR signaling. Next, we verify that Tat synergize with L. amazonensis in PKR expression by promoter activation, and increases of mRNA and total protein, in addition to PKR phosphorylation. We also demonstrated the involvement of NF- B in context of HIV-1 Tat and L. amazonensis. EMSA and reporterLuciferase assays reinforce that cells infected with L.amazonensis before the treatment with Tat was able to increase the same complex activated in cells infected only with Leishmania, and reversed the NF- B promoter activation in a PKR-dependent manner, respectively. Taken together, we demonstrated that Tat stimulates Leishmania replication via induction of PKR synthesis and activation. In conclusion, we identified, for the first time, the molecular mechanism promoted for an HIV-1 molecule that synergize with host kinase protein and favoring multiplication of a pathogenic protozoan.
- CLINICAL AND EXPERIMENTAL IMMUNOLOGY [873] P 184 - DIFFERENCES IN THE LEISHMANIA MAJOR-SPECIFIC ANTIBODY REPERTOIRE OF BALB/C AND C57BL/6 MICE DEVELOPED AFTER INJECTION OF LIVE OR KILLED PARASITES. SASSI, A.1; OUALHA, R.1; KAAK, O.1; BORCHANI, L.2; GUIZANI, I.1 1.LMEEP (LR11IPT04). INSTITUT PASTEUR DE TUNIS, TUNIS, TUNISIA; 2.LVT. INSTITUT PASTEUR DE TUNIS, TUNIS, TUNISIA.
Keyword:leishmania major; antibody repertoire; mhc class ii alleles
Abstract: The murine model using BALB/c, sensitive to infection with Leishmania major, and C57BL/6, resistant mice, is the most investigated system to understand the mechanisms of resistance to Leishmania parasites and to unravel effective serodiagnostic tools and vaccines. The kinetics of leishmanial-specific antibodies IgG2a and IgG1, which indirectly reflect the Th1 (resistance, IFN-gamma production) and Th2 (susceptibility, IL-4 production) cellular responses respectively, were previously used as markers of the T helper type induced in L. major-infected mice. We aimed in this work to analyze by ELISA and Western blotting the kinetic of IgG1 and IgG2a development after injection of L. major live amastigotes and their antigenic reactivity with leishmanial crude preparations. Results were compared with those obtained when both mice strains developed Th1 immune response after an injection of killed amastigotes. Our data showed wide qualitative and quantitative differences in the recognition of leishmanial crude extracts by mice sera collected from day 17 to 142. BALB/c mice developed higher levels of IgG1 but also of IgG2a antibodies than C57BL/6. Interestingly, in BALB/c mice IgG1/IgG2a ratios, representative of Th2 response, were at their highest around 23-37 days post infection, whereas IgG2a/IgG1 antibody ratios, representative of a Th1 response, peaked at the healing period for C57BL/6 mice around 115-142 days post infection. Mice injected with killed parasites had lower antibody titers than infected ones, and both mice strains produced similar levels of IgG1 and IgG2a antibodies with a predominance of the IgG1 isotype. Moreover, western blotting analysis showed that serum reactivity with leishmanial protein fractions was different between the two mouse strains during either the infection or the immunization experiments. Observed differences in the antibody repertoires developed during L. major infection are not due only to their susceptibility/resistance but also to their MHC genetic background. Hence, selection of leishmanial proteins to be used in serodiagnosis/vaccine trials investigations based on infection experiments alone should be interpreted with caution. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [874] P 185 - T CELLS IMMUNE RESPONSE INDUCED BY CYCLOPHILIN A, SUPEROXIDE DISMUTASE AND AMINOPEPTIDASE L. INFANTUM RECOMBINANT PROTEINS BOLAS, A.S.1; RODRIGUES, M.A.1; TEIXEIRA, F.2; SANTOS-MATEUS, D.1; CARVALHO, S.2; TOMĂ S, A.M.2; SANTOS-GOMES, G.M.1
1.CMDT- INSTITUTO DE HIGIENE E MEDICINA TROPICAL, UNIVERSIDADE NOVA DE LISBOA, LISBON, PORTUGAL; 2.IBMC - INSTITUTO DE BIOLOGIA MOLECULAR E CELULAR, UNIVERSIDADE DO PORTO, PORTO, PORTUGAL.
Keyword:cellular immunity; recombinant proteins; cytokines
Abstract: Intra-macrophage Leishmania parasites lead to the development of complex processes involving both innate and adaptive immune mechanisms crucial for the success of infection. It is universally accepted that the nature of T-cell response is one of the key factors controlling leishmaniosis. Resistance has been mediated by the activation of Th1 cells producing interferon (IFN)-gamma and interleukin (IL)-12. In contrast, Th2 cells releasing IL-10 promote
macrophages deactivation, helping intracellular parasite growth and disease progression. T-cell differentiation from na誰ve T cells to either to Th1- or Th2-effector cells depends chiefly on the priming. IL-12 is a heterodimeric cytokine that plays a central role in promoting Th1 cell differentiation and enhancing lymphocyte-activated cytotoxic T cells. Therefore this study aims to investigate cellular immune response induced by cyclophilin A (FA), superoxide dismutase (SOD) and aminopeptidase (AMP) L. infantum recombinant proteins by evaluating cytokine gene expression in CD4+ and CD8+ T cell subpopulations during L. infantum infection. Mice immunize with FA, SOD or AMP were challenged with L. infantum virulent promastigotes. One month after infection, CD4+ and CD8+ T cell subsets were isolated from spleen and RNA extracted. The gene expression of IL-12, IFN-gamma and IL-10 was quantified by real time PCR. Mice immunized with FA and AMP showed accumulation of IL-12, IFN-gamma and IL10 mRNA by CD8+ T cells pointing to the activation of cytotoxic T cells associated to a reduction in parasite load. Immunization with SOD restrained parasite replication and induced the development of an inflammatory immune response predominantly generated by CD4+ T cells. These recombinant proteins confer partial protection to infected mice exerting a differential regulation on helper and cytotoxic T cells differentiation. This work was funded by the Portuguese Foundation for Science and Technology the European Union (FEDER) through projects PTDC/CVT/70275/2006 and PTDC/CVT/113121/2009. Rodrigues A. hold the PhD Scholarship SFRH/BD/73386/2010 awarded by the Portuguese Foundation for Science and Technology.
- CELLULAR AND MOLECULAR BIOLOGY [879] P 186 - CHARACTERIZATION OF REPLICATION ORIGIN OF LEISHMANIA DONOVANI SINGH, T.; SRIVASTAVA, P.; CHAKRAVARATY, J.; SUNDAR, S. INSTITUTE OF MEDICAL SCIENCES, BANARAS HINDU UNIVERSITY, VARANASI, INDIA.
Keyword:replication initiation point; switch region; replication machinery
Abstract: Chromosomal origins of DNA replication in eukaryotic cells not only are crucial for understanding the basic process of DNA duplication but also provide a tool to analyze how cell cycle regulators are linked to the replication machinery. Moreover, they could be a potential therapeutic and diagnostic targets. During the past decade much progress has been made in identifying replication origins in eukaryotic genomes but unfortunately genus Leishmania is exception where it is not yet known. More recently, replication initiation point (RIP) mapping has allowed us to detect start sites for DNA synthesis at the nucleotide level and thus to monitor replication initiation events at the origin very precisely. No centromeric sequences or replication origins have been identified in any species of genus Leishmania. Though some studies bioinformatically suggest that transcription switch region (AT rich region) may be the actual site for replication origins, but no one has mapped it exactly. This pathogen is extremely important although poorly understood from the basic biology point. Not a single effective vaccine is available for either of these pathogens. There are reports of drug resistant strains too. Our aim is to find the key regulators in DNA replication processes so that we can identify potential targets for therapy.We are testing the switch region and non transcribed spacer region of leishmania chromosome as probable site of initiation of replication.(experiments are going on). The DNA replication process offers various important and interesting targets for new inhibitors development. Understanding the mechanism of initiation of DNA replication in L. donovani will be useful in finding new targets for screening
new antibiotics to eradicate these pathogens. Parasite DNA replication and cell cycle regulation are topics of intense current interest not only from the perspectives of eukaryotic DNA transactions but also of Cancer Biology.
- CLINICAL AND EXPERIMENTAL IMMUNOLOGY [880] P 187 - RECALL IMMUNOLOGICAL RESPONSES IN HUMAN CUTANEOUS LEISHMANIASIS. CRISTAL, J.R.1; COSTA, J.2; SANTANA, G.2; NARDY, V.2; SALGADO, V.2; BRODSKYN, C.2; ANDRADE, D.2; BARRAL-NETTO, M.1; DE OLIVEIRA, C.I.2; BARRAL, A.1 1.FACULDADE DE MEDICINA DA BAHIA, SALVADOR, BA, BRAZIL; 2.CENTRO DE PESQUISAS GONÇALO MONIZ FIOCRUZ, SALVADOR, BA, BRAZIL.
Keyword:cutaneous leishmaniasis ; immune response; delayed-type hypersensitivity
Abstract: The involvement of cell-mediated immunity in the protection against cutaneous leishmaniasis (CL) is well established, however, persistence of cellular immunity has been questioned. It has been argued that parasite persistence is associated with immunological memory but where parasites persist, following clinical cure is also unclear. In the present report we took advantage of a cohort of individuals from an endemic area for CL. We have obtained blood samples as well as performed delayed-type hypersensitivity (DTH) at 2006 and repeated this evaluation in 2011. During this period, individuals were clinically examined for the presence of cutaneous ulcers. We then established three groups of individuals based on their status at the 2006 evaluation: individuals with a previous history of CL and a positive DTH (DTH+); individuals without a previous history of CL and DTH+; individuals without previous CL and a negative DTH (DTH-). During the second evaluation, in 2011, each group was split in two, based on the positivity of the DTH reaction: CL+ DTH+ DTH+ (n=8); CL+ DTH+ DTH- (n=6); CLDTH+DTH+ (n=8); CL-DTH+DTH- (n=3); CL-DTH-DTH+ (n=15) and CL-DTH- DTH(n=12). At this time point, PBMCs were obtained from all groups and PBMCs were stimulated in vitro to determine cytokine production. In 2011, after five years of the initial evaluation (2006) individuals with or without a previous DTH+ produced higher IFN- levels than those who were consistently DTH-. Similar findings, albeit not as marked, were observed for TNFand for IL-10 production. Of note, IFN- levels at the 2nd evaluation (2011) were similar for the groups of individuals who had DTH+ at 2006 with or without a previous history of clinical CL. We can suggest that DTH positivity may act a marker of persisting anti-Leishmania cellmediated immunity. We also are currently evaluating the presence of parasite DNA in these groups of individuals. - GENETICS, EVOLUTION AND TAXONOMY [883] P 188 - GENETIC VARIABILITY AMONG SPANISH LEISHMANIA INFANTUM MON-1 ISOLATES REVEALED BY A SIMPLE APPROACH: ITS AND K26 GENE TYPING LLANES-ACEVEDO, I.P.; CHICHARRO, C.; CRUZ, I. WHO COLLABORATING CENTER FOR LEISHMANIASIS, INSTITUTO DE SALUD CARLOS III, MAJADAHONDA, SPAIN.
Keyword:molecular typing; its; k26
Abstract: Sequence analysis of the ribosomal internal transcribed spacers (ITS)-1 and -2 has been applied by other authors to infer phylogenetic relationships among different groups and to characterize strains of the L. donovani complex, showing that this complex had the lowest degree of ITS
sequence polymorphism when compared with other Leishmania complexes (Kuhls et al. 2005). Interestingly almost no differences in the ITS sequences were found within L. infantum strains, only two sequence types (A and B, or the combined A/B) were found in strains from the Mediterranean region. Although that paper was the most comprehensive study by that date based on sequence analysis of the ITS regions of strains of the L. donovani complex, it did only include 6 Spanish L. infantum strains (only two from the MON-1 zymodeme, both typed as ITS type A). The study of the amplicon size of the k26 gene has also been applied for typing strains of the L. donovani complex (Haralambous et al. 2008), this method was able to identify higher variability among Mediterranean L. infantum strains; however, it did include only 5 L. infantum strains (only two from the MON-1 zymodeme, both typed as k26 group 1b, 626 bp). Given that zymodeme MON-1 is the most frequently isolated in Spain, and taking into account that this country has shown the highest degree of variability for L. infantum at the zymodeme level, we attempted to ascertain whether these two approaches were able to identify further variability among Spanish MON-1 strains in a bigger sample size. For that purpose we selected from our collection an assembly of 96 MON-1 strains isolated between 1987-2005, 71 from the peninsula (P) and 25 from the Balearic Islands (BI), 18 from HIV- and 70 from HIV+ individuals, 8 unknown HIV status. Analysis of the ITS regions identified 3 different sequence types; 89 strains presented the ITS type A, while two other not-previously-described types were identified for 6 and 1 strains. All strains from the BI were typed as ITS type A, while one of the notpreviously-described ITS type was identified only in Madrid, and the second in Alicante. The analysis of the k26 gene amplicon size returned 6 different genotypes; the previously described 626 bp size was identified in 75 strains, being the most frequent in the P and the BI, the other genotypes were not associated to any particular region. By combining data from the ITS sequence and the k26 amplicon size we generated 8 different combined genotypes. We show that this simplified approach identifies a high variability among MON-1 isolates, this not observed before using the same approach, very likely because of the small sample size, a point to be considered in further studies. Funding: IP Llanes-Acevedo is supported in part by FUNDACIÓN COLFUTURO (Colombia) - GENETICS, EVOLUTION AND TAXONOMY [886] P 189 - LACK OF ASSOCIATIONS OF GENETIC VARIANTS OF SLC11A1 (NRAMP1) TO CUTANEOUS LEISHMANIASIS IN THE AMAZONAS SOUZA, L.M.1; BENTES, P.1; TALHARI, A.1; NOZAWA, S.2; GUERRA, J.1; SARDINHA, F.1; MIRA, M.1; TALHARI, S.2; RAMASAWMY, R.3 1.FUNDAÇÃO DE MEDICINA TROPICAL DR. HEITOR VIEIRA DOURADO, MANAUS, AM, BRAZIL; 2.UNIVERSIDADE NILTON LINS, MANAUS, AM, BRAZIL; 3.FUNDAÇÃO DE MEDICINA TROPICAL DR. HEITOR VIEIRA DOURADO. UNIVERSIDADE NILTON LINS, MANAUS, AM, BRAZIL.
Keyword:leishmania guyanensis; genetics; amazonas
Abstract: T helper cell type 1-mediated immunity is essential for human defense against Leishmaniainfection in tegumentary leishmaniasis. However, an exaggerated type 1 immune response is associated with mucosal leishmaniasis (ML). ML follows cutaneous leishmaniasis (CL). In the Amazonas, mostly Leishmania guyanensis caused tegumentary leishmaniasis. Only a subset of individuals infected with L. guyanensis develop chronic disease such as cutaneous or mucosal. Familial aggregation of CL or ML in areas of endemicity indicates that susceptibility may be genetic. Genetic variants of SLC11A1 (NRAMP1) have been linked to visceral leishmaniasis. Here, we assessed whether variants (rs7576974, rs2276631, rs2290708, rs3816560 and rs2279015) of SLC11A1 are associated with CL. Allelic identification of the variants in 302 patients with CL and 302 healthy individuals was performed by RT-PCR with TaqMan technology. All the variants studied were in Hardy-Weinberg equilibrium. However, the
distribution of allelic or genotypic frequency in both cases and controls were similar. Our results suggest that the above variants are not associated with susceptibility to CL. However, to exclude definitely SLC11A1 as a susceptibility locus to CL, further studies are needed from other endemic area of CL. Acknowledgement: This study was financed by FAPEAM and CNPq. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [888] P 190 - TROPISM OF LEISHMANIA (L.) CHAGASI FOR DIFFERENT ORGANS IN DOGS SHOWING CANINE LEISHMANIASIS. BANDARRA, M.B.; FERNANDO, F.S.; ANDRÉ, M.R.; MOREIRA, P.R.R.; MONTASSIER, H.J.; VASCONCELOS, R.O. UNESP - UNIVERSIDADE ESTADUAL PAULISTA, JABOTICABAL, SP, BRAZIL.
Keyword:leishmania chagasi; dog; qpcr
Abstract: The immune response developed by dogs showing canine leishmaniasis (CL) varies among different organs. The knowledge of this sort of immune response compartimentalization is contributing to a better understanding of mechanisms of evasion/modulation of the immune response to L. chagasi. The present work aimed at quantifying, by qPCR, the parasite load in several organs collected from L. chagasi-naturally infected dogs from Araçatuba city, São Paulo state, an endemic city for CL. Spleen, liver, popliteal and pre-scapular lymph nodes samples were collected from 16 L.chagasi-infected dogs immediately after death, and stored at 80ºC. In order to perform absolute quantification of parasite load, serial 10-fold dilutions of a plasmid (pIDTSMART-AMP vector - IDT) containing a 90 bp fragment of a L. chagasi-single-copynumber DNA polymerase gene were used as standards in a qPCR. The software generated a standard curve by plotting the threshold cycle (CT) against the values of each plasmid-standard dilution. Amastigotes were predominantely found in the spleen, followed by popliteal and prescapular lymph nodes. Liver was the organ where the lowest parasite load was found. The parasite load was significantly different (p<0.05) between spleen and lymph node, and spleen and liver samples. Additionally, popliteal and pré-scapular lymph nodes showed different parasite load when compared to liver samples (p<0.05). The present work showed that the spleen is the organ where a higher parasite load is found, followed by peripheral lymph nodes, indicating a parasite tropism to these organs. After L. chagasi penetrates in the organism, it is drained, remaining in by lymph nodes. After amastigotes reaching the blood stream, they are collected by spleen. This finding may be a possible explanation why this parasite is predominantely found in spleen. In the other hand, it seems that liver is capable of controlling the parasite load through the activity of Kuppfer and natural killer cells, which could eliminate the majority of L.chagasi-infected macrophages. In conclusion, the quantification of the parasite load in different organs from naturally infected dogs may contribute to a better understanding of the immunopathology of the disease. Financial support: Fapesp (Fundação de Amparo à Pesquisa do Estado de São Paulo; Process n° 2010/16030-8) - VACCINE DEVELOPMENT AND TRIALS [889] P 191 - IMMUNE RESPONSES AND PROTECTION INDUCED BY LEISHMANIA L. DONOVANI A2 ANTIGEN WHEN EXPRESSED IN A NONVIRULENT STRAIN OF TRYPANOSOMA CRUZI PEREIRA, D.H.D.1; MACHADO, L.F.M.2; GIUSTA, C.J.3; COSTA, B.H.3; SERUFO, A.V.2; DAMASCENO, L.M.4; COELHO, E.A.5; GAZZINELLI, R.T.4; FERNANDES, A.P.S.M.2
1.DEPARTAMENTO DE GENÉTICA, INSTITUTO DE CIÊNCIAS BIOLÓGICAS - UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL; 2.DEPARTAMENTO DE ANÁLISES CLÍNICAS E TOXICOLÓGICAS, FACULDADE DE FARMÁCIA – UNIVERSIDADE FEDERAL DE, BELO HORIZONTE, MG, BRAZIL; 3.LABORATÓRIO DE IMUNOPATOLOGIA,
CENTRO DE PESQUISAS RENÉ RACHOU – FIOCRUZ, BELO HORIZONTE, MG, BRAZIL; 4.DEPARTAMENTO DE BIOQUÍMICA E IMUNOLOGIA. INSTITUTO DE CIÊNCIAS BIOLÓGICAS. UNIVERSIDADE FEDERAL DE, BELO HORIZONTE, MG, BRAZIL; 5.DEPARTAMENTO DE PATOLOGIA CLÍNICA, COLÉGIO TÉCNICO. UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL.
Keyword:live vaccine ; a2; trypanosoma cruzi
Abstract: The A2 gene plays an important role in the virulence and visceralization of Leishmania species. Also, the characterization of the immune response and ability to induce protection of the A2 protein has already been proven in several models and through different immunization protocols. It is well known that the use of non-virulent organisms as antigen delivery systems is very effective, since they mimic the natural course of the infection and present a wide set of antigens maintaining a durable immunity. Hence, we expressed the Leishmania donovani A2 antigen in a non-pathogenic strain of Trypanosoma cruzi for evaluation of the protective efficacy as a live vaccine against L. infantum challenge. The CL-14 strain was transfected with pRockNEO vector containing a 729bp synthetic A2 gene and sites for integration in T. cruzi genome. Expression of A2 in the CL-14 T. cruzi strain was confirmed by PCR, Western Blot and immunofluoresence. BALB/c mice were then immunized i.p. with 107 parasites through a homologous prime-boost protocol. Sera and splenocytes of mice were collected and tested in ELISA and ELISPOT, respectively. Immunized mice were checked for IFN- production. Mice immunized with recombinant protein A2 plus MPL-A or alumen produced significantly more anti-A2 antibodies as compared to T. cruzi CL14-A2 immunized or control mice. In contrast, increased numbers of IFN- producing cells were detected in T. cruzi CL14-A2 immunized mice upon stimulation with either rA2 or A2-derived CD4 and CD8 peptides. Mice were then challenged subcutaneously with 107 L. chagasi promastigotes and the parasite burden of liver and spleen was checked after 30 days. Significant decrease in tissue parasitism was observed in animals vaccinated with rA2 or T. cruzi CL14-A2 as compared to PBS control mice. However, non-transfected T. cruzi CL14 also induced significant reductions in tissue parasitism. These results confirm the potencial of the A2 antigen as a vaccine candidate and suggest that the use of the A2-expressing T. cruzi is safe and may improve immunogenicity and protection against visceral leishmaniasis. - OTHER SPECIAL TOPICS [890] P 192 - OCULAR EXPERIMENTAL LEISHMANIASIS IN C57BL/10 AND BALB/C MICE INDUCED BY LEISHMANIA AMAZONENSIS INFECTION AND IN VITRO EVALUATION OF RETINAL EPITHELIAL CELLS CALABRESE, K.S.; SILVA, L.S.; CARVALHO, L.O.P.; HARDOIM, D.J.; ALMEIDA, M.S.; MORTARA, R.A.; DE SOUZA, C.S.F.; SILVA, A.L.A. FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:ocular leishmaniasis; retinal pigment epithelial (rpe) cells; murine model
Abstract: Ocular lesions are the one of the various manifestations of leishmaniasis and are characterized by injury of the anterior chamber of the eye and the resistance to specific treatments. The retinal pigment epithelial (RPE) cells participate in pathogen-induced intraocular inflammatory processes. The purpose of this study was to describe experimental ocular leishmaniasis caused by Leishmania amazonensis in C57BL/10 and BALB/c mice comparing the different inoculation routes and evaluate the parasite-host interaction and their impact on cells of the RPE. We demonstrated, in vitro, that L. amazonensis adhere to RPE cells in the 1st hour of infection, whereas parasite internalization was only observed after 6 hours. After 96 hour of infection, no parasites were observed intra or extracellular. These results suggest that Leishmania can infect
RPE cells although these cells are able to clear the infection. The influence of extracellular matrix (EM) proteins (Fibronectin and laminin) on L. amazonensis–RPE interaction was also investigated. Confocal analysis showed no substantial alterations in fibronectin presence in RPE-infected or RPE-non infected cells, whereas laminin levels increased three times 10 hours after L. amazonensis infection. After this time, laminin levels decreased in infected cells. These results suggest that L. amazonensis–RPE infection induces increased production of laminin in the beginning of infection which may facilitate parasite–host cell interactions. Histopathological eye analysis showed that mice inoculated by the intravitreal route developed more severe lesions than those noted in mice inoculated by instillation, presenting parasites in the anterior region of the eye 60 days after infection. The C57BL/10 mice presented cells containing parasitophorous vacuoles associated with pigmented cells and inflammatory infiltrate, which included mast cells. Ninety days after infection no parasites could be found in either mouse strain, which led us to hypothesize that parasites had been eliminated. In this context, we show that both intravitreal and instillation routes were effective in promoting ocular leishmaniasis infections in C57BL/10 and BALB/c mice. There were no differences in the parasite infection between the two mouse models and it mimicked the ocular lesions described in symptomatic dogs in endemic areas of visceral leishmaniasis. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [892] P 193 - IL-1B, IL-6, IL-8 AND IL-10 CYTOKINES EXPRESSION IN THE CLINICALIMMUNOLOGICAL SPECTRUM OF HUMAN LEISHMANIA (L.) INFANTUM CHAGASIINFECTION IN AMAZONIAN BRAZIL RAMOS, P.K.1; CARVALHO, K.I.2; ROSA, D.S.3; LIMA, L.R.1; CAMPOS, M.B.1; GOMES, C.C.4; LAURENT, M.D.4; CORBETT, C.P.4; SILVEIRA, F.T.1 1.INSTITUTO EVANDRO CHAGAS, ANANINDEUA, PA, BRAZIL; 2.FACULDADE DE MEDICINA DA UNIVERSIDADE DE SÃO PAULO- LABORATÓRIO DE IMUNOLOGIA CLÍNICA E ALERGIA, SÃO PAULO, SP, BRAZIL; 3.UNIVERSIDADE FEDERAL DE SÃO PAULO, SÃO PAULO, SP, BRAZIL; 4.FACULDADE DE MEDICINA DA UNIVERSIDADE DE SÃO PAULO - LABORATÓRIO DE PATOLOGIA EM DOENÇAS INFECCIOSAS, SÃO PAULO, SP, BRAZIL.
Keyword:il-1b, il-6, il-8 and il-10 cytokines; l. (l.) i. chagasi; human infection
Abstract: Background: The clinical-immunological spectrum of human L. (L.) i. chagasi-infection in Amazonian Brazil has been defined in five profiles with basis on the IFAT-IgG and DTH reactions: three profiles of asymptomatic individuals - Asymptomatic infection (AI: DTH+/++++/IFAT-), Subclinical resistant infection (SRI:DTH+/++++/IFAT+/++) and Indeterminate initial infection (III: DTH-/IFAT+/++), and two of symptomatic ones Symptomatic infection (SI=AVL) and Subclinical oligosymptomatic infection (SOI), both with the same (DTH-/IFAT+++/++++) reaction profile. The innate immune response of these profiles was investigating by the Il-1b, Il-6, Il-8 and Il-10 cytokine expression. Methods: There were examined by flow cytometry 85 serum samples of individuals (both genders and with 1 year old) from an AVL endemic area in Barcarena municipality, Pará state, Brazil, belonging to the clinical-immunological profiles: SI=AVL treated cases (9), SI=AVL non-treated (6), III (15), SRI (6), SOI (3), AI (9) and, 37 non-infected (DTH-/IFAT-) individuals (control group). The flow cytometry cytokine analysis was realized by a BDTM system Cytometric Bead Array (CBA) Human inflammation Cytokine kit (BD Biosciences, San Diego, CA) in a FACS CantoII. Results: There were no differences concerning the IL-1b and IL-8 expressions among the III, SRI, AI and SI=AVL (non-treated) profiles; but IL-1b expression of SI=AVL (non-treated) profile was higher than those of SI=AVL (treated) and the control group. There were higher IL6 and IL-10 expressions in SI=AVL (non-treated) profile compared to the other profiles.
Conclusion: IL-6 and IL-10 cytokines seem to play a crucial role on the way of the susceptibility of human L. (L.) i. chagasi-infection.
- VACCINE DEVELOPMENT AND TRIALS [896] P 194 - ANTI-LEISHMANIA PHAGOCYTIC ACTIVITY AND NITRIC OXIDE PRODUCTION IN MONOCYTES AND NEUTROPHILS FROM PERIPHERAL BLOOD OF DOGS IMMUNIZED AGAINST LEISHMANIASIS. MOREIRA, M.L.1; PEREIRA, C.C.1; MARTINS-FILHO, O.A.1; MARTELETO, B.H.1; GIUNCHETTI, R.C.2; FRANÇA-DIAS, M.H.1; ALVES, M.L.R.1; RIBEIRO, V.M.3; ARAÚJO, M.S.S.1 1.CENTRO DE PESQUISA RENÉ RACHOU / FIOCRUZ, BELO HORIZONTE, MG, BRAZIL; 2.UNIVERSIDADE FEDERAL DE MINAS GERAIS - UFMG, BELO HORIZONTE, MG, BRAZIL; 3.CLÍNICA VETERINÁRIA SANTO AGOSTINHO, BELO HORIZONTE, MG, BRAZIL.
Keyword:canine visceral leishmaniasis; leishmune; nitric oxide
Abstract: Visceral Leishmaniasis (LV), a severe disease caused by Leishmania chagasi, is transmitted by the bite of phlebotomine sandflies. Its reservoirs include wild foxes and domestic dogs. For this reason, the elimination of seropositive dogs is recommended in Brazil as the current strategy for controlling the disease. The use of a canine protective vaccine would represent the better control tool, reducing the availability of parasites to the sand fly vectors and consequently reducing the LV cases. The Leishmune®, a second-generation vaccine, have been recently licensed in Brazil and become commercially available. Its formulation proved to be safe protective and highly immunogenic for dogs, but there are a few studies about duration of protective immunity responses. Accordingly, the main goal of the current work was to investigate if the time-frame of one year, proposed for protective immunity following vaccination with Leishmune®, is accompanied by supportive immunological events. Those included cytokine profile change, nitric oxide synthesis, and phagocytosis in peripheral blood. Our preliminary results identified that Leishmune® was able to induce an increased phagocytic capacity in monocytes associated with the NO production by these cells, reaching a peak at six months after vaccination. In neutrophils, the higher long lasting phagocytic capacity triggered was not accompanied by enhanced NO production. The vaccination induced a long lasting blockage of IL-4 and an increase in the IL-8 production by monocytes along with higher IFN-g production by T-cells upon L. chagasi challenge in vitro. The evidences presented suggested that Leishmune® was able to induce a long lasting change in the immune response mediated by the interface of adaptive/innate immunity with impact on the monocyte functional properties. This is consistent with a potential anti-Leishmania activity stimulated by Leishmune® vaccine against CVL. Financial support: CNPq, CAPES, FAPEMIG and FIOCRUZ - GENOMICS, TRANSCRIPTOMICS, PROTEOMICS, METABOLOMICS [902] P 195 - DIFFERENT EXPRESSION OF PROTEINS IN A PATIENT WITH LETHAL VISCERAL LEISHMANIASIS BY MASS SPECTROMETRY. SILVA, V.C.1; COSTA, D.L.1; MELO, J.A.T.2; DE ALMEIDA LEITE, J.R.S.1; JÚNIOR, C.B.2; COSTA, C.H.N.1
1.UNIVERSIDADE FEDERAL DO PIAUÍ, TERESINA, PI, BRAZIL; 2.EMBRAPA RECURSOS GENÉTICOS E BIOTECNOLOGIA, BRASÍLIA, DF, BRAZIL.
Keyword:lethal visceral leishmaniasis; virulence factors; proteomics
Abstract: Background: Visceral leishmaniasis (VL) caused by Leishmania infantum kills ~10% of patients even after diagnosis and treatment, usually by bacterial co-infection or by hemorrhagic complications. We decided to investigate the parasite side of host/parasite relationship to check if different expression of proteins could be related to a bad outcome. Methods: A proteomic analysis of metacyclic promastigotes of L. infantum obtained from bone marrow aspiration of patients with lethal and non-complicated VL was performed. We did absolute and relative analysis via the acquisition of dependent data (DDA) combined with independent data (MSE). Results: Sixty-six proteins were precisely identified and classified by biological processes (Gene Ontology). Several molecules described as candidates as virulence factors were identified in the patient with the lethal outcome. A post-translation modification (acetylation) not previously described was obtained in the KMP-11 protein (AA Lys-45). Molecules such as TXNPx, ATPase beta subunit, hypothetical protein A4ICV2, among others, were found much more represented in the isolate from the patient who died. Conclusions: The proteomic techniques associated to Label-free nanoLC-MSE e LC-MALDITOF/TOF MS/MS allowed the identification of several proteins of promastigotes of L. infantum. The identification of molecules in the lethal case may help the selection of diagnostic markers as well as vaccine models. New analyses are under way to estimate quantitatively the presence of virulence factors in organisms derived from severe cases of VL. Supported by Universidade Federal do Piauí, Rede Nordeste de Biotecnologia-RENORBIO and FAPEPI/CAPES. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [903] P 196 - T CELLS IMMUNOPHENOTIPES IN BLOOD AND CEREBROSPINAL FLUID OF DOGS WITH VISCERAL LEISHMANIASIS GRANO, F.G.; DE MELO, G.D.; SILVA, J.E.S.; SCHWEIGERT, A.; MACHADO, G.F. FACULDADE DE MEDICINA VETERINÁRIA - UNIV. ESTADUAL PAULISTA, ARAÇATUBA, SP, BRAZIL.
Keyword:cd4+; cd8+; lymphocytes
Abstract: Infection caused by Leishmania spp. results in a disease that presents a variety of clinical manifestations both in humans and dogs. These manifestations can range from observation of asymptomatic infected dogs to disorders that can result in the death of the host. Symptomatic infected dogs may show deficits in the cellular immunological response, characterized by reduction of the lymphoproliferative response to Leishmania antigens and a reduction in the number of CD4+ and CD8+ cells. In order to evaluate the mechanisms involved in the immunopathology of this disease, it was proposed to characterize the T lymphocytes subsets in the blood and cerebrospinal fluid (CSF) of dogs with visceral leishmaniasis (VL). Blood collection was performed in ten infected dogs by puncture of the jugular vein and samples of CSF were collected by puncturing of the atlanto-occipital articulation. Analyses of the cerebrospinal fluid and blood were taken by flow cytometry to assess CD3, CD4 and CD8. The results were analyzed by Student's t test at the 5% level. The average values, in percentage, of the lymphocyte subpopulations in the peripheral blood mononuclear cells were 9,7%±10,96 of CD3+CD4+ and 11,1%±11,5 of CD3+CD8+ cells. For the CSF it was observed 1,6%±1,2 of CD3+CD4+ and 1,2%±0,8 of CD3+CD8+ cells. There was statistical difference (P <0.05) in the subsets of lymphocytes in the CSF when compared with the blood, where a smaller population
of CD3+CD4+ and CD3+CD8+ was observed in the CSF. However, no statistical difference was verified in the subpopulation of CD3+CD4+ cells in relation to CD3+CD8+ within the same type of sample. The results also indicate that the lymphocytes subpopulations were significantly reduced in animals with VL compared with healthy groups previously described in the literature, both in blood and CSF. The evolution of the VL can be determined by immune response mediated by T cells against the Leishmania parasite. Therefore, studies with immunophenotyping of these cells should be more realized, considering that T cells have a central function in the immunity against the leishmaniasis by influencing the production of cytokines, interaction with infected macrophages and activation of the humoral response. - VACCINE DEVELOPMENT AND TRIALS [905] P 197 - IMMUNOGENICITY AND PROTECTION LEVELS EMPLOYING LBSAP, LEISHMUNE® AND LEISH-TEC® VACCINES MENDONÇA, L.Z.1; RESENDE, L.A.1; LANNA, M.F.1; ROATT, B.M.1; AGUIAR-SOARES, R.D.O.1; CASTRO-OLIVEIRA, R.2; VIANA, K.F.3; REZENDE, S.A.2; SILVEIRA-LEMOS, D.3; MARTINS-FILHO, O.A.4; REIS, A.B.2; GIUNCHETTI, R.C.3 1.NÚCLEO DE PESQUISAS EM CIÊNCIAS BIOLÓGICAS, UNIVERSIDADE FEDERAL DE OURO PRETO, OURO PRETO, MG, BRAZIL; 2.PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS FARMACÊUTICAS, UNIVERSIDADE FEDERAL DE OURO PRETO, OURO PRETO, MG, BRAZIL; 3.INSTITUTO DE CIÊNCIAS BIOLÓGICAS, UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL; 4.CENTRO DE PESQUISAS RENÉ RACHOU, FUNDAÇÃO OSWALDO CRUZ, BELO HORIZONTE, MG, BRAZIL.
Keyword:immunogenicity; visceral leishmaniasis; vaccine
Abstract: In the last years, several researchers have been employed efforts to develop a vaccine against Leishmania infection using mice as a model for understanding the mechanisms of immunogenicity and protection against visceral leishmaniasis (VL). In the present study, we performed a pre-clinical vaccine trial, including analysis of immunogenicity and protection levels, in different groups, as follows: LBSap (composed by L. braziliensis antigens plus saponin), Leishmune® and Leish-Tec®. BALB/c mice were inoculated with three doses of each vaccine and challenged with L. chagasi. Thirty days post-challenge all animals were submitted to euthanasia. At the times before of first dose (T0), after third dose (T1; 14 days after the last dose) and 30 days after L. chagasi challenge (T3) the blood was collected in order to evaluate the hemogram and ex vivo immunophenotyping whole blood leukocytes by flow cytometry. The spleen and liver were collected at T3 for analyzing parasite load quantified by Real-time PCR. Preliminary results demonstrated at T1 a increased of total lymphocytes in Leish-Tec® and LBSap groups. The ex vivo immunophenotypic analyzes demonstrated an increase (P<0.05) in the CD3+ and CD4+ T-lymphocytes in LBSap group, besides tendency to increase in the CD4+ T-cells in the Leish-Tec® group. Moreover, LBSap group presented reduction (P<0.05) in the levels of CD19+ B-cells. After L. chagasi challenge (T2) Leishmune® and LBSap groups displayed increased levels of CD3+ and CD4+ T-cells, reduction of CD19+ B-cells and increase of CD3-CD49+ NK-cells in all vaccine groups. The Real-time PCR used for quantifying parasite load demonstrated in the splenic tissue reduction of 64% (Leishmune®), 42% (LBSap) and 36% (Leish-Tec®). The same analysis performed in the liver showed reduction in the parasite load of 71% (Leishmune®), 62% (LBSap) and 48% (Leish-Tec®). We concluded that mice immunized by LBSap and Leishmune® vaccine exhibited a strong immunogenicity profile reflecting in the reduction of parasite levels. Supported by FAPEMIG, CNPq, CAPES, CPqRRFIOCRUZ, UFOP and UFMG. - CELLULAR AND MOLECULAR BIOLOGY [910] P 198 - COMPARATIVE ANALYSIS OF LEISHMANIA BRAZILIENSIS ISOLATED FROM TWO DISTINCT CLINICAL MANIFESTATIONS
ALVES FERREIRA, E.V.C.1; FERREIRA, T.R.1; PINZAN, C.1; CAVALCANTE DE OLIVEIRA, A.1; BARRAL, A.2; NETO, M.B.2; CRUZ, A.K.1 1.UNIVERSIDADE DE SÃO PAULO, RIBEIRÃO PRETO, SP, BRAZIL; 2.FUNDAÇÃO OSWALDO CRUZ, SALVADOR, BA, BRAZIL.
Keyword:leishmania braziliensis; mucosal lesion; virulence
Abstract: Leishmania Viannia braziliensis is a major etiological agent of cutaneous leishmaniasis in Brazil. The severe and metastatic mucosal form of the disease affects about 5% of patients infected by L. braziliensis. To investigate possible phenotypic modifications between parasites from mucosal and cutaneous sites we evaluated the proteome profile differences of paired mucosal and cutaneous isolates of L. V. braziliensis. Parasites recovered concomitantly from cutaneous (LBrC) and mucosal sites (LBrM) of the same patient were investigated. We observed that the molecular karyotypes of all isolates were undistinguishable. The proteome comparative analysis revealed among other genes, the differential pattern of expression of LbrM.31.2410 (prostaglandin f2-alpha synthase) which was uniquely expressed in LBrC1 (patient 1) and was 1.7 times more expressed in LBrC2 (patient 2) when compared with LBrM1 and LBrM2, respectively. Histological analysis from cutaneous tissue suggests that the LbrC isolates induce a stronger inflammatory response than LbrM isolates in BALB/c mice and hamsters. In silico analysis using the TritrypDB and TDR Target Database showed that PGF2S has been identified in the secretome of L. V. braziliensis and in the exosome of L. L. major and that it has a high potential as a drug target. We could not detect, by Northern Blotting, PGF2S transcript levels differences between the paired isolates evaluated. Based on the recent study suggesting a positive correlation between the presence of a single stranded RNA virus (LRV) in L. V. guyanensis and the metastatic behavior of subgenus Viannia strains, we searched, by qRTPCR, for the virus in LbrM and LbrC isolates with no success. In vitro and in vivo infection assays revealed that LbrC is more infective than LbrM and we are currently conducting experiments to quantify cytokine release (IL-4, IL-10, IL-2, IFN- and IL-12) and parasite load after infection. A L. braziliensis overexpressing PGF2S was generated and displayed increased ability to survive within the macrophage after 24-48h post-infection when compared to the control transfectant. - VACCINE DEVELOPMENT AND TRIALS [911] P 199 - LBSAPSAL VACCINE INDUCED TYPE 1 IMMUNE RESPONSE IN DOGS EVIDENCED BY LOWER LEVELS OF IL-4 AND TGF-B, INCREASED LEVELS OF IL-12, IFN-G AND ENHANCED NITRIC OXIDE RESENDE, L.A.1; ROATT, B.M.1; AGUIAR-SOARES, R.D.O.1; LANNA, M.F.1; VIANA, K.F.2; MENDONÇA, L.Z.1; MARTINS-FILHO, O.A.3; REIS, A.B.1; GIUNCHETTI, R.C.2 1.NÚCLEO DE PESQUISAS EM CIÊNCIAS BIOLÓGICAS, UNIVERSIDADE FEDERAL DE OURO PRETO, OURO PRETO, MG, BRAZIL; 2.INSTITUTO DE CIÊNCIAS BIOLÓGICAS, UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL; 3.CENTRO DE PESQUISAS RENÉ RACHOU, FUNDAÇÃO OSWALDO CRUZ, BELO HORIZONTE, MG, BRAZIL.
Keyword:canine visceral leishmaniais; vaccine; immunogenicity
Abstract: Dogs represent the most important domestic reservoir of Leishmania chagasi (syn. L. infantum), and a vaccine against canine visceral leishmaniasis (CVL) would be an important tool in the control of human visceral leishmaniasis (HVL) by decreasing the infection pressure of L. chagasi. Herein, we investigated the immunogenicity of LBSapSal vaccination in dogs. In this sense, dogs were inoculated with: saline (C), sand fly gland extract (SGE) of Lutzomyia longipalpis (Sal), L. braziliensis promastigotes protein plus SGE (LBSal), L. braziliensis promastigotes protein and saponin together with SGE (LBSapSal). Cytokines (IL-10, TNF- , IL-4, TGF- , IL-12, IFN- and nitric oxide (NO) were evaluated in the in vitro context from
supernatants of peripheral blood mononuclear cells (PBMC) cultures at four different times: previously the vaccine protocol (T0), after the third vaccine dose (T3) and at the times 90 (T90) and 885 (T885) days after experimental challenge (dac). Our major results demonstrated that LBSapSal group presented lower levels of IL-4 (T90) and TGF- (T90 and T885) after Leishmania stimulation. In addition, higher levels of IL-12 were observed in LBSapSal group (T3, T90 and T885). Likewise, increased levels of IFN- were related to LBSapSal (T3, T90 and T885) group. Cultures stimulated with SLcA of the group LBSapSal showed increased levels of NO (T885). These results obtained from the analysis of NO levels confirmed the hypothesis that this vaccine induces a potential resistance profile against Leishmania infection. In conclusion, the results of this study encourage the continuation of investigations related to parasitological and immunological events after challenge of this new vaccine against canine visceral leishmaniasis and the establishment of new biomarkers of immunogenicity. Supported by FAPEMIG, CNPq, CAPES, CPqRR-FIOCRUZ, UFOP and UFMG. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [914] P 200 - LEISHMANIA (LEISHMANIA) AMAZONENSIS AND LEISHMANIA (LEISHMANIA) INFANTUM INFECTION AND CO-INFECTION EXPERIMENTAL MODEL (MESOCRICETUS AURATUS) MOURA, A.P.V.; CELESTE, J.L.L.; SILVA, S.O.; MELO, M.N.; ANDRADE, H.M. UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL.
Keyword:leishmania chagasi, l.amazonensis; co-infection; experimental model
Abstract: The leishmaniasis presents a broad clinical spectrum determined by factors of the parasite and the host immune response. In Brazil, the visceral form is mainly associated with infection by L. infantum and cutaneous form by L. braziliensis, L. guyanensis and L. amazonensis among others. Areas of overlap species are frequent and have been isolated L. amazonensis in dogs with visceral leishmaniasis. This study aimed to perform clinical, parasitological and histopathological groups of hamsters (Mesocricetus auratus) infected separately and simultaneously by L. amazonensis (strain IFLA/BR/1967PH8) and L. infantum (strain MHOM/BR/1972/BH401). Clinical assessments were performed weekly for 90 days, where it was observed clinical signs of leishmaniasis as dermatitis, ulceration of the skin, alopecia, lymphadenopathy, weight loss, ascites and hepatosplenomegaly. Then the animals were euthanized to collect fragments of skin, spleen and liver for parasitological and histopathological evaluations. The parasitological assessment was made by direct visualization of amastigotes by microscopy, isolation of the parasite in culture, quantitative PCR and PCR-RFLP, while histopathological evaluation through observation of slides containing paraffin sections of skin, liver and spleen, stained by the hematoxylin eosin (H & E). Histopathological analysis was performed in a semi-quantitative, and evaluated the following criteria: granuloma, portal inflammation, degenerative diseases, inflammation and thickening of the capsule, sinus congestion, and hemosiderin deposition. This work will contribute to the understanding of the biology of the parasite in experimental models especially in cases of co-infection. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [916] P 201 - BRAZILIAN PEMPHIGUS PATIENTS DO NOT RECOGNIZE LEISHMANIA VIANNIA BRAZILIENSIS ANTIGENS SANTOS, P.V.W.G.; NASCIMENTO, M.P.; MACHADO, A.R.S.R.; DE PAULA, N.A.; ROSELINO, A.M. UNIVERSITY OF Sﾃグ PAULO, FACULTY OF MEDICINE OF RIBEIRﾃグ PRETO, RIBEIRﾃグ PRETO, SP, BRAZIL.
Keyword:leishmaniasis; pemphigus; elisa
Abstract: Being the northeastern region of São Paulo state (Brazil) endemic for both pemphigus foliaceus (PF) and pemphigus vulgaris (PV), as well as for American tegumentary leishmaniasis (ATL) mainly caused by L. (Viannia) braziliensis, our group has been studying the immune genetic relationship between them. Our studies have correlated both diseases by showing that salivary antigens of hematophagous insects are recognized by antibodies from patients with PF, as well as by identifying antibodies against desmogleins in patients with ATL. Thus, this study intends to demonstrate the humoral response in serum samples from pemphigus patients against two different species of leishmanias. 33 serum samples from pemphigus (17 PF and 16 PV), and 71 from ATL patients (45 cutaneous and 26 mucosal form) were compared to 64 controls by ELISA (Enzyme-linked immunosorbent assay) tests. There were used an in house ELISA test, prepared with L. (V.) braziliensis (ELISAb), and a commercial L. mexicana (ELISAm) test. Antigens from promastigotes of L. (V.) braziliensis were prepared with Trizol and with a protein extraction commercial kit and, as the protein extraction with the commercial kit was much better, this was the one chosen for ELISAb. The cellular response was tested by Montenegro´s intradermal reaction (MIR) in 5 pemphigus patients. The ODs from ATL resulted higher when compared to pemphigus and control groups on ELISAm (median 0.2410, 0.1440 and 0.1680, respectively). In relation to ELISAb the result was similar (0.3310, 0.0360 and 0.0310, respectively). There was no statically difference between pemphigus and control groups for both leismania species. The MIR resulted negative in all pemphigus patients. While we have demonstrated that serum samples from PF patients react with maxadilan and others salivary antigens from Lutzomyia longipalpis the actual results discharge the immune reaction against two species of leishmanias by pemphigus patients. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [919] P 202 - HAIRLESS MICE AS EXPERIMENTAL MODEL FOR CUTANEOUS LEISHMANIASIS INDUCED BY LEISHMANIA (LEISHMANIA) AMAZONENSIS DE ARAUJO, V.C.; QUEIROZ, D.S.; RIZK, Y.S.; BOSQUIROLI, L.S.; COSTA, E.C.; RODRIGUES, P.O.; OSHIRO, E.T.; ARRUDA, C.C. UNIVERSIDADE FEDERAL DE MATO GROSSO DO SUL, CAMPO GRANDE, MS, BRAZIL.
Keyword:leishmania (leihsmania) amazonensis; hairless mice; experimental infection
Abstract: Murine cutaneous leishmaniasis arose as a model for the study of development and control of cell-mediated immune response, and the susceptibility to Leishmania is genetically determined among the different strains of mice. Hairless mice have been widely used as models in order to test products which reduce skin aging, damage caused by UVB light and skin alterations caused by treatment with acids. Due to the absence of hair, which would facilitate the study of cutaneous lesions, and because of their peculiar immunological characteristics, it was investigated their use as a model in experimental cutaneous leishmaniasis induced by Leishmania (Leishmania) amazonensis. Hairless mice and BALB / c (used as controls) were infected with 10 L. amazonensis promastigotes in the right hind footpad. 30, 60 and 90 days after infection, thickness of the infected paws was determined by measuring with a caliper; parasitolological analysis and histopathological study were performed in the site of infection, popliteal lymph node, spleen and liver, as well as the weighing of these organs. Amastigote forms were observed in the footpads of both mice 30, 60 and 90 days after infection. Signals of visceralization were confirmed by the presence of the parasite in the lesion draining lymph node, as well as in the spleen and liver at different points of observation. Regarding the footpad swelling due to the cutaneous lesions, BALB / c mice presented an statistically significant increase over the observation points; the same did not occur in Hairless mice. Despite the absence of increased thickness in the footpads, Hairless mice presented a large number of
parasites associated to a mild mononuclear inflammatory infiltrate at the site of infection, while BALB/c presented an intense inflammatory infiltrate. Hairless mice had an statistically significant increase in the weight of infected footpad and draining lymph nodes among the three points of analysis. In BALB/c mice, the weight of all analyzed organs had an statistically significant increase. These results show that Hairless mice are susceptible to L. amazonensis infection, suggesting that these strain may be promising as a model to the study of experimental cutaneous leishmaniasis. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [920] P 203 - DENDRITIC CELLS AND T CELL TRANSCRIPTION FACTORS IN HUMAN MUCOCUTANEOUS LEISHMANIASIS TAPIA, F.J.; RODRIGUEZ, O.L.; LUGO, D.A.; ORTEGA, M.E.; CABRERA, M.; ZERPA, O.; SANCHEZ, M.A.
INSTITUTO DE BIOMEDICINA, UNIVERSIDAD CENTRAL DE VENEZUELA, MINISTERI DE SALUD, CARACAS, VENEZUELA.
Keyword:dendritic cells; t cells; transcription factors
Abstract: Mucocutaneous leishmaniasis (MCL) is characterized by an exacerbated inflammatory response and parasites spread from a primary lesion to distant sites, resulting in destructive secondary metastatic lesions, especially in the nasopharyngeal mucosa. Dendritic cells (DCs), myeloid and plasmacytoid (mDCs and pDCs) are involved in the activation of naive T cells; however, there is scarce information on the role of peripheral DCs subtypes inducing specific T helper (Th) responses in human leishmaniasis. The aim of this study was to correlate the activation of DCs with the expression of T cell transcription factors associated with distinct effector T cell functions in peripheral blood of patients with mucocutaneous leishmaniasis (MCL) using flow cytometry. Biological identity was based on characterization with specific cell probes, as follows: mDCs (CD11c +), pDCs (CD123 +), activated DCs (CD83), mature DCs (CD86 +), Tbet (Th1), GATA-3 (Th2), ROR-gamma t (Th17) and FOXP-3 (Treg). Analysis was made on peripheral blood of patients with LCM 1(n=13); endemic control (EC, n=8) and non-endemic healthy control (HC, n=7) individuals. The results demonstrated that 69.2% and 38.5% of MCL patients showed strong CD83 expression, respectivelly, but regarding CD86 expression in mDCs was absent and a merely 7.69% in pDCs. In MCL patients with activated mDCs their T cell expressed ROR-gamma t = 88%, FOXP-3 = 80%, GATA-3 = 67% and T-bet = 50%, whereas patients with non-activated-mDCs T cells were significantly different (p <0.05) showing ROR- gamma t = 75%, FOXP-3 = 100%, GATA-3 = 50% and T-bet = 0%. Comparison of activated vs non-activated pDCs in MCL patients, gave significant differences (p <0.01) on ROR-gamma t = 80% vs 100% and T-bet = 20% vs 38%. A multiple variance analyses showed that mDCs CD83+ in MCL are associated with ROR-gamma t, FOXP-3 and GATA-3, whereas pDCs CD86+ is associated with T-bet. These results suggest a higher activation of mDCs than pDCs in MCL patients, which is associated with a Th1/T-bet response rich in IFN-gamma, whereas, activation of pDCs is mainly associated with Th17- ROR-gamma t. Financed by CDCH UCV PG-09-7429-2008. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [921] P 204 - CD16: A POSSIBLE TARGET IN CUTANEOUS LEISHMANIASIS TREATMENT ? COSTA, R.1; PASSOS, S.1; MENEZES, T.1; MACHADO, P.1; GUIMARﾃウS, L.H.1; MOSSER, D.2; CARVALHO, E.M.1; SCOTT, P.3; CARVALHO, L.P.1 1.FEDERAL UNIVERSITY OF BAHIA, SALVADOR, BA, BRAZIL; 2.UNIVERSITY OF MARYLAND, WASHINGTON DC, UNITED STATES; 3.UNIVERSITY OF PENNSYLVANIA, PHILADELPHIA, UNITED STATES.
Keyword:monocytes; cd16; cutaneous leishmaniasis
Abstract: Ulcer development in cutaneous leishmaniasis (CL) patients is associated with exaggerated inflammatory response with high levels of TNF. CD16+ cells include a monocyte subset known to produce high levels of TNF. Most studies have focused on T cell responses little attention has been given to the contribution of other leucocytes to the immunopathology observed in human CL. Here, we analyzed the frequency and the contribution of monocyte subsets to the inflammatory response observed in patients with CL due to Leishmania braziliensis infection. Peripheral monocytes in human were recently shown to be heterogeneous according to CD14 and CD16 expression. We found that early after infection the frequency of CD16+ monocytes (intermediate (CD14+CD16+) and non-classical (CD14dimCD16+)) are increased in peripheral blood from CL patients compared to healthy controls. Ex vivo analysis showed that intermediate monocytes expressed significantly more MHC class II and Metalloproteinase-9, a protease involved in basal membrane disruption and extracellular matrix degradation, then the other monocyte subsets in these patients. Also, when pulsed with soluble Leishmania antigen or LPS, intermediate monocytes were the main source of TNF. Circulating classical and intermediate subsets from CL patients expressed more CCR2, what make us believe that at least one of these two cell subset differentiate into phagocytes with non-classical phenotype at lesion site, once this third population was more prevalent at CL lesions. These data show that intermediate monocytes are the main inflammatory cell in CL patients and since this population expresses CD16, this molecule may serve as target in immunotherapy studies. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [931] P 205 - EVALUATION OF THE TH17, TH1 AND TH2 RESPONSE IN PATIENTS WITH AMERICAN CUTANEOUS LEISHMANIASIS DE ALMEIDA, A.F.; DE CASTRO, M.C.A.B.; DE OLIVEIRA, A.P.; DE ALMEIDA, T.M.; SOUZA, M.A.; DE OLIVEIRA, B.C.; PEREIRA, V.R.A. CENTRO DE PESQUISAS AGGEU MAGALHÃES, FUNDAÇÃO OSWALDO CRUZ, RECIFE, PE, BRAZIL.
Keyword:flow cytometry; leishmania; cellular immune response
Abstract: Leishmania infections induce specific host responses, specially characterized by T cell expansion. CD4+ and CD8+ T cells act as cytokine sources involved in macrophage activation, and the balance between them is important in the healing process. Also, the classical TH1 and TH2 subsets have been joined recently by the TH17 lineage. IL-17 producing T cells were identified as important drivers of autoimmune pathology and allergic inflammation. The aim of this study is to access the role of TH17, TH1 and TH2 cytokines in the outcome of American cutaneous leishmaniasis (ACL). A peripheral blood mononucleated cells (PBMC) ring was obtained from 7 patients with active lesions (AT) and 10 healthy controls (CT). The cells were washed twice with PBS, centrifuged and resuspended in culture medium. The cells were then adjusted to a concentration of 0,5x106 per well and then incubated with PMA and Ionomycin for 6 hours. Brefeldin was added to the culture 4 hours prior to the end of the incubation period. After the incubation time, EDTA was added to the culture and incubated for 10 minutes. The cells were washed and added to polystyrene tubes containing monoclonal antibodies anti-CD4 and anti-CD8, both labeled with FITC, and incubated for 30 minutes. The cells were then permeabilized and stained with cytokine specific antibodies against IFN- , TNF- , IL-4, IL-10, IL-17, IL-21, IL-22 and IL-23 labeled with PE, PeCy7, APC and AF647. The cells were washed and analyzed using a FACSCalibur cytometer. A significant increase in the production of IL-4 by CD4+ and CD8+ T lymphocytes was observed in AT in comparison to CT (p=0,0002 and p=0,0020, respectively). It was also shown an increase in the production of IL-10 by CD8+ cells in AT in comparison to CT (p=0,0136). As to the TH17 response, it was observed an increase in the production of IL-17 and IL-23 in AT in comparison to CT (p=0,0136 and p=0,0004,
respectively). The significant increase in the production of IL-4 by CD4+ and CD8+ T lymphocytes suggest that this cytokine plays an important role in the imunopathology of ACL. IL-4 induces a TH2 response in Leishmania infections, an immune profile related with the resistance to the disease. IL-17 and other cytokines from the TH17 response are known to contribute to the disease control, being increased in the early response to pathogens. Additional studies are under development to further contribute in the immunologic knowledge about the mechanisms involved in the cure of ACL. - GENOMICS, TRANSCRIPTOMICS, PROTEOMICS, METABOLOMICS [934] P 206 - IMMUNOPROTEOMIC CHARACTERIZATION OF ANTIGENS OF LEISHMANIA (LEISHMANIA) CHAGASI, L. (L.) AMAZONENSIS AND L. (VIANNIA) BRAZILIENSIS DETECTED BY IMMUNOHISTOCHEMICAL TEST RODRIGUES, B.S.; PIRES, S.F.; ANDRADE, H.M.; TAFURI, W.L. UFMG, BELO HORIZONTE, MG, BRAZIL.
Keyword:leishmania ; immunohistochemical; proteomic
Abstract: Currently, leishmaniasis are among the main endemic diseases of the world. They are caused by flagellate parasite of the genus Leishmania (Kinetoplastida: Trypanosomatidae). Clinical form of Leishmaniasis are determinated by parasite’s species and host immune response, in: cutaneous, diffuse cutaneous, mucocutaneous and finally, visceral leishmaniasis, which is the most severe form of leishmaniasis, may be fatal if left untreated. Different methods associated diagnosis are used to confirmation of the disease, among which we can mention: clinical, epidemiological, parasitological and immunological. However, the definitive diagnosis is the parasitological, but, they present some difficulties, either because there are few parasites in the sample, difficulty in recognizing the same, need for experienced technical staff, among other factors. The immunohistochemical detection of Leishmania amastigotes in tissue sections fixed in is routinely used in many laboratories. Thus, Tafuri et al., 2004 demonstrated a method of parasitological diagnosis by use of an alternative technique of immunohistochemistry, according to the authors, efficient and less expensive and still be able to diagnose LV and LTA. Therefore, this work is justified for a better understanding of this methodology by identifying reactive proteins that makes this test able to be used in both clinical forms: ATL and VL. For this, we used protein extracts of the species cited fractionated in 2-DE gel followed by western blot using antibodies used in the test Tafuri et al.,. Were selected to identification the reactive spots test in each species and all species simultaneously. The protein identification was carried using MSMS. Among the proteins identified, two were common to the three species studied: heat shock protein 70 (hsp 70) and -tubulin, and 6 were specific to L. amazonensis, 8 to L. braziliensis and 3 to L. chagasi. Some of these proteins have been described in the literature, for its antigenicity, whereas others appear to be new antigens, which could be targets of future studies for developing alternative diagnostic test. The identification of these antigens could contribute to the development of serological tests with greater specificity. Financial support: CAPES - CELLULAR AND MOLECULAR BIOLOGY [935] P 207 - CHANGES IN INTEGRIN CONFORMATION AND CELL SPREADING IN LEISHMANIA-INFECTED MONONUCLEAR PHAGOCYTES FIGUEIRA, C.P.1; ALMEIDA, R.A.1; HERMIDA, M.D.1; SANTOS, T.C.1; CARVALHAL, D.G.F.1; ROBERT, P.2; BONGRAND, P.2; DOS-SANTOS, W.L.C.1 1.FUNDAÇÃO OSWALDO CRUZ, CENTRO DE PESQUISAS GONÇALO MONIZ, SALVADOR, BA, BRAZIL; 2.UNIVERSITÉ DE LA MÉDITERRANÉE, PARC SCIENTIFIQUE ET TECHNOLOGIQUE DE LUMINY, MARSEILLE, FRANCE.
Keyword:macrophage; integrin; cell migration
Abstract: Leishmania infection decreases the adherence of mononuclear phagocytes to connective tissue. Since adhesion and desadhesion may constitute essential steps in cell migration, changes in cell adherence caused by Leishmania infection may interfere with parasite dissemination or permanence in the infection sites. In order to study the changes in adherence of infected mononuclear phagocytes to the connective tissue we performed two sets of experiments: (1) We compared the expression of the high affinity beta-2 integrin epitope by non-infected and by Leishmania-infected human peripheral blood monocytes, using the p24 mAb in flow cytometry. (2) We used scanning electron microscopy to examine cytoplasm spreading of non-infected and Leishmania-infected J774 cells over fibronectin-coated coverslips. As controls for cytoplasm spreading we used EDTA-treated (low level of cytoplasm spreading), Mn++-treated (high level of cytoplasm spread) cells. The percentage of uninfected monocytes positive for the high affinity beta-2 integrin epitope was 67±14% and of Leishmania-infected cells was 31±15%. Cytoplasm spreading was similar among infected (61.74 m2) and uninfected (60.51 m2) J774 cells. The cytoplasm spreading of these cells were intermediate between EDTA (60.09 m2) and Mn++ (81.51 m2) treated cells. In conclusion, there is a shift towards lower affinity of integrins expressed by Leishmania-infected cells in comparison with uninfected cells. This change in integrins does not affect cell spreading over extracellular matrix substrates. CAPES-COFECUB, CNPQ. cpf@bahia.fiocruz.br - VACCINE DEVELOPMENT AND TRIALS [939] P 208 - USE OF CHITOSAN MICROPARTICLES AS DELIVERY SYSTEMS IMPROVES THE LACK DNA INTRANASAL VACCINE EFFICACY AGAINST LEISHMANIA AMAZONENSIS MURINE INFECTION COSTA-SOUZA, B.L.S.1; BEZERRA, I.P.S.1; MATOS-GUEDES, H.L.1; SCHWEDERSKY, R.P.2; MARTINEZ, A.M.B.2; RÉ, M.I.3; ROSSI-BERGMANN, B.1 1.IBCCF - UFRJ, RIO DE JANEIRO, RJ, BRAZIL; 2.UFRJ, RIO DE JANEIRO, RJ, BRAZIL; 3.CENTRE RAPSODEE, ALBI, FRANCE.
Keyword:leishmania amazonensis; lack; intranasal vaccine
Abstract: We have previously shown that an intranasal vaccine consisting of a plasmid carrying the LACK gene (LACK-DNA) partially protects both mice and hamsters against cutaneous and visceral leishmaniasis, respectively. In this work, the use of chitosan microparticles (CM) as delivery systems to improve the LACK-DNA intranasal vaccine efficacy was investigated. Besides being a mucoadhesive biopolymer, the cationic nature makes chitosan suitable for the mucosal delivery DNA. First, the CM were prepared by spray drying technique and then crosslinked with glyceraldehyde (CMC-Gly) to prevent hygroscopic swelling. To evaluate the systemic toxicity of intranasal CMC-Gly, two doses of 2 mg were given to BALB/c mice with a 7 day interval. Serum levels of AST, ALT and creatinine were quantified 24 hours after the second dose. We found no alterations in the toxicity markers in relation to untreated controls. In vitro LACK DNA – CMC-Gly complexation studies indicated an optimal relation of 30 µg of LACK-DNA to 1.2 mg of CMC-Gly. To assess the mucoadhesiveness and penetration onto the nasal mucosa, mice were given 15µg of LACK-DNA/CMC-Gly in 10 µl of PBS in each nostril. After 15 minutes, the animals were euthanized and their nose were decalcified and processed for histology. CMC-Gly were found not only adhered to the surface of nasal mucosa, but also intercalated between epithelial cells and deeper inside the subepithelial region. These results confirms the mucoadhesiveness and mucosal penetration of LACK-DNA/CMC-Gly, supporting the notion that CMC-Gly can serve as efficient delivery systems for intranasal DNA vaccines. To evaluate its adjuvant effect in LACK-DNA vaccination, mice were intranasally immunized
with 2 doses of LACK-DNA/CMC-Gly with a 7 day interval between them. Controls received free LACK DNA, empty CMC-Gly or PBS. One week after immunization, mice were challenged in the footpads with L. amazonensis of high or low virulence. The CMC-Gly improved the LACK-DNA intranasal vaccine efficacy by promoting a better lesion growth control and a lower parasite burden in the footpads compared to LACK-DNA alone, even in high virulence challenge. These data show that CMC-Gly is a promising delivery system adjuvant for LACK-DNA, and possibly other DNA intranasal vaccines. - CLINICAL LEISHMANIASIS [941] P 209 - ANTIMONY CARDIOTOXICITY IN A PATIENT TREATED FOR MUCOSAL LEISHMANIASIS RODRIGUES, A.M.1; CARVALHO, T.B.F.2; MARCOS, S.F.2; LARANJEIRA, E.T.S.2; BRASIL, H.H.2; SILVA, E.V.L.P.2; LIMA, C.A.A.2; SILVA, T.C.2; MOREIRA, M.R.2; ROCHA, M.O.C.1 1.UFMG, BELO HORIZONTE, MG, BRAZIL; 2.FACIMED, CACOAL, RO, BRAZIL.
Keyword:antimony; leishmaniasis; cardiotoxicity
Abstract: INTRODUCTION Antimonial compounds are the cornerstone therapy for leishmaniasis in Brazil the only antimonial compound used in this country. This drug has several adverse effects, and cardiotoxicity is one of major concern due to repolarization abnormalities and prolongation of QT interval leading to cardiac arrhythmias and sudden death. We describe a case of fatal arrhythmia resulting from long QT syndrome induced by antimony compound in a patient with mucosal leishmaniasis. CASE REPORT. Black retired male, 82 years old, had a diagnosis of mucosal leishmanisis because of mild nasal symptoms plus positive intradermal leishmanin test. After a clinical and laboratorial evaluation and a rest 12 lead ECG reveling sinus rhythm, mid sinusal bradicardia and QTc of 370 msec. the patient received meglumine antimoniate and was schedule to repeat an rest ECG after first dose of antimony. Patient became well until 18th dose of antimony whem he presented with malaise, nรกusea and dyspnea. On the 20th day of antimony therapy he was admitted in another hospital receive intravenous fluids and a new ECG was recorded revealing QTc of 720msec, but the antimony therapy was continued. In 24th day of treatment he was referred to Hospital Regional de CacoalRO for evaluation with an infectious diseases consultant. At that time he presented with hypotension , hemodynamic instability and arrhythmia. A new ECG track reveal ventricular arrhythmia, QT of 640 msec and QTc of 682 msec. The patient was admitted in the intensive care unit immediately and the dose of antimony by body weight was calculate in 21mg/kg/dia. After three days in ICU the patient died because severe ventricular arrhythmia. DISCUSSION Severe cardiotoxicity and sudden death are described during antimony therapy and one major concern during treat of cutaneous and mucosal leishmaniasis with such group of drugs. Proper dosage of antimony and monitoring of QT/QTc interval is useful but must be done by skilled health professionals, and if prolongation beyond 500 msec occur suspension of antimony is warranted. Careful clinical and electrocardiography monitoring is necessary during antimony therapy, mainly in elderly and patients with cardiac conditions.
- CLINICAL AND EXPERIMENTAL IMMUNOLOGY [946] P 210 - EVIDENCES OF SIMULTANEOUS MODULATORY EFFECTS IN A MICE MODEL OF CO-INFECTION WITH LEISHMANIA SPP. AND PLASMODIUM YOELII PINNA, R.A.; DE LUCA, P.M.; SILVA, L.A.; DORES, T.L.; TINOCO, N.A.B.; FINKELSTEIN, L.C.; FERREIRA, J.O.; BANIC, D.M. FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:co-infection; leishmaniasis; malaria
Abstract: Introduction: cutaneous leishmaniasis and malaria are highly endemic in several regions of the world. In many of these areas the distribution of both diseases are overlapped, like in the Brazilian Amazon. Although there are no data of co-infection by a dermotropic leishmania and plasmodia in humans, great evidences indicate that concomitant infections can alter the course and the immune response to different diseases. Objectives: determine if concurrent infection with dermotropic Leishmania and Plasmodium yoelii influences the course and load of parasites in a mouse model of co-infection. Material and methods: BALB/C mice were divided into 7 groups: G1-P. yoelii; G2-L. braziliensis; G3-L. major; G4-L. amazonensis; G5-L. braziliensis + P. yoelii; G6-L. major + P. yoelii and G7-L. amazonensis + P. yoelii). Firstly, BALB/c mice of G2 to G7 were infected with one of the Leishmania spp. (L. braziliensis: inoculum of 1x105 parasites; L. major and L. amazonensis: inoculum of 1x104 parasites). P. yoelli 17XNL (nonlethal strain) infection was performed 3 days later on groups G1, G5-G7(inoculum of 1x106 infected erythrocytes). Lesions sizes were monitored weekly with a digital caliper for a period of 11 weeks. Time of appearance and number of ulcers in lesions was observed. Parasite load of infected ears was determined using a quantitative limiting-dilution assay at days 8, 13, 20, 28 and 80 post Leishmania spp. infection. P. yoelli infection was monitored through blood strains stained with Giemsa every 2 days. Results: development of leishmanial lesions in co-infected mice was delayed in comparison to those only infected with Leishmania. It was also observed a decrease in the number of ulcerated lesions in mice belonging to G7 and G8 and a transient reduction of the load of Leishmania spp. parasites during patent malaria parasitaemia in coinfected groups. P. yoelii infection was similar in mono- and co-infected mice. The mean peak parasitaemia was higher in G2 than in G5 and in G6. There was no difference in parasitaemia peak levels between G2 and G8, although G8 presented a delay in the clearance of P. yoelii. G7 and G8 had a significant increase in mortality during malaria infection. No mono-infected mice died during follow-up. Conclusions: P. yoelii infection seems to influence the course of leishmaniasis in BALB/c mice, presumably in consequence of systemic immune response elicited during malaria acute stage. Leishmaniasis may also influences the course of malaria in co-infected mice as seen in L. major and L. amazonensis infections in which lethality were increased. - CLINICAL LEISHMANIASIS [947] P 211 - CHEMICAL AND CLINICAL PANCREATITIS DURING TREATMENT FOR CUTANEOUS LEISHMANIASIS RODRIGUES, A.M.1; SILVA, E.V.L.P.2; CARVALHO, T.B.F.2; MARCOS, S.F.2; LARANJEIRA, E.T.S.2; BRASIL, H.H.2; ARAUJO, L.H.3; ROCHA, M.O.C.1
1.UFMG, BELO HORIZONTE, MG, BRAZIL; 2.FACIMED, CACOAL, RO, BRAZIL; 3.AMBULATORIO ESPECIALIZADO, CACOAL, RO, BRAZIL.
Keyword:antimony; pancreatitis; leishmaniasis
Abstract: Introduction. Cutaneous leishmaniasis is a common infectious tropical diseases in Brazil. Antimony treatment is the cornerstone therapy. Antimony compounds have several adverse effects, among them; pancreatic toxicity is described. Asymptomatic elevation of amylase is one of more common, although cases of clinical pancreatitis are reported. Objective. Describe occurrence of pancreatitis and the evolution of amylase levels during treatment of cutaneous leishmaniasis with antimony therapy for cutaneous leishmaniasis. Material and methods. We evaluate 15 patients without any other pathology during antimony therapy for cutaneous leishmaniasis. All patients had dermal lesions and parasitological or immunological diagnosis of cutaneous leishmaniasis and receive meglumine antimoniate according orientation of Brazilian ministry of health. All patients have amylase levels measured before treatment and in the 5th, 10th, 15th, and after finish of antimony use in the 35th day after beginning of treatment. All patients were clinically evaluated for clinical evidence of pancreatitis. Results. The group was composed by 12 males (80%), average (±SD) age of 36,5(±7,7) years, and receive average(±SD) dose of antimony of 15,9(±1,8) mg/kg. All patients receive 50% or more of the total dose prescribed of antimony. One case of clinical pancreatitis (6,7%), was registered in a female in the 26th day of treatment and receive conservative treatment with analgesics and intravenous fluids with complete resolution of symptoms and normalization of laboratory values. Amylase levels rise in the beginning of treatment and return to normal just after the treatment, pre-treatment average(±SD): 69,4±25,2, 5th day:113,2±52,6 (p=0,007), 10th day:107,0±57,1(p=0.027), 15th day:102,6±44,0 (p=0,017) and 35th day:67,7±25,0(p=0.854). Discussion. Our results demonstrated that chemical pancreatitis or asymptomatic elevation of amylase is common during antimony use, and the levels return to normal after suspension of the drug. Clinical pancreatitis is not common but must be consider as an important adverse effect of antimony therapy even in health and young patients under treatment of cutaneous leishmaniasis with antimony compounds.
- CLINICAL AND EXPERIMENTAL IMMUNOLOGY [950] P 212 - IMMUNOPATHOLOGICAL EVALUATION IN THE LESION SITE OF PATIENTS WITH AMERICAN CUTANEOUS LEISHMANIASIS, CAUSED BY LEISHMANIA (VIANNIA) SP, IN THE AMAZONIAN REGION OF MARANHÃO STATE, BRAZIL RODRIGUES, F.M.D.1; BEZERRA DE MENEZES, J.G.P.1; COELHO NETO, G.T.2; CAMPOS, M.B.3; GAMA, M.E.A.2; GONÇALVES, E.G.R.2; SILVA, A.R.2; LAURENTI, M.D.1; CORBETT, C.E.P.1; SILVEIRA, F.T.4; GOMES, C.M.C.1; GOMES, C.M.C.1 1.LABORATORY OF PATHOLOGY OF INFECTIOUS DISEASES, MEDICAL SCHOOL OF SÃO PAULO UNIVERSITY, SÃO PAULO, SP, BRAZIL; 2.FEDERAL UNIVERSITY OF MARANHÃO, SÃO LUIS, MA, BRAZIL; 3.EVANDRO CHAGAS INSTITUTE, BELÉM, PA, BRAZIL; 4.EVANDRO CHAGAS INSTITUTE AND FEDERAL UNIVERSITY OF PARÁ, ~BELÉM, PA, BRAZIL.
Keyword:american cutaneous leishmaniasis; immunopathology; leishmania(viannia) sp
Abstract: The interaction between Leishmania species and host response results in a wide spectrum of clinical, histopathological and immunopathological presentations in human infection. In Brazil, American Cutaneous Leishmaniasis (ACL) represents a serious public health problem and the Maranhão state is responsible for 13% of the brazilian cases. The aim of this study was to characterize the immunopathology of lesions from 22 biopsies of patients with ATL, caused by Leishmania (Viannia) sp, from Maranhão State. Immunohistochemistry was carried out by incubating skin biopsies with monoclonal mouse anti- human CD68, CD1a, XIIIa factor, CD4, CD8, IL-10 and IFN-gama and polyclonal rabbit anti- human Lisozima, Foxp3 and TGF-ß. Analysis of skin biopsies demonstrated a dense inflammatory infiltrate in the dermis, characterized by the presence of mononuclear cells, mainly lymphocytes, macrophages, plasma cells and granulomas in most samples. Suggestive forms of amastigotes were detected in 90.9% samples. Immunopathological analyses showed high levels of activated macrophages (4824 cell/mm²) when compared to inactive macrophages (3459 cells/mm²). In the epidermis, the densities of Langerhans cells (CD1a+) were 310 cells/mm², while in the dermis the densities of dermal dendritic cells (factor XIIIa+) were 634 cells/mm². High density of CD8 T+ lymphocytes (2374 cell/mm²) has been found compared to CD4+ T lymphocytes (1268 cell/mm²). The cellular density of Treg (Foxp3+) was 301 cells/mm². Among cell markers there were positive correlations between TGF-ß-producing cells and CD8+ T lymphocytes and between TGF-ßproducing cells and dendritic dermal cells, as well as a negative correlation between TGF-ßproducing cells and IFN-gama producing cells. Patients infected with L. (Viannia) sp. presented increased densities of Treg cells, IL-10 and IFN-gama- producing cells compared to L. (V.) braziliensis infected patients. The density TGF-ß producing cells was similar in both groups of patients. Evaluation of immunological markers according to the time of infection showed no differences in the densities of immunostained cell. Our results suggest that the profile of immunopathological response is associated to the control of infection, although the presence of a regulatory response at the site of infection can be responsible to the persistence of parasite in skin. Supported by: LIM50/HC FMUSP; FAPESP (06/56319-1)
- VACCINE DEVELOPMENT AND TRIALS [952] P 213 - CHARACTERIZATION OF NOVEL VACCINE ANTIGENS IN VISCERAL LEISHMANIASIS SESANA, A.M.; DAMASCENO, L.; MELO, M.N.; ANDRADE, H.M.; GAZZINELLI, R.T. UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL.
Keyword:visceral leishmaniasis; vaccine; immune response
Abstract: The evolutionary cycle of Visceral Leishmaniasis (VL) involves the insect vector, man and dog as a reservoir of the parasite. It is therefore of great importance to control the disease development of effective vaccines for dogs, which are critical in transmission and dissemination of VL to humans. The insufficient knowledge about the complexity of the immune response and the low number of immunogenic proteins known so far have hampered the development of new vaccine formulations against VL. Currently, two vaccines for canine VL are commercially available in Brazil: consisting of extracts of the parasite and recombinant protein specific amastigote. However, the efficacy of these vaccines remains partially, being necessary to develop novel formulations aimed more effectively. Whereas the amastigote is stage of the parasite found in the mammalian host, proteins expressed in this form of development represent
potential candidates for vaccine development. Hypothetical proteins identified in the study of Costa et al (2011) showing an increase in expression in amastigotes relative to promastigotes, high percentage of epitopes to CD8 T cells and promiscuity for binding to different HLAs were selected for this work. These new antigens recombinantly produced form and their immunological characterization was performed in an experimental study of protection in BALB/C challenged with L.infantum (MHOM/BR/1972/BH401). Groups of 10 BALB/C mice were immunized for each protein pure or associated with the saponin adjuvant subcutaneously, using 3 doses at 15-day intervals. After 30 days of immunizations, part animal was sacrificed for the tests to assess the cellular and humoral immune responses and the other was challenged with promastigotes of L.infantum for testing protection. Analysis of the cellular immune response was performed by evaluating cytokine release by spleen cells before and after the test challenge, and they -IFN, IL-12, IL-4 and IL-10 as well as the involvement of CD8 and CD4 T cells using the ELISPOT assay. In the analysis of humoral immune responses, serum samples from immunized and challenged animals were used for the quantitation of total IgG, IgG1 and IgG2a using the ELISA method. Evaluation of the parasite load after immunization and challenge test was performed by limiting dilution. Since the hypothetical proteins used to study highly conserved among different species of Leishmania sp., can constitute a candidate to form a panLeishmania vaccine.
- VACCINE DEVELOPMENT AND TRIALS [957] P 214 - FIELD RANDOMIZED DOUBLE-BLIND TRIAL TO EVALUATE THE EFFICACY OF A VACCINE AGAINST CANINE VISCERAL LEISHMANIASIS IN PORTEIRINHA MUNICIPALITY, SOUTH-EASTERN BRAZIL MACHADO-COELHO, G.L.L.1; TIBÚRCIO, A.M.L.2; FRANÇA-SILVA, J.C.3; REGINA-SILVA, S.4; MONTEIRO, E.M.4; ANDRADE, H.M.3; FERNANDES, A.P.S.M.5; DIAS, E.S.4 1.ESCOLA DE MEDICINA, UNIVERSIDADE FEDERAL DE OURO PRETO, OURO PRETO, MG, BRAZIL; 2.HERTAPE CALIER SAÚDE ANIMAL, JUATUBA, MG, BRAZIL; 3.INSTITUTO DE CIÊNCIAS BIOLÓGICAS, UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL; 4.CENTRO DE PESQUISAS RENÉ RACHOU DA FUNDAÇÃO OSWALDO CRUZ, BELO HORIZONTE, MG, BRAZIL; 5.FACULDADE DE FARMÁCIA, UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL.
Keyword:efficacy; canine visceral leishmaniasis; recombinant vaccine
Abstract: Brazil is an endemic country for zoonotic visceral leishmaniasis (ZVL) and regularly conducts prophylactic and epidemiologic control programs involving treatment of human cases, insect vector control and the removal of seropositive dogs. Dogs have a fundamental role in urbanization and in the maintenance of the disease in endemic areas. In the urban and rural areas of municipal of Porteirinha, a city near of Montes Claros, north of Minas Gerais state of Brazil, were accomplished a field trial set out in randomized blocks to evaluate the efficacy of LeishTec® vaccine against canine visceral leishmaniasis (CVL). Blood samples were collected of 2,629 dogs distributed in all 14 urban areas (2,015 dogs) and six of 11 rural districts (614 dogs). The prevalence of CVL were 20.6% (9.1-55.3) and 73.3% (35.9-92.8) in the urban and rural areas, respectively. After serologic survey, a sample of 865 negative dogs by ELISA (Biomanguinhos), and IFAT (Biomanguinhos) were selected to receive three doses and one booster of Leish-Tec® vaccine or placebo. In the highest infection areas, 150 negative prevaccinated (or placebo) beagle dogs were domiciled as sentinels’ dogs. These 1,015 dogs were follow-up during 527 days after the third dose. Dogs seropositive by ELISA, IFAT and Kalazar Detect rapid test (KD-rK39) (IMBIOS) were removed from the area for necropsy and parasites diagnosis (direct exam, marrow culture, PCR of tissue and xenodiagnosis). The protective effect for the transmission of Leishmania in sandfly was 40% in animals vaccinated with Leish Tec ® that produced antibodies A2. Considering only the parasitological diagnosis results, the
protective effect of the vaccine Leish-Tec® in this population was 71% (34.9-87.3; pvalue=0.001), and 81% (CI: 37.6-94.1, p-value=0.001) when the analysis was stratified by individual response of the dog to the antigen A2, i.e., the efficiency of the vaccine is increased in those animals seropositive for anti-A2. These results demonstrate that the vaccine LeishTec® could potentially be employed for vaccination in conjunction with other health strategies, such as serologic detection and euthanasia of infected dogs. Supported by Hertape Calier Saúde Animal S.A. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [962] P 215 - INFLUENCE OF INOCULUM IN THE CLINICAL AND IMMUNOPATHOLOGICAL COURSE OF GOLDEN HAMSTER (MESOCRICETUS AURATUS) EXPERIMENTALLY INFECTED WITH LEISHMANIA (VIANNIA) BRAZILIENSIS RIBEIRO-ROMÃO, R.P.; GOMES-SILVA, A.; COUTO, L.S.; PINTO, E.F.; DA-CRUZ, A.M. IOC/FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:golden hamster (mesocricetus auratus); leishmania (viannia) braziliensis; immunopathology
Abstract: American tegumentary leishmaniasis (ATL) is widely spread in Brazil, represents a public health problem and is one of the most important neglected diseases in our country. In this context, Leishmania (Viannia) braziliensis stands out as the most prevalent species and is responsible for most cases of ATL. Studies of infectious diseases in experimental model are of paramount importance to evaluate immunopathogenesis and efficacy of vaccine candidates and new drugs. Golden hamster appears as the most susceptible experimental model to this Leishmania species, presenting lesions similar to those observed in humans at clinical and histopathological level. This study aimed to define the influence of inoculum size in the clinical and immunological parameters presented by golden hamster (Mesocricetus auratus) infected with L. braziliensis. For this, sixty hamsters in four independent experiments were intradermally infected with different inocula (104, 105 e 106 stationary promastigotes of L. braziliensis) and parameters such as lesions development, parasite load, lymphocyte proliferation response (LPR), immunoglobulin level and histopathology of skin were analysed. Statistically significant differences were seen in lesions size (104 versus 105, p<0,001; 104 versus 106, p<0,001; 105 versus 106, p<0,02; n=60) and positive correlation between: inoculum size and lesion development (r=0,60, p< 0,02, n=60), parasite load and lesion development (r=0,74, p<0,001; n=53); LPR and lesion development (r=0,60, p<0,05, n=15); IgG levels and lesion development (r=0,71, p<0,01, n=15); parasite load and LPR (r=0,79, p<0,001; n=15); IgG levels and LPR (r=0,69, p<0.05; n=15). 104 inoculum generated more benign lesions but with uncertain in terms of lesion outcome (8 in 20 animals didn’t develop lesions) and 106 generated more severe lesions, pointing the 105 in intermediate level. Histopathology of skin lesions showed extensive areas with granulomatous reaction and Shaumann’s bodies, related with presence of parasites and lesion severity since they were more frequently in 106 infected animals than in 105; 104 showed no Shaumann’s bodies. These data report the main clinical and immunopathological parameters associated with infection with L. braziliensis in hamster model and indicate that inoculum size contribute with disease development, pointing 105 the best to be used in immunopathological studies, vaccine and new drugs candidates trials. Support: IOC/FIOCRUZ, FAPERJ, CAPES, CNPq. - CELLULAR AND MOLECULAR BIOLOGY [964] P 216 - CHARACTERIZATION OF CYTOSKELETAL HIGH MOLECULAR WEIGHT PROTEIN IN LEISHMANIA DIAS, S.G.; MOREIRA, B.P.; BAQUI, M.M.A.
FACULDADE DE MEDICINA DE RIBEIRÃO PRETO, RIBEIRÃO PRETO, SP, BRAZIL.
Keyword:flagellar pocket; cytoskeleton; giant protein
Abstract: The cytoskeleton of eukaryotic cells is essential to maintain cell structure and to optimize membrane dynamics, intracellular transport, cell division and locomotion. In higher eukaryotes, the cytoskeleton is mainly composed of actin microfilaments, intermediate filaments and microtubules. In the Trypanosomatidae family, it was demonstrated the existence of a new class of high molecular weight phosphoproteins (1000-4000kDa) in the cytoskeleton, besides having a structural role, have been implicated in the regulation and organization of the cytoskeleton. These proteins are partially located in the flagellar pocket, but they differ for each genus analyzed, and that may be involved with the way of life of these parasites. However, elements that interact with these proteins in the cytoskeleton are still unknown and little is known about the giant proteins of the pathogenic trypanosomatid. In many species of Leishmania we identified a giant protein, which comprises 1500 kDa in the cytoskeletal fraction. The gel slice relative to the L. tarentolae giant protein was injected in BALB/c mice to produce specific antibody. The immunopurified antibody recognizes by western blotting the Leishmania giant protein in the cytoskeletal fraction as well in the isolated flagella. Immunofluorescence assay revealed this protein in the flagellar pocket and along the flagella of the L. major and L. tarentolae. Microtubule Associated Proteins (MAPs) interact with microtubules to regulate their function. It has been reported that some high molecular mass proteins like MAP 2 of brain and MARPs of T. brucei are not affected when high ionic strength cytoskeletal fractions are submitted to heat treatment, whereas tubulin is thermodenatured. Heat treatment of the 1 M NaCl cytoskeletal extracts of L. major and L. tarentolae shows that both proteins partially remain stable in solution, whereas the majority is thermodenatured. Therefore, using antibodies and pull-down techniques we will identify proteins in the cytoskeleton that interact with high molecular weight proteins. Also, a partial amino acid sequence of this protein will be determined by MS mass spectrometry to know the nature of these proteins. - CELLULAR AND MOLECULAR BIOLOGY [965] P 217 - LEISHMANIA (LEISHMANIA) AMAZONENSIS AP-ENDONUCLEASE GENE (LAMAP) RESTORES THE SURVIVAL OF ESCHERICHIA COLI MUTANTS STRAINS CHALLENGED WITH MENADIONE DE OLIVEIRA, M.Y.K.1; VILLELA, É.V.2; DA-CRUZ, A.M.1; BELLO, A.R.2 1.LABORATÓRIO INTERDISCIPLINAR DE PESQUISAS MÉDICAS, INSTITUTO OSWALDO CRUZ, FUNDAÇÃO OSWALDO CRUZ, R, RIO DE JANEIRO, RJ, BRAZIL; 2.LABORATÓRIO DE BIOLOGIA MOLECULAR- DEPARTAMENTO DE MICROBIOLOGIA, IMUNOLOGIA E PARASITOLOGIA-FACULDA, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:leishmania (l.) amazonensis; lamap ; ap-endonuclease
Abstract: The life cycle of Leishmania spp involves two distinct stages: one that occurs inside the vertebrate host (intracellular stage as amastigote) and the stage inside an invertebrate host (the promastigote extracellular form). During the intracellular stage, the parasite is constantly challenged with reactive oxygen species (ROS), produced by phagocytic cells, which can damage the DNA and consequently act as a leishmanicidal mechanism. Some of these damages can generate apurinic or apyrimidinic abasic sites through the parasite genome and must be repaired to assure the survival of this organism. AP-endonuclease family enzymes are actively involved in the repair of some of these lesions, have already been described in Leishmania major and Trypanosoma cruzi. Our preliminary work showed that the lamap gene, an APendonuclease homologue of Leishmania (L.) amazonensis, coded an enzyme able to complement Escherichia coli mutants deficient in the repair of oxidative lesions (mostly
Endonuclease III) and alkylating lesions. In this work, the BW535 E. coli mutant strain (xth-, nfo-, nth-) was transfected with pLamap, a plasmid carrying the lamap gene cloned into the TOPO vector and challenged with menadione sodium bisulfite (MSB) a superoxide-generating agent. The non transfected strain presented minimal growth at 62.5 ÂľM of MSB displaying hypersensitivity to this drug. On opposition, the transfected ones exhibited a higher survival rate, growing at 10.0 mM of MSB, a concentration 80x higher than that observed for the non transfected ones, although a decrease was observed when compared to the control ones. We have extended the scope of action of the lamap gene, showing that its expression increases significantly the survival of E. coli deficient strains challenged with MSB. Peroxidation of lipids and DNA damage are intrinsically associated with the mode of action of MSB. It is also well established that trypanothione, a glutathione analog in trypanosomatids displays an important role in the antioxidant defense. In this regard, the completion of this panel of functional studies carried out momentarily in E. coli strains, as Leishmania mutants are not yet readily available, might suggest novel responses of these trypanosomatids when submitted to oxidative stress. Financial Support: IOC/FIOCRUZ, FAPERJ, CNPq - CELLULAR AND MOLECULAR BIOLOGY [966] P 218 - IDENTIFICATION OF A LEISHMANIA BAX INHIBITOR-1-RELATED PROTEIN INVOLVED IN CELL DEATH REGULATION. GENES, C.1; FASEL, N.2; GAGO, F.1; JIMENEZ-RUIZ, A.1 1.UNIVERSITY OF ALCALA DE HENARES, ALCALA DE HENARES, SPAIN; 2.UNIVERSITY OF LAUSANNE, LAUSANNE, SWITZERLAND.
Keyword:programmed cell death; mitochondria; regulation
Abstract: The relevance of programmed cell death (PCD) on Leishmania parasites has been a controversial issue during the last ten years. Some recent evidences suggest that the apoptoticlike death showed by this organism may be essential for the establishment and maintenance of its pathogenic behavior and for survival in to the vertebrate host. Although several putative effector molecules such as EndoG and metacaspases have been identified and characterized, knowledge about Leishmania PCD is still far to be completed. The role of the single mitochondrion and that of specific molecules involved in regulation of this pathway are important issues yet to be clarified. Apoptosis is classically regulated by the Bcl-2 family of proteins; however comparative genomic studies and conventional bioinformatics tools have failed to detect ortholog Bcl-2 members in unicellular organisms. Recently, the Bax Inhibitor-1 protein (BI-1) in humans has been shown to participate in apoptosis regulation. This protein is the founding member of a family of highly hydrophobic proteins localized in diverse cellular membranes that seem to be evolutionarily conserved, since they are present in a wide diversity of species including bacteria, protozoa and plants. Here we describe the identification of a protein related to the BI-1 family that is highly conserved among Leishmania spp, Trypanosoma cruzi and Trypanosoma brucei. In Leishmania infantum parasites this protein is located in the mitochondrion and its overexpression causes protection against staurosporine-induced cell death. The mechanism responsible for this protection seems to be related to mitochondrial stability since, in contrast with that observed in wild type individuals, parasites overexpressing the Leishmania BaxI-1 like protein (LB1-L) are able to maintain a stable mitochondrial transmembrane potential ( m) and low mitochondrial superoxide anion production during staurosporine treatment. Mitochondrial instability has been well characterized as one of the early apoptotic markers whereas m decrease is strongly
associated with the release of Cytochrome C and proapoptotic nucleases from the mitochondria and apoptosis progression. Thus, LB1-L emerges as one of the first proteins involved in cell death regulation in protozoan parasites. Analyses to further characterize the mechanisms causing death-protection as well as generation of knocked–out parasites are currently in process.
- CLINICAL LEISHMANIASIS [969] P 219 - CLINICAL SIGNS AND LABORATORY ABNORMALITIES ASSOCIATED WITH LEISHMANIA BRAZILIENSIS INFECTION IN DOGS FROM NORTH-EASTERN BRAZIL FIGUEREDO, L.A.; DE PAIVA-CAVALCANTI, M.; ALMEIDA, E.L.; BRANDÃO-FILHO, S.P.; DANTAS-TORRES, F. CENTRO DE PESQUISAS AGGEU MAGALHÃES, RECIFE, PE, BRAZIL.
Keyword:dogs; leishmaniasis; clinical signs
Abstract: In South America, Leishmania infantum and Leishmania braziliensis are the most common etiological agents of visceral and cutaneous leishmaniasis in dogs, respectively. Infection by L. braziliensis in dogs has been reported frequently, being characterized clinically by local, selfhealing ulcers or ulcerated lesions on the ears, scrotum, feet, nipple, snout, and less frequently, on nasal mucosa. However, little is known about the clinical signs and haematological findings of dogs infected by L. braziliensis. This study aims to evaluate the clinical and haematological findings of 17 dogs PCR-positive for L. braziliensis from a rural community of north-eastern Brazil. All dogs were physically examined and haematological findings recorded. The most common haematological findings were thrombocytopenia (82.4%), anaemia (70.6%), low packed cell volume (52.9%) and eosinophilia (41.2%). Twelve (70.6%) dogs displayed at least one evident physical alteration; 11 dogs (64.7%) presented skin lesions, four (23.5%) had weight loss and two (11.8%) onychogryphosis. L. braziliensis-infected dogs present clinical and haematological signs often observed in dogs infected by other pathogens. This suggests that the diagnosis of cutaneous leishmaniasis in dogs cannot be made on a clinical basis only. The clinical and haematological findings observed in dogs infected by L. braziliensis might be suggestive, but are not specific. This fact requires attention of veterinarians and public health workers, in order to avoid misdiagnosis and subsequent elimination of dogs infected by L. braziliensis in Brazil endemic areas. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [970] P 220 - TRANSFORMING GROWTH FACTOR BETA EXPRESSION IN PATIENTS WITH ACTIVE AMERICAN TEGUMENTARY LEISHMANIASIS SOUZA, M.A.; DE ALMEIDA, T.M.; DE CASTRO, M.C.A.B.; DE OLIVEIRA, A.P.; DE ALMEIDA, A.F.; DE OLIVEIRA, B.C.; PEREIRA, V.R.A. CPQAM, RECIFE, PE, BRAZIL.
Keyword:cutaneous leishmaniasis; cytokines; mrna
Abstract: INTRODUCTION: Leishmania infections promote an immune response, mainly characterized by the participation of CD4+ T lymphocytes, and healing or progression of the disease may happen by influence of cytokines produced by immune cells. Once transforming growth factor
beta (TGF- ) is involved in immunomodulation processes, as well as in the development of Th17 subtype, the aim of this study was to characterize the expression of this mediator in peripheral blood mononuclear cells (PBMC) from active american tegumentary leishmaniasis (ATL). MATERIAL AND METHODS: PBMC from twelve patients and five healthy individuals (control group) were incubated during 24 hours in the presence of phytohemagglutinin (5,0µg/ml) and L. (V.) braziliensis soluble (AgSol, 1,25µg/ml) or insoluble (AgIns, 2,5µg/ml) antigens. TGF- expression was evaluated through real time quantitative PCR, with the results obtained by the comparative Ct method. Data were considered significant when p < 0,05 (Wilcoxon and Mann-Whitney tests). RESULTS: Under mitogenic stimulus, TGF- were similarly expressed in ATL patients and control group. TGF- was 2,51-fold more expressed in patients stimulated by AgSol ( Ct = 11,85 ± 2,29) and 2,08-fold under AgIns stimulus ( Ct = 11,67 ± 2,05), even without statistical difference in relation to control group ( Ct AgSol = 13,18 ± 0,22; Ct AgIns = 12,73 ± 0,25). Patients exhibited a 0,88-fold higher TGF- expression in response to AgIns comparing to AgSol, although without statistical significance. MAIN CONCLUSIONS: TGF- expression indicates the development of cellular immune response against L. (V.) braziliensis soluble or insoluble antigens, even without statistical significance in active disease. Other studies involving treated patients and other cytokines are being developed, in order to clarify its role in cellular immune response against L. (V.) braziliensis. - CLINICAL LEISHMANIASIS [972] P 221 - RENAL INVOLVMENT IN HUMAN ACTIVE CHRONIC VISCERAL LEISHMANIASIS POMARES, C.1; JEANDEL, P.Y.2; MARTIS, N.2; VASSALLO, M.3; MORANNE, O.4; HASSEINE, L.5; LAMOTHE, J.6; FUZIBET, J.G.2; ROSENTHAL, E.2; MARTY, P.1 1.CENTRE HOSPITALIER UNIVERSITAIRE DE NICE, UNIVERSITÉ DE NICE-SOPHIA ANTIPOLIS, C3M INSERM 1065, NICE, FRANCE; 2.MÉDECINE INTERNE, CHU ARCHET 2, NICE, FRANCE; 3.CENTRE HOSPITALIER DE CANNES, CANNES, FRANCE; 4.NÉPHROLOGIE - TRANSPLANTATION, CHU PASTEUR, NICE, FRANCE; 5.CENTRE HOSPITALIER UNIVERSITAIRE DE NICE, LABORATOIRE DE PARASITOLOGIE MYCOLOGIE, NICE, FRANCE; 6.CLINIQUE VÉTÉRINAIRE, CARROS, FRANCE.
Keyword:renal involvment; human; active chronic visceral leishmaniasis
Abstract: Introduction Zoonotic leishmaniasis due to Leishmania infantum is endemic in Mediterranean area. Renal involvement is common in dog but rarely in human. Patients and Methods We report three cases of active chronic visceral leishmaniasis (ACVL) with renal impairment. Observation Two 35 and 48-year-old men infected with HIV presented a visceral leishmaniasis (VL) without hepatitis B (HBV) and C viruses (HCV) co-infection. Parasitic disease under the form of ACVL progressed over a period of 10 years along with persistent immune deficiency (with CD4<100/mm3) despite highly active antiretroviral treatment (HAART). Both presented a glomerular damage diagnosed 9 years after the diagnosis of VL. Within this period, several clinical episodes of VL occurred with parasitemia despite primary and prophylactic treatment by liposomal amphotericin B (L-Amb). The first patient revealed an extramembranous glomerulonephritis with Leishmania positive PCR in situ. The second patient developed an impure nephrotic syndrome solely attributed to VL in the absence of HIV nephropathy. Histopathology was not performed in the latter case as the patient died from iatrogenic
pancreatitis. A third man, 53 years old, with no infection with HIV, HVB and HVC and with no underlying immunosuppression presented an ACVL treated 4 times in 5 years by L-Amb. He developed a glomerular proteinuria with microscopic hematuria 4 years after the VL diagnosis, with type 2 cryoglobulinemia. Renal biopsy histology showed a membranoproliferative glomerulonephritis probably due to cryoglobulinemia with Leishmania PCR positive in the cryoglobulin. Discussion During VL, renal involvement is rarely described in HIV-infected patients. There is no report in non immunocompromised patients. Along with the extended life span of HIV-infected people, a new clinical entity, termed ACLV, has been described. ACLF is defined by the presence of relapses over a period of several years together with the continuous blood circulation of the parasite. ACVL appears close to canine leishmaniasis in which renal involvement is frequent.
- CELLULAR AND MOLECULAR BIOLOGY [973] P 222 - THE KILLING OF LEISHMANIA MAJOR BY MACROPHAGES IN RESPONSE TO NEUTROPHIL ELASTASE AND TLR4 DEPEND ON PROTEIN KINASE R DE MACEDO, M.S.F.1; SILVA, T.C.1; VIVARINE, Ă .C.1; TORRES, B.1; COSTA, T.1; MOTTRAM, J.2; LOPES, U.G.1; LIMA, A.P.C.A.1 1.UFRJ/IBCCF, RIO DE JANEIRO, RJ, BRAZIL; 2.WELLCOME TRUST, GLASGOW, UNITED KINGDOM.
Keyword:leishmania major; macrophage; pkr
Abstract: Leishmania major has three genes encoding ecotin-like serine peptidase inhibitors, ISP1-3, one of which, ISP2, is required for parasite survival and growth in macrophages. ISP2 inhibits neutrophil elastase (NE) at the surface of macrophages, preventing the activation of Toll-like receptor 4 (TLR4) that results in parasite death. L. major lacking ISP2 and ISP3 ( isp2/3) survive poorly in murine macrophages and display delayed intracellular growth. Here, we report that the activation of macrophage Double-stranded RNA-activated kinase, PKR, downstream of NE-TLR4 is responsible for isp2/3 killing. PKR activation followed by prodcution type-1 IFN is required for the survival of L. amazonensis in macrophages. In contrast, we show that in RAW cells stably expressing a dominant-negative (DN) PKR construct or macrophages from PKR knockout 129Sv/Ev mice (pkr-/-), isp2/3 survive and grow normally. The killing of isp2/3 in control RAW cells was reversed by 2-aminopurine, a pharmacological kinase inhibitor. Cell treatment with the PKR ligand, synthetic double-stranded RNA poly[I:C], promoted the death of wild type L. major while it did not further enhance killing of isp2/3. The addition of recombinant type-1 IFN to macrophages infected with WT L. major affected the parasites’ ability to multiply, while it did not further affect the delayed growth of isp2/3. Infection by isp2/3 promoted the secretion of TNF- and nitric oxide by RAW, but not by DNPKR RAW. Neutralizing antibodies to TNF- added to RAW cells after the uptake of isp2/3 reverted parasite killing. In vivo analysis revealed that parasite burden in the footpads of mice 48 h after infection with isp2/3 is higher in pkr-/-mice when compared to background mice. Collectively, those observations suggest that in the absence of ISP2, the activation of PKR by L. major through NE and TLR4 contribute to parasite killing in macrophages through TNF and type-1 IFN.
- VACCINE DEVELOPMENT AND TRIALS [976] P 223 - ACIDIC RIBOSOMAL AND HISTONES PROTEINS OF LEISHMANIA INFANTUM AS POTENTIAL CANDIDATES FOR VACCINES AGAINST CUTANEOUS LEISHMANIASIS AND ITS LIMITATIONS/IMPLICATIONS FOR VISCERAL LEISHMANIASIS PEREIRA, L.S.1; ABBEHUSEN, M.M.1; CUNHA, J.M.1; BARRAL, A.1; NETTO, M.B.1; DE OLIVEIRA, C.I.1; DOS SANTOS, W.L.1; SOTO, M.2; BRODSKYN, C.I.1 1.CENTRO DE PESQUISAS GONÇALO MONIZ, FUNDAÇÃO OSWALDO CRUZ, RUA WALDEMAR FALCÃO, 121, CANDEAL, SALVADOR, BA, BRAZIL; 2.CENTRO DE BIOLOGÍA MOLECULAR SEVERO OCHOA, UNIVERSIDAD AUTÓNOMA DE MADRID, CAMPUS DE CANTOBLANCO, MADRID, SPAIN.
Keyword:histones; acidic ribosomal protein; leishmania
Abstract: It has already been demonstrated that immunization with histones (HIS) and acidic ribosomal protein (LiP0) of Leishmania infantum are able to protect mouse against an infection by L.braziliensis and L. major. In this study, we evaluated the ability of immunization with DNA vaccines or recombinant proteins of HIS and LiP0 employing homologous or heterologous prime booster schedules, in protecting against an infection by L. infantum in the hamster. Two to 4 months male hamsters (15 per group) were immunized with plasmids carrying the four genes of HIS (pcDNA3LiH2A-H3, pcDNA3LiH2B-H4) and/or proteins of HIS (rH2A, rH2B, rH3 e rH4) plus 1nM of CpG. Other group of animals were immunized with plasmid of LiP0 (pcDNA3LiP0) and/or (rLiP0) plus 1nM of CpG, three times, at fifteen days of interval. After last immunization, sera and draining lymph node were collected for serologic tests and quantification of cytokine, respectively. Animals were challenged by intradermal route with 105 metacyclic mastigotes of L.infantum plus 0,5 pair of Lutzomya longipalpis salivary gland. Two, 5 and 7 months after challenge, draining lymph node, spleen and liver were obtained to evaluate parasite load during infection by limiting dilution. Only animals that were immunized with heterologous strategy of vaccination showed significantly IgG antibodies anti-His (p<0,0079) and anti-LiP0 (p<0.0041) analyzed by ELISA, when compared to non-immunized hamsters. In cytokine analysis by Real Time PCR, we observed that hamsters immunized with antigen HIS employing homologous strategy presented significant higher expression of IL-10 (p<0,0147) and IFN- (p<0,022) when compared to heterologous strategy. Concerning the group of hamsters immunized with LiP0 in homologous or heterologous strategies, animals, showed a low expression of TGF- and IL-10, but no difference in IFN- was observed when compared with the control group. No difference was observed in the parasite load in the spleen, liver and lymph node between immunized hamsters and controls at all points of evaluation. There was no protection in immunized animals with histones or LiP0 suggesting that are not able to induce a strong Th1 response that would protect against an infection more severe of leishmaniasis as seen in visceral leishmaniasis. - CELLULAR AND MOLECULAR BIOLOGY [977] P 224 - CHARACTERIZATION OF LEISHMANIA SP. FROM SÃO GABRIEL OF CACHOEIRA, AM-BR PINHEIRO, F.G.1; DA CRUZ, P.F.1; ORTIGÃO, M.R.2; FRANCO, A.R.1
1.ALABORATÓRIO DE LEISHMANIOSE E DOENÇA DE CHAGAS, INSTITUTO NACIONAL DE PESQUISAS DA AMAZÔNIA, MANAUS, AM, BRAZIL; 2.DEPARTMENT OF ENTOMOLOGY KANSAS STATE UNIVERSITY, MANHATTAN, UNITED STATES.
Keyword:trypanosomatids; lutzomyia; leishmaniasis
Abstract: The different Leishmania species are morphologically indistinguishable, however molecular and biochemical methods have greatly importance in their identification. The geographical
distribution in the transmission of leishmaniasis in Brazil is clear, in the 80's was notified on 19 federal units while in 2003, were confirmed indigenous cases, in almost all states. Despite this importance in the setting of public health in Brazil, few studies related to the characteristics and variability of parasites in relation to border area in the north region was conducted. The aim of this study was to characterize by molecular method samples of trypanosomatids isolated from sandflies and human of the municipality of São Gabriel da Cachoeira (SGC), border area between the Amazon and two other countries: Venezuela and Colombia. The samples were collected from phlebotomines and biopsies were obtained from lesions with positive diagnosis of human leishmaniasis. Eleven solates were obtained, eight of sandflies and three from humans. Of the seven sandflies samples were from Lutzomyia dendrophyla and L. shanonni. We carried out the sequencing of the internal transcribed spacer region of the ribosomal RNA gene with primers described by Hernandez et al. (1990). Evaluation of the sequences obtained with the editing program and alignment in NCBI website (National Center for Biotechnology Information) of the DNA fragments sequenced, there was the formation of two main and distinct subgenus, nine isolates with biological characteristics similar to subgenus Viannia (two groups A and B) and two isolates for the subgenus Leishmania (C group). Of sequences that grouped the subgenus Viannia, two were human, five of L. dendrophyla and one of L. shanonni. For subgenus Leishmania, each sample was isolated from human and L. dendrophyla. This result suggest the participation of L. dendrophyla in a cycle of Leishmania specie in Amazon region and others trypanosomatids species. Financial support: FAPEAM, FINEP and INPA
- CLINICAL LEISHMANIASIS [981] P 225 - GENETIC DIVERSITY IN LEISHMANIA (VIANNIA) BRAZILIENSIS POPULATIONS FROM BRAZIL IS CORRELATED TO CLINICAL VARIATIONS OF CUTANEOUS LEISHMANIASIS QUARESMA, P.F.; DE BRITO, C.F.A.; FREIRE, J.M.; DE CASTRO, E.M.; REGO, F.D.; RUGANI, J.M.N.; GONTIJO, R.C.; MELO, M.N.; GONTIJO, C.M.F. CENTRO DE PESQUISAS RENÉ RACHOU, BELO HORIZONTE, MG, BRAZIL.
Keyword:cutaneous leishmaniasis; leishmania braziliensis; variabilidade genética
Abstract: American Tegumentary Leishmaniasis (ATL) is a complex parasitic disease presenting significant clinical pleomorphism and epidemiological diversity. Clinical manifestations and course of infection depend on factors such as Leishmania species/strain involved, its virulence and interaction with the host. The contribution of the parasite to the broad spectrum of cutaneous leishmaniasis variation has been supported by some studies that demonstrated a correlation between genetic variants of Leishmania and clinical forms. This study analyzed different isolates of Leishmania (Viannia) braziliensis from patients with ATL residents in the Indigenous Reserve Xacriabá, Minas Gerais, Brazil, an area of particular interest for presenting different clinical forms as well as a variety of reservoirs and vectors of Leishmania.. The molecular characterization of the parasite was performed using analyses of MLEE, hsp70 PCRRFLP and hsp70 sequencing to compare samples obtained from patients with atypical clinical manifestations of the disease or typical lesions. All of the samples analyzed by MLEE showed similar electromorphic profiles with L. (V.) braziliensis reference strain. When the DNA extracted from promatigotes was submitted to hsp70 PCR-RFLP, the analysis showed that the restriction profile obtained for most samples was different from that expected for L. braziliensis and also for other Leishmania species tested. Of all 38 samples tested, one (2.6%) showed a result that corresponds to L. braziliensis and 37 (97.4%) produced a variant profile of L.
braziliensis. The PCR-RFLP performed with DNA extracted directly from lesions fragments resulted in 09 (16.3%) samples with a identical profile of L. braziliensis and 46 with different profile of L. braziliensis. The 1300pb fragment of the hsp70 was sequenced to clarify the identification of the parasite. All obtained sequences aligned to the sequence of reference strains of L. braziliensis, but some single nucleotide polymorphisms (SNPs) were detected at several sites. Furthermore, in our study we observed a strong association between two variants of L. braziliensis and clinical manifestations. Parasites that showed a profile similar to the reference strains of L. braziliensis were related to typical ulcer, while the parasites that showed variant profile were associated with atypical lesions. Studies with other molecular markers are being performed to prove these observations. "Acknowledgements Centro de Pesquisas René Rachou, FIOCRUZ, Fapemig.")
- VACCINE DEVELOPMENT AND TRIALS [985] P 226 - PROTECTION INDUCED BY IMMUNIZATION WITH A LEISHMANIA-SPEFICIC HYPOTHETICAL PROTEIN AGAINST MURINE VISCERAL LEISHMANIASIS COSTA, L.E.1; MARTINS, V.T.2; CHÁVEZ-FUMAGALLIA, M.A.1; DAMASCENO, L.M.; LAGE, P.S.1; LAGE, D.P.3; NASCIMENTO, D.V.3; DUARTE, M.C.1; RIBEIRO, T.G.4; REGIS, W.C.5; DA ROCHA, W.D.6; SOTO, M.7; FERNANDES, A.P.8; TAVARES, C.A.2; COELHO, E.A.1 1.PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE: INFECTOLOGIA E MEDICINA TROPICAL, UFMG, BELO HORIZONTE, MG, BRAZIL; 2.DEPARTAMENTO DE BIOQUÍMICA E IMUNOLOGIA, ICB, UFMG, BELO HORIZONTE, MG, BRAZIL; 3.DEPARTAMENTO DE PATOLOGIA CLÍNICA, COLTEC, UFMG, BELO HORIZONTE, MG, BRAZIL; 4.PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS FARMACÊUTICAS, FACULDADE DE FARMÁCIA, UFMG, BELO HORIZONTE, MG, BRAZIL; 5.PUC MINAS AND MINASFUNGI DO BRASIL LTDA, BELO HORIZONTE, MG, BRAZIL; 6.DEPARTAMENTO DE BIOQUIMICA, UFPA, CURITIBA, PR, BRAZIL; 7.CENTRO DE BIOLOGIA MOLECULAR SEVERO OCHOA, CSIC-UAM, MADRID, SPAIN; 8.DEPARTAMENTO DE ANÁLISES CLÍNICAS E TOXICOLÓGICAS, FACULDADE DE FARMÁCIA, UFMG, BELO HORIZONTE, MG, BRAZIL.
Keyword:vaccine; leishmania infantum; hypotetical protein
Abstract: Experimental vaccine models have been developed to protect against visceral leishmaniasis (VL) using the BALB/c mice immunized with candidate immunogens, and late challenge with different Leishmania species; however, an effective vaccine still does not exist. The present study aims to evaluate a non-described hypothetical Leishmania-specific protein (namely LiHyp4), which was identified by a recent immunoproteomic approach performed in Leishmania infantum, in an attempt to select a new candidate antigen to compose an vaccine against VL. The LiHyp4 DNA sequence was cloned and the recombinant protein (rLiHyp4) was purified, and used to immunize BALB/c mice. In this context, the immunogenicity and protective efficacy of rLiHyp4 plus saponin, as adjuvant, was evaluated in BALB/c mice challenged subcutaneously with 1x107 virulent L. infantum promastigotes. rLiHyp4 plus saponin vaccinated mice showed a specific-Th1 immune response, which was primed by high levels of IFN- , IL-12, and GM-CSF cytokines, after in vitro stimulation with the recombinant protein. Immunized and infected mice, as compared to the control (saline and saponin) groups, showed significant reduction in the parasite load in the liver, spleen, bone marrow, and in the draining lymph nodes in the paws of the animals. Protection was associated with an IL-12dependent production of IFN- , which activate macrophages to eliminate the parasites. In these mice, a decrease in the parasite-mediated IL-4 and IL-10 responses was also observed. Therefore, the present study showed that a non-described hypothetical Leishmania-specific protein, LiHyp4, when combined with saponin as adjuvant, can be useful in the effective protection against murine VL.
Financial support: FAPEMIG, CNPq and INCT-NanoBiofar - CELLULAR AND MOLECULAR BIOLOGY [990] P 227 - DOK PROTEINS ARE RECRUITED TO THE PHAGOSOME AND CLEAVED BY THE PROTEASE GP63 DURING LEISHMANIA MAJOR INFECTION DUPLAY, P.; DE CELIS, H.A.; GOMEZ, C.P.; DESCOTEAUX, A. INRS- INSTITUT ARMAND-FRAPPIER, LAVAL, CANADA.
Keyword:macrophage; protease; signaling
Abstract: Three adaptor molecules of the Dok family, Dok-1, Dok-2 and Dok-3 are expressed in macrophages and are implicated in the negative regulation of signaling in response to LPS and various cytokines and growth factors. We investigated the role and the fate of those Dok proteins during FcR-dependent phagocytosis and following infection with Leishmania major promastigotes in macrophages. The protozoan parasite L. major causes cutaneous leishmaniasis and is renowned for its capacity to alter host-cell signaling and function. Dok-1/Dok2-/- bone marrow-derived macrophages displayed normal phagocytosis of IgG-coated beads and entry of L. major promastigotes. Moreover, activation of ERK1/2 and Akt induced following the entry of the parasites or after stimulation via FcR was not modulated by Dok-1 and Dok-2 proteins. We next showed that Dok proteins were recruited to phagosomes containing latex beads and that Dok remained associated to phagosomes during the maturation process. By contrast, Dok-1 was absent from phagosomes containing Leishmania promastigotes. However, in the absence of the virulence GPI-anchored metalloprotease GP63, Dok-1 was recruited to phagosomes containing L. major promastigotes. Further biochemical analysis revealed that similar to Dok-1, Dok-2 and Dok-3 are substrate of GP63. Moreover, we showed that the production of NO and TNF by Dok-1/Dok-2-/- IFN- -primed BMM upon infection with WT or gp63 L. major promastigotes was reduced compared to WT BMM. These results suggest that Dok proteins may be important regulator of macrophage responses to Leishmania infection. Supported by grants from the Canadian Institutes of Health Research
- CLINICAL AND EXPERIMENTAL IMMUNOLOGY [994] P 228 - PERSISTENT T-CELL ACTIVATION CAN ACCELERATE THE IMMUNOSENESCENCE AND CONTRIBUTE TO DISTURBANCES OF TCRV REPERTOIRE IN VISCERAL LEISHMANIASIS-HIV-1 CO-INFECTED PATIENTS SANTOS-OLIVEIRA, J.R.1; COTA, G.F.2; MARQUES-PAULO, E.1; COUTO, L.S.1; GIACOIAGRIPP, C.B.3; BERTHO, A.L.4; MORGADO, M.G.3; RABELLO, A.2; DA-CRUZ, A.M.1 1.LABORATÓRIO INTERDISCIPLINAR DE PESQUISAS MÉDICAS, INSTITUTO OSWALDO CRUZ-FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 2.CENTRO DE PESQUISAS RENÉ RACHOU, FIOCRUZ, BELO HORIZONTE, MG, BRAZIL; 3.LABORATÓRIO DE AIDS E IMUNOLOGIA MOLECULAR, INSTITUTO OSWALDO CRUZ, FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 4.LABORATÓRIO DE IMUNOPARASITOLOGIA, INSTITUTO OSWALDO CRUZ, FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:cellular activation; immunosenescence; tcrv
Abstract: Visceral leishmaniasis-HIV-1/AIDS patients (VL/HIV-1) have enhanced cellular activation levels, independently of clinical phase of VL, antiretroviral (ART) and anti-leishmanial (ALT) therapies. Indeed, we have shown that Leishmania infection and the plasmatic lipopolysaccharide (LPS), probably of luminal origin, were associated with the heightened cellular activation in VL/HIV-1. Considering this, we investigated whether persistent cell
activation observed in co-infected patients can accelerate the immunosenescence and disturb the T-cell repertoire. Ten VL/HIV-1 were followed-up during active disease, at the end and at sixth month after ALT. Sixteen HIV-1/AIDS patients, 6 VL monoinfected cases and 10 healthy subjects (HS) were included. All HIV-1 cases were under ART therapy and those Leishmaniaco-infected were submitted to secondary chemoprophylaxis to VL with amphothericin B. The immune status was assessed by CD4+T-cell counts, cellular activation degree (CD38+HLADR+) and senescence (CD57+CD27-), as well as by the characterization of the TCRV repertoire. VL/HIV-1 presented low levels of CD4+T-cells (<200cells/mm3) throughout the study. Higher levels of CD38+/HLA-DR+ on CD8+T cells were seen independently of VL clinical phase and even long-term after treatment when compared to VL cases alone (p<0.05). Low or undetectable RNA copies/mL were observed and had no influence on CD38+/HLADR+ or CD4+T counts. Considering all the time points evaluated, VL/HIV-1 presented similar levels of senescent cells on CD8+T (33%) compared to HIV-1/AIDS cases (38%), but higher than HS (12.6%,p<0.05). The degree of immunosenescence was higher on CD8+T cells compared to CD4+T lymphocytes. Consistent with the cellular activation state, a positive correlation between CD38+/HLA-DR+ and CD57+/CD27+ on CD8+T cells was verified. The V families more expressed by CD4+T cells on VL/HIV-1 were the same used in VL and HIV1 cases alone. However, all V chains were used for less than 5% of CD8+T cells from VL/HIV-1, suggesting a different and altered repertoire. Our results suggest that the T-cell activation persistence, despite the control of viral load, ALT and even of the chemoprophylaxis, can contribute to the immunosenescence phenomenon and account for the TCRV repertoire disturbances. As consequence, CD4+T depletion and probably CD8+T function are worsened, impairing the immune status of co-infected patients. Support:MS/PNDSTAids/CNPq/FAPERJ/IOC/FIOCRUZ. - CELLULAR AND MOLECULAR BIOLOGY [995] P 229 - PARTICIPATION OF ARP2, WASP, GALECTIN-3 IN INVASION AND MULTIPLICATION OF LEISHMANIA AMAZONENSIS. MACHADO, F.C.; TEIXEIRA, T.L.; DE ARAÚJO, K.C.L.; QUINTAL, A.P.N.; TEIXEIRA, S.C.; DA SILVA, C.V. UNIVERSIDADE FEDERAL DE UBERLÂNDIA, UBERLÂNDIA, MG, BRAZIL.
Keyword:cytoskeleton; promastigotes; small harpin rna
Abstract: Leishmania amazonensis is the agent responsible for the cutaneous form of leishmaniases. In the vertebrate host, such as humans, L. amazonensis keeps its cycle inside leukocytes, mostly, macrophages. Engulfment of Leishmania promastigotes by macrophages is conducted actively by the cytoskeleton of the host cell. During the intracellular traffic, the cytoskeleton leads several endocytic vesicules forming the phagolysosome which allows replication for the amastigotes forms. Here, we wanted to verifiy the importance of several host cytoskeleton molecules during crucial points of invasion and intracellular multiplication using knockdown macrophages for ARP2 (Actin-Related Protein 2), WASP (Wiskott–Aldrich Syndrome Protein), Galectin-3. These cells lineages were already obtained by transduction of wild-type macrophages with lentiviral particles (Santa Cruz Biotechnology, Inc.) carrying a small harpin RNA capable of interfering in the expression of those molecules. Now, invasion (2 hours) and multiplication (24, 48 and 72) assays are being realized using these new lineages. For final analysis, 100 of total cells will be counted for invasion score and 100 infected cells for multiplication score. Financial support: FAPEMIG/UFU/CNPq.
- OTHER SPECIAL TOPICS [997] P 230 - IN VIVO GROWTH OF LEISHMANIA INCREASES THE INFECTIVITY IN MICE. MÜLLER, K.E.1; REBBESTAD, K.1; TELLEVIK, M.G.2; SØRNES, S.1; NÆSS, A.1; SJURSEN, H.1; NERLAND, A.H.3 1.INSTITUTE OF MEDICINE, UNIVERSITY OF BERGEN, BERGEN, NORWAY; 2.NATIONAL CENTRE FOR TROPICAL INFECTIOUS DISEASES, HAUKELAND UNIVERSITY HOSPITAL, BERGEN, NORWAY; 3.THE GADE INSTITUTE, UNIVERSITY OF BERGEN, BERGEN, NORWAY.
Keyword:leishmania major; virulence; infection
Abstract: Background – The virulence of Leishmania is dependent on the growth status of the parasites, which includes how many passages the parasites have been propagated in vitro. Objectives – To study the effect of in vivo passages in mice on Leishmania (L.) major. Materials and methods - L. (L.) major promastigotes were grown in RPMI medium at 26 °C. The medium was changed every 7 days. 10 L (106/mL) parasites from stationary phase of growth were inoculated into one of the hind-feet of female Balb/c mice. 60 days post infection, parasites harvested from the footpads were transferred to RPMI medium and propagated 4 weeks in vitro, and then used to infect new mice. The passages through mice were repeated 4 times. By parallel inoculation in mice (as described above), parasites propagated 4 cycles in mice were compared to parasites that had only been propagated in vitro. Then the mice were euthanized, footpads, spleen and liver were harvested. Blood samples were taken by heart puncture, and swelling and weight of footpads was registered. To determine the quantity of parasites in the footpad, a qPCR technique was used. To investigate differences in phagocytosis of promastigotes by neutrophils, leukocytes were obtained from heparinized venous blood from healthy human donors. The parasites, which had or had not been propagated in mice, were incubated with pooled human serum. Then total leucocytes and promastigotes were incubated for 7.5 min. for opsonisation and 15 min. for phagocytosis and processed for flow cytometric analysis. The phagocytosis related oxidative burst capacity was evaluated by the conversion of non-fluorescent dihydrorhodamine 123 to fluorescent rhodamine 123. Multiplex array analysis (Life Technologies) was performed to detect serum cytokine levels (GM-CSF, IFN- , IL-1 , IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, TNF- ) in control mice and infected mice. Results – The qPCR results indicate that there is a significant difference between the parasite load in mice infected with parasites propagated in vivo and parasites propagated in vitro. However, our functional studies suggest that there may not be any changes in phagocytosis of promastigotes by neutrophils. No significant difference was observed in serum cytokine levels. - OTHER SPECIAL TOPICS [1000] P 231 - EVALUATION OF MITOCHONDRIAL PATHWAY OF SIGNAL TRANSDUCTION OF APOPTOSIS IN MACROPHAGES INFECTED WITH LEISHMANIA (LEISHMANIA) INFANTUM BASAGLIA, F.C.1; LAULETTA LINDOSO, J.A.1; DE SOUZA, E.B.1; CACERE, C.R.2 1.INSTITUTO DE MEDICINA TROPICAL DA UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL; 2.LABORATÓRIO DE DERMATOLOGIA E IMUNODEFICIÊNCIAS DO INSTITUTO DE MEDICINA TROPICAL, SÃO PAULO, SP, BRAZIL.
Keyword:visceral leishmaniasis – genetics; apoptosis; macrophages
Abstract: During Leishmania infection, the immune response triggers important role in the control or progression of infection by different mechanisms. A possible mechanism for evasion of the response is inhibition of apoptosis in main host cell (macrophage) as already demonstrated that cells infected with Leishmania spp. are protected from death by apoptosis even when induced death, however the mechanism of signal transduction protection apoptosis is not clear. It is speculated that the mitochondrial death pathway is inhibited during Leishmania infection, as it could inhibit the production of pro-apoptotic proteins and induce the production of antiapoptotic proteins, as mechanism of escape from host immune response. Also heat shock proteins during infection produced by Leishmania could bind APAF-1, inhibiting the binding there of caspase 9 and there by inhibiting the formation of apoptossoma that would be critical for cleavage of caspase 3 death effector. In this study we evaluated the protection of apoptosis in J774 and THP-1 infected with Leishmania (L.) infantum and the mitochondrial pathway of signal transduction involved. For this, cells were infected with Leishmania (L.) infantum and induced to apoptosis using camptothecin as inductor. To marker of apoptosis it was measured the expression of phosphatidyl serine and cleaved caspase-3. Aiming to evaluate the behavior of the mitochondrial pathway, we measured the expression of Bcl-2. It was observed in infectedcells inhibition of apoptosis, even when cells were induced with camptothecin. Increased of Bcl2 expression was observed when infected cells were protected from death by apoptosis. Our data suggest that infection of cell lines with Leishmania (L.) infantum inhibits the death of the host cell, probably due to increased expression of anti-apoptotic protein and consequently inhibiting the alteration of mitochondrial membrane potential. - GENOMICS, TRANSCRIPTOMICS, PROTEOMICS, METABOLOMICS [1008] P 232 - RNASEQ ANALYSIS OF LEISHMANIA DONOVANI: A HIGH-THROUGHPUT SCREENING FOR VIRULENCE DETERMINANTS GUERFALI, F.Z.1; PESCHER, P.2; KEDRA, D.3; NOTREDAME, C.3; SPÄTH, G.F.2 1.INSTITUT PASTEUR DE TUNIS, TUNIS, TUNISIA; 2.INSTITUT PASTEUR, PARIS, FRANCE; 3.CENTER FOR GENOMIC REGULATION, BARCELONA, SPAIN.
Keyword:leishmania donovani; rna, deep sequencing; virulence, stress
Abstract: RNASeq analysis of Leishmania donovani: a high-throughput screening for virulence determinants Fatma Z. Guerfalia,b, Pascale Pescherb, Darek Kedrac, Cedric Notredamec, and Gerald F. Späthb a
Institut Pasteur de Tunis, Laboratoire de Transmission, Contrôle et Immunobiologie des Infections, Tunisie; bInstitut Pasteur and CNRS URA258, Unité de Parasitologie moléculaire et Signalisation, Paris, France; cCenter for Genomic Regulation (CRG) and UPF, 08003 Barcelona, Spain. Abstract text: Human visceral leishmaniasis is caused by infection with different species of the Leishmania (L.) donovani complex. Although immunological, genomics, transcriptomics and proteomics studies revealed parasite factors involved in disease progression and intracellular parasite survival, our understanding on Leishmania virulence mechanisms remains very limited, and the
search for virulence-associated determinants applying novel technologies remains an important priority to generate new insight into Leishmania pathogenesis. Here we used RNAseq analysis to profile the transcriptomes of L. donovani LD1S avirulent axenic and virulent hamster-derived amastigotes. Significantly, both strains were derived from the same Sudanese field isolate but were maintained for several decades in different environments, i.e. either exclusively propagated in infected hamsters, or adapted for in vitro culture and axenic amastigote differentiation. Thus our analysis informs on evolution and adaptation of L. donovani gene expression under different environmental constraints. About 30 million reads were generated per transcriptome and mapped on the recently released L. donovani BPK282A1 genome (http://www.genedb.org/Homepage/Ldonovani_BPK282A1) covering almost all reported genes in this species. Using a combined statistical and fold change analysis we revealed a set of 257 genes showing significant differential transcript abundance. A global pathway analysis of these genes showed a significant functional enrichment in axenic amastigotes of transcripts related to the GO terms ‘stress response’ and ‘chaperone function’ indicating that axenic differentiation fails to fully adapt the parasite to growth in acidic pH and elevated temperature, in contrast to bona fide animal-derived amastigotes. Moreover, our analysis revealed in non-virulent axenic amastigotes the existence of whole genomic regions with higher read coverage, potentially due to large duplication events, but also genes without read coverage compared to animal-derived amastigotes likely reflecting gene deletions. Our analysis allowed new insight into mechanisms associated with loss and maintenance of virulence during in vitro culture and in vivo infection, respectively, and revealed the presence of regulatory processes that adapt the parasite transcription profile to different growth environments despite absence of transcriptional regulation and largely constitutive gene expression characteristic for trypanosomatids. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1021] P 233 - LEUKOCYTE BEHAVIOR IN MESENTERIC MICROCIRCULATION OF BALB/C MICE IN EXPERIMENTAL INFECTION WITH LEISHMANIA SPECIES ABREU, H.C.N.; CAETANO, R.C.S.; REINHOLD-CASTRO, K.R.; NOGUEIRA DE MELO, G.A.; CUMAN, R.K.N.; LONARDONI, M.V.C. UNIVERSIDADE ESTADUAL DE MARINGÁ, MARINGÁ, PR, BRAZIL.
Keyword:leishmania; infection; leukocytes migration
Abstract: After Leishmania infections, leukocytes migrate to the infection site to eliminate, isolate or dilute the parasite. With the evolution of stasis in small blood vessels, leukocytes are moved to the plasma zone with greater contact with the endothelium and start the rolling; depending on the stimulus, adhere to endothelium and migrate to interstice. In this study the number of leukocyte rolling, and adhesion to endothelial wall of mesenteric vessels of microcirculation in BALB/c females infected by Leishmania (Viannia) braziliensis, Leishmania (Leishmania) amazonensis and Leishmania (Leishmania) major were evaluated. The objective was understood the cellular recruitment in the earlier stages of infection, and the directing of the immune response that generates different clinical forms of disease or controls infection. The animals were kept without food for 3 hours before surgery. Five animals were used per group: control (PBS), L. (V.) braziliensis, L. (L.) amazonensis and L. (L.) major and, 2 x 107 promastigotes were inoculated intraperitoneally. Animals were anesthetized, and right lateral incision was made to expose the mesentery 3, 6, 12 and 24 hours after inoculation of parasites. The preparation was kept moist by irrigation with Locke-Ringer solution, on a heated plate of optical microscope coupled to digital camera and computer. Were selected postcapillary venules with
23 to 30 µm diameters and extension of 75 µm. The determinations were performed at intervals of 5 minutes. The Leishmania infection resulted in increased numbers of rolling and leukocyte adhesion compared to the control group (190). The mice with L. (V.) braziliensis showed increased number of rolling at 3 hours (222), with peak at 6 hours (362) and gradual decreasing to baseline within 24 hours (177). In animals infected with L. (L.) amazonensis and L. (L.) major the increase was higher at 3 hours (524 and 613, respectively) with reduction in 24 hours (333 and 245, respectively), however, remaining increased at all times. The Leishmania intraperitoneal inoculation promoted an increase in leukocyte rolling and adhesion to endothelium of mesenteric microcirculation, with peak at 6 hours to L. (V.) braziliensis and 3 hours to L. (L.) amazonensis and L. (L.) major. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1022] P 234 - T-BET/GATA-3 RATIO IS INVERSELY CORRELATED WITH TIME OF HEALING IN LEISHMANIA (VIANNIA) BRAZILIENSIS INFECTED PATIENTS. FAGUNDES-SILVA, G.A.1; GOMES-SILVA, A.1; OLIVEIRA-NETO, M.P.2; JUNIOR, S.F.3; DACRUZ, A.M.1 1.LABORATÓRIO INTERDISCIPLINAR DE PESQUISAS MÉDICAS, INSTITUTO OSWALDO CRUZ, FUNDAÇÃO OSWALDO CRUZ, RIO DE JANEIRO, RJ, BRAZIL; 2.INSTITUTO DE PESQUISA CLÍNICA EVANDRO CHAGAS, FUNDAÇÃO OSWALDO CRUZ, RIO DE JANEIRO, RJ, BRAZIL; 3.ALLERGY AND IMMUNOLOGY DIVISION, FEINBERG SCHOOL OF MEDICINE, NORTHWESTERN UNIVERSITY, CHICAGO, UNITED STATES.
Keyword:t-bet\gata-3; spontaneously healed subjects; time of healing
Abstract: The modulation of anti-Leishmania immune response achieved after lesion healing of American tegumentary leishmaniasis is important to the maintenance of clinical cure. Thus, the comprehension of the role of transcriptional factors (TF) involved on the cytokines regulation is of paramount importance once those cytokines levels can be associated with immunopathogenesis or Leishmania control. Our aim was to investigate whether the gene expression of type 1 (T-bet, IFN-gama, IL-12A, IL-18), type 2 (GATA-3, IL-4, IL-5) and regulatory immune response molecules (Foxp3, IL-10), were differentially expressed in clinically cured cutaneous leishmaniasis patients (CCL) after antimonial treatment or in spontaneously healed subjects (SH), and if the time of healing influences the expression of these TFs and cytokines. Cutaneous leishmaniasis (CL) patients with less than two years of clinical cure after therapy (CCL <2y; n=11), from two to five years of clinical cure after therapy (CCL 2-5y; n=10), or that evolved to spontaneous healing without therapeutic intervention (n=05, time of healing >10 years) were investigated. The control group was composed of healthy individuals without history of leishmaniasis (HI; n=08). Total RNA was extracted from peripheral blood mononuclear cells after in vitro stimuli with L. braziliensis antigens. The extracted RNA was used to quantify specific gene transcription by real time PCR. There was no difference at number of RNA copies to T-bet, GATA-3, Foxp3 between CCL groups. However, SH had higher number of mRNA copies for all analyzed TF and cytokines (p<0.05) when compared with CCL groups. The T-bet/GATA-3 ratio was more elevated in CCL<2y (median =1.0; range: 0.8 to 3.8) than CCL 2-5y (median = 0.5; range: 0.2 to 2.7), although this difference was not significant. Interestingly, SH subjects presented the lowest T-bet/GATA-3 ratio (median = 0.1; range: 0.07 to 0.2) in comparison to CCL groups. A significant inverse correlation between Tbet/GATA-3 ratio and the period of healing of lesions was observed (r = -0.62, p<0.01). Taken together, our results suggest that the time of clinical cure can be determinant for establish a Tbet/GATA-3 favorable ratio that sustain the clinical cure conditions. This parameter may represent a pattern of well modulated protective immune response against the parasite. Support: IOC/FIOCRUZ, CNPq, FAPERJ.
- VACCINE DEVELOPMENT AND TRIALS [1029] P 235 - INTRANASAL VACCINATION AGAINST LEISHMANIA (VIANNIA) BRAZILIENSIS INFECTION IN THE GOLDEN HAMSTER (MESOCRICETUS AURATUS) COUTO, L.S.; RIBEIRO-ROMÃO, R.P.; GOMES-SILVA, A.; DA-CRUZ, A.M.; PINTO, E.F. LABORATÓRIO INTERDISCIPLINAR DE PESQUISAS MÉDICAS - INSTITUTO OSWALDO CRUZ - FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:nasal vaccine; leishmania (v.) braziliensis; golden hamster
Abstract: Previously result have shown that intramuscular and subcutaneous administration of adjuvantfree whole Leishmania amazonensis antigens (LaAg) promoted the disease aggravation in mice. Not only oral but also nasal immunization with LaAg protected BALB/c mice against L. amazonensis infection. However, most mice strains are resistant to infection by species of the subgenus Viannia (L. braziliensis, L. guyanensis), responsible for the mainly cutaneous leishmaniasis in the Americas. Recently, it was demonstrated that golden hamster (Mesocricetus auratus) is a suitable experimental model for L. braziliensis infection since it develops chronic lesions resembling humans in both clinical and histopathological aspects. Herein, we investigated the efficacy of the vaccine LaAg administered intranasally to protect against L. braziliensis infection in the hamster model. We used 56 hamsters in three independents experiments, divided in 3 groups (n=19). The animals received one dose containing 10µg or 25µg of LaAg intranasally (IN group) or intramuscularly (IM group), respectively. A booster vaccination was performed 14 days later. Controls received phosphate buffer saline (PBS group). Ten days after the last dose, the animals were infected in the footpad with 1x105 L. braziliensis stationary phase promastigotes. The lesion growth was monitored up to 110 days after infection, at the end parasite loads were measured by limiting dilution assay. AntiLeishmania antibodies levels were measured by ELISA. The IN group presented a significantly lesion reduction (1.2±0.9;median=1.3) compared with IM (1.96±1.09;median=2.3;p<0.01) or PBS (2.0±1.1;median=2.5;p<0.01) groups. There was no significant difference in lesion development between IM and PBS groups. In addition, parasite load was lower in IN group than IM or PBS groups, but without statistical significance. ELISA anti-Leishmania IgG levels were lower in IN group (0.3±0.3;median=0.4) than PBS group (0.9±0.2;median=0.9;p<0.05). Clinical and parasitological aspects showed that nasal immunization with LaAg significantly protected hamsters from L. braziliensis infection. To sum up, these results demonstrate for the first time that the protective effect of nasal immunization with adjuvant-free whole antigens of L. amazonensis promastigotes (LaAg) may be extended to L. braziliensis. Infection. Support: FAPERJ/CNPQ/FIOCRUZ. - GENETICS, EVOLUTION AND TAXONOMY [1042] P 236 - GENETIC VARIABILITY OF NYSSOMYIA NEIVAI POPULATIONS OF PARANÁ STATE, BRAZIL REINHOLD-CASTRO, K.R.1; GASPAROTTO, J.C.1; COSTA-RIBEIRO, M.C.V.2; SOCCOL, V.T.3; CASTRO, E.A.2; LIEBEL, S.M.R.S.2; NEITZKE-ABREU, H.C.1; CRISTOVÃO, E.C.4; TEODORO, U.1 1.UNIVERSIDADE ESTADUAL DE MARINGÁ, MARINGÁ, PR, BRAZIL; 2.UNIVERSIDADE FEDERAL DO PARANÁ, CURITIBA, PR, BRAZIL; 3.UNIVERSIDADE POSITIVO, CURITIBA, PR, BRAZIL; 4.NÚCLEO DE ENTOMOLOGIA, PORTO RICO, PR, BRAZIL.
Keyword:nyssomyia neivai ; genetic variability; ndh4 mitochondrial gene
Abstract: Investigation on the genetic structure of sandfly populations is essential for understanding dispersal patterns of species and development of control strategies, given the importance of
these insects on public health. Thus, in order to understand the genetic structure and gene flow of Nyssomyia neivai species were performed the sequencing of NDH4 mitochondrial gene from population of municipalities of Doutor Camargo, Lobato, Japira, and Porto Rico, Paraná state, Brazil. Sandfly collections were made with Falcão light traps installed in the peridomicile, from 20:00 to 24:00 hours. Females were preserved, one by one, in tubes containing ethanol 80% until DNA extraction with solution of Chelex resin 5%. Were used the primers NDH4 Forward and NDH4 Reverse. The products were submitted to electrophoresis in agarose gel in transilluminator. After quantification, the sequencing was performed on automated sequencer ABI 3130. The sense and antisense strands obtained were examined in the Staden package software version 1.5, which produced a consensus strand for each sample. The sequences alignment was performed using the ClustalX program version 1.83. Were sequenced 80 Ny. neivai specimens, 20 from each municipality, getting 269 base pair. Were detected a total of 27 haplotypes, 28 polymorphic sites, haplotypic diversity of 0.80696 and a nucleotide diversity of 0.00567. H5 haplotype, with 33 specimens, was the most common among the four populations. H5 and H7 haplotypes were the only ones present in the four sandfly populations. Ny. neivai population from Doutor Camargo presented the higher genetic diversity. Just this population shared haplotypes with the other municipality population; it shared the highest number of haplotypes with Lobato and also had the highest number of exclusive haplotypes. There was no significant correlation between genetic and geographic distances from studied population. However, were observed the highest genetic and geographic distance, and the lower gene flow among Japira and Porto Rico. The Neighbor-Joining (NJ) dendograms. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1047] P 237 - CELLULAR CYTOTOXIC PROFILE IN THE IMMUNOPATHOGENESIS OF CUTANEOUS LEISHMANIASIS CUNHA, C.F.1; FERRAZ, R.1; SCHUBACH, A.2; PIMENTEL, M.I.F.2; GOMES-SILVA, A.3; DACRUZ, A.M.3; LYRA, M.R.4; BERTHO, Á.L.5 1.LABORATÓRIO DE IMUNOPARASITOLOGIA, INSTITUTO OSWALDO CRUZ, FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 2.SERVIÇO DE REFERÊNCIA EM LEISHMANIOSES, INSTITUTO DE PESQUISA CLÍNICA EVANDRO CHAGAS, FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 3.LABORATÓRIO INTERDISCIPLINAR EM PESQUISAS MÉDICAS, INSTITUTO OSWALDO CRUZ, FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 4.SERVIÇO DE REFERÊNCIA EM LEISHMANIOSES - INSTITUTO DE PESQUISA CLÍNICA EVANDRO CHAGAS - FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 5.LABORATÓRIO DE IMUNOPARASITOLOGIA INSTITUTO OSWALDO CRUZ - FUNDAÇÃO OSWALDO CRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:cutaneous leishmaniasis; cytotoxicity; flow cytometry
Abstract: The clinical course of cutaneous leishmaniasis (CL) depends on the interaction between the parasite and the immune response of patients. Is well known that CD8+ T lymphocytes play a key role in this response due to their cytotoxic potential and cytokine production. Nevertheless, natural killer cells (NK), natural killer T cells (NKT) and a cytotoxic subpopulation of CD4+ T lymphocytes present similar functions, and few studies point to the roles of these cell populations in the pathogenesis in CL. The aim of this study is to evaluate the frequency and the cytotoxic profiles of these cell populations, by immunophenotyping and the assessment of the expression of CD107a, in peripheral blood samples from active disease (AD) and after antimonial therapy (AT) CL patients. For this, samples were obtained from healthy individuals (HI) and from the two cohorts of CL patients. Cells were submitted to a flow cytometry analysis to assess the distributions of CD4+ or CD8+ T lymphocytes, NK and NKT cells, and their cytotoxicity profiles by identifying the CD107a expression. Results have allowed us to observe that the percentage of CD8+ T lymphocytes is lower in AD patients compared to HI and AT patients, in which the frequencies were similar. CD4+ T lymphocytes showed higher percentages in HI compared to the groups of patients, in which the frequencies were similar. Conversely, NK and NKT cells had lower percentages in HI compared to patients, of which higher rates were seen in those with active disease. Furthermore, concerning about the citotoxic
profiles (CD107a+), higher percentages of CD4+ and CD8+ T lymphocytes and NKT cells were observed in AT patients compared to AD patients and, most dramatically, to HI. In contrast, percentages of CD107a+ NK cells were lower in AT patients compared to HI and AD patients. These results point in particular to an important role of CD8+ T lymphocytes in disease resolution, showing not only a higher frequency of these cells but also a remarkable cytotoxic profile after clinical cure of CL. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1056] P 238 - LEISHMANIA BRAZILIENSIS-REACTIVE T CELLS ARE DOWN-REGULATED IN LONG-TERM CURED CUTANEOUS LEISHMANIASIS, BUT THE RENEWAL CAPACITY OF T EFFECTOR MEMORY COMPARTMENTS IS PRESERVED PEREIRA-CARVALHO, R.1; MENDES-AGUIAR, C.O.1; OLIVEIRA-NETO, M.P.2; FAGUNDESSILVA, G.A.1; COVAS, C.J.F.1; BERTHO, A.L.3; DA-CRUZ, A.M.1; GOMES-SILVA, A.1 1.LABORATÓRIO INTERDISCIPLINAR DE PESQUISAS MÉDICAS/IOC/FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 2.IPEC/FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 3.LABORATÓRIO DE IMUNOPARASITOLOGIA/IOC/FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:leishmania (vaiannia) braziliensis; t cell memory; immuneregulation
Abstract: Leishmania (Viannia) braziliensis control and tissue damage are relate to the effector immune response, which in turn affects clinical outcome. Leishmania reactive CD4+ and CD8+ T cells are expanded in long-term healed cutaneous leishmaniasis (hCL) patients but their functional characteristics remain to be determined. This study investigates antigen-specific recall in longterm healed CL caused by L. braziliensis infection. Healed CL subjects were grouped according to the time elapsed since the end of therapy: less than two years and two to five years. Activation, cytotoxity, and subpopulations of memory T cell phenotypes were quantified in blood mononuclear cells ex vivo and after Leishmania antigens stimuli. A reduction in the percentage of activated (CD69+) Leishmania-responder CD4+ and CD8+ T cells in hCL was associated with the time elapsed since clinical cure (r= -0.61, p<0.05, n=13; and r= -0.56, p<0.05, n=14, respectively). Percentage of CD69+ in TCD4+ and TCD8+ cells were negatively correlated with IL-10 levels (r= -0.71, p<0.05, n=08; and r= -0.80, p<0.01, n=09, respectively). Ex vivo analyses showed contracted T effector memory (TEM) CD4+ (4.4%±4.4%, median=3.1%, n=15) and TEM CD8+ (3.3%±4.2%, median=2.3%, n=15) compartments, although renewal of these compartments was observed following in vitro exposure to leishmanial stimuli (18.6%±17.6%, median=8.4%, n=12, p<0.05; and 13.7%±11.7%, median=9.4%, n=12, p<0.01, respectively). Finally, this increase of TEM CD8+ under leishmanial stimuli was positively correlated with the frequency of activated TCD8+ cells (r= 0.66, p<0.05), although not positively associated with cytotoxic phenotype. Our results show that healed L. braziliensis infected patients exhibit a recall response to Leishmania antigens with evident expansion of effector memory T cells. Regulated leishmanial-specific response seems to emerge only about two years after initial contact with the parasite antigens. These results raise the question whether clinical trials aimed at determining vaccine efficacy being conducted too early. Suport: IOC/FIOCRUZ; CNPq; FAPERJ. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1062] P 239 - COMPLEMENT SYSTEM ACTIVITY IN THE SERUM OF DOGS NATURALLY INFECTED WITH LEISHMANIA INFANTUM NASCIMENTO, A.A.S.1; ROATT, B.M.2; DA SILVA, S.M.3; ARAUJO, R.N.1; PEREIRA, M.H.1; FUJIWARA, R.T.1; GONTIJO, N.F.1 1.UNIVERSIDADE FEDERAL DE MINAS GERAIS - UFMG, BELO HORIZONTE, MG, BRAZIL; 2.UNIVERSIDADE FEDERAL DE OURO PRETO - UFOP, OURO PRETO, MG, BRAZIL; 3.UNIVERDIDADE FEDERAL DE UBERLANDIA - UFU, UBERLANDIA, MG, BRAZIL.
Keyword:complement system; dogs; hemolysis
Abstract: The complement system is part of the innate immune system and is comprised of three different pathways: the classical, the lectin and the alternative pathway. It recognizes patterns present on the surface of different pathogens, such as Leishmania infantum, the causative agent of visceral leishmaniasis in Americas, promoting phagocytosis and / or lysis of these organisms. This study aimed to compare the activity of the complement system (classical and alternative pathways) in dogs naturally infected with Leishmania infantum with the activity present in non-infected dogs. Different dilutions of the sera were incubated during thirty minutes at 37°C with a suspension of sheep erythrocytes previously sensitized with anti-sheep erythrocyte antibody (for assay of the classical pathway) or with a suspension of rabbit erythrocytes (for assay of the alternative pathway). After incubation, reactions were stopped by addition of cold 0.9% NaCl solution. The samples were centrifuged and the supernatant was transferred to a 96 wells plate for subsequent reading of the optical densities at 414nm in order to evaluate the hemolysis. Spontaneous and total hemolysis were also measured. The activity of the complement was expressed by the serum dilution required to achieve 50% of hemolysis. For the classical and alternative pathways, the amount of serum required to achieve 50% of hemolysis was significantly lower in the group of dogs naturally infected with Leishmania infantum when compared to the healthy dogs. Thus, unlike what happens in other parasitic diseases, which promotes a decrease in the activity of the complement system; visceral leishmaniasis might cause an increase in the activity of this system in infected dogs. Despite the increased activity of the complement, the dogs remain highly susceptible to visceral leishmaniasis. - VACCINE DEVELOPMENT AND TRIALS [1067] P 240 - EVALUATION OF LEUKOCYTE MIGRATION IN IMMUNIZED MICES WITH POTENTIAL VACCINE ADJUVANTS AND ITS EFFECT ON EXTRACELULLAR MATRIX OLIVEIRA, L.A.M.; DE MOURA, T.C.M.; DA SILVA, M.R.; DE MIRANDA, M.B.; DE SOUZA, J.V.; MOREIRA, N.D.; REIS, A.B.; DE MOURA, S.A.L. UNIVERSIDADE FEDERAL DE OURO PRETO, OURO PRETO, MG, BRAZIL.
Keyword:leukocyte migration; adjuvants; extracellular matriz
Abstract: Intradermic immunization occurs in skin’s superficial layers, rich sites of defense cells. The comprehension of structural elements and supporting stroma biomolecules after the administration of vaccine components in murine model is extremely important regarding the establishment of pattern and evolution of triggered response. The study was developed in two steps: in the first one, it was determined the enzymes activity of activated leukocyte MPO (neutrophils' Myeloperoxidase) and NAG (macrophages' N-Acetyl-ß-D-glucosaminidase) to each group of vaccine adjuvant used, and evaluated the histopathological changes in comparison to the control group. In the second step, it was morphometric characterized the participation of extracellular matrix with qualitative and quantitative analyses of the adhesion proteins laminin and fibronectin. Through histopathological evaluation and analyses of the enzymes MPO and NAG, we observed the active participation of adjuvants SAP, AIF and MPL in inflammatory kinetic. The first results indicate a more balanced and efficient performance from SAP regarding the studied mechanisms. It seems to activate neutrophils moderately, promoting a reasonable initial response to inflammatory stimulation, associated with an important macrophage activation, which cooperates with other immune system components and enhance the resolution process. In addition, SAP showed a coherent immunohistochemical expression of laminin and fibronectin, which support inflammatory response. With these results we target to
obtain the profile from the studied adjuvants, contribute to vaccinology field and support the definition of these substances immunogenic potential. Therefore, we shall provide cientific and technical tools to future tests with potential vaccine candidates that would maximize vaccination immunizing action and decrease possible adverse effects. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1068] P 241 - ASSOCIATION OF ANTIBIOTIC TO ANTI-LEISHMANIAL THERAPY CAN CONTRIBUTE TO DECREASE THE POSSIBLE EFFECTS OF PLASMATIC LPS IN THE IMMUNOPATHOGENESIS OF EXPERIMENTAL VISCERAL LEISHMANIASIS MARQUES-PAULO, E.1; SANTOS-OLIVEIRA, J.R.1; SOARES, S.R.C.2; LINDOSO, J.Â.L.2; DACRUZ, A.M.1 1.LABORATÓRIO INTERDISCIPLINAR DE PESQUISAS MÉDICAS, INSTITUTO OSWALDO CRUZ-FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 2.LABORATÓRIO DE SOROEPIDEMIOLOGIA E IMUNOBIOLOGIA, INSTITUTO DE MEDICINA TROPICAL DE SÃO PAULO-IMT, SAO PAULO, SP, BRAZIL.
Keyword:lipopolisaccharide ; antimicrobial therapy; anti-leishmanial therapy
Abstract: Visceral leishmaniasis (VL) is caused in Brazil by Leishmania (Leishmania) infantum. The active phase of VL is characterized by parasite-specific immunosuppression along with low levels of CD4+T cells. Despite this, an intense cellular activation and pro-inflammatory cytokine response is observed. In other T-cell deficient diseases such as HIV-1/AIDS, the lipopolisaccharide (LPS) probably from gut microbial translocation has a crucial role on immune activation. Our recent results showed that LPS along with parasite antigens are implicated in T-cell activation and pro-inflammatory cytokines in VL patients. In this context, our aim was to evaluate whether plasma LPS levels were increased in experimental VL and the possible association with the clinical aspects. Also, we investigated if the specific antileishmanial and/or gram-negative antimicrobial treatment could influence the LPS levels. For this, 117 golden hamsters (Mesocricetus auratus) were divided into 8 groups: without infection, infected with L. infantum; infected and uninfected group treated with anti-leishmanial therapy (antimonial); infected and uninfected animals treated with the antibiotic amikacin; and infected and uninfected animals treated with both drugs concomitantly. The clinical evaluation included animal weighting as well as spleen and the liver weight. Physical aspects of the infected animals and anatomical alterations in the small intestine were also evaluated. Parasite load was quantified by the number of stained amastigotes in the spleen imprint. Plasma LPS levels were measured using a commercial kit. Infected group presented a significant increase of the median spleen weight 101 days post-infection, compared to infected and treated group. The lowest spleen weight was observed in those animals treated with both drugs. Consistent with this, infected and treated animals presented lower number of amastigotes/mg compared to infected group. No parasite load differences were observed between antimonial and antimonial plus antibiotic group. Also, the small intestines of infected animals were visually altered compared to uninfected group, due to a decrease in the number of Peyer’s patches. Small intestines of treated animal were macroscopically similar to uninfected ones. However, clinical signs of VL were not observed throughout the experiment. After 45 days post-infection, the plasmatic LPS levels were increased in infected group in comparison to uninfected. The LPS levels tended to decrease after the treatment, especially in the group under antibiotic use. Our results suggest that LPS can be involved in immunopathogenesis of experimental VL. Indeed, small intestine damage can be related to LPS gut origin. Further studies are needed to unequivocally demonstrate the possible benefits of concomitant use of antibiotic and anti-leishmanial therapy. Support: IOC/FIOCRUZ, CAPES, CNPq, IMT/USP.
- GENOMICS, TRANSCRIPTOMICS, PROTEOMICS, METABOLOMICS [1070] P 242 - DISCOVERY OF NEW TARGETS FOR VACCINE AGAINST CANINE VISCERAL LEISHMANIASIS USING REVERSE VACCINOLOGY DE BRITO, R.F.1; OLIVEIRA, M.M.1; REZENDE, A.M.2; OLIVEIRA, L.A.M.3; REIS, A.B.3; CORRÊA-OLIVEIRA, R.4; RUIZ, J.C.2; BOUILLET, L.É.M.1; RESENDE, D.M.1
1.LABORATÓRIO DE PESQUISAS CLÍNICAS, UNIVERSIDADE FEDERAL DE OURO PRETO, OURO PRETO, MG, BRAZIL; 2.LABORATÓRIO DE PARASITOLOGIA CELULAR E MOLECULAR, CENTRO DE PESQUISAS RENÉ RACHOU, BELO HORIZONTE, MG, BRAZIL; 3.LABORATÓRIO DE IMUNOPATOLOGIA, UNIVERSIDADE FEDERAL DE OURO PRETO, OURO PRETO, MG, BRAZIL; 4.LABORATÓRIO DE IMUNOLOGIA CELULAR E MOLECULAR, CENTRO DE PESQUISAS RENÉ RACHOU, BELO HORIZONTE, MG, BRAZIL.
Keyword:reverse vaccinology; canine visceral leishmaniasis; leishmania infantum
Abstract: Canine Visceral Leishmaniasis (CVL) is a zoonose in Europe and Latin America and dogs play a key role in the urban cycle of Leishmania infantum. Time and cost are limitations for classic methodology for vaccine development. Reverse vaccinology arose in this context with epitope predictions for T and B cells, important for protective immune responses, allowing the design of vaccines at reduced cost and time. Previous studies showed the feasibility of making epitope prediction in proteins of protozoa using open source algorithms. Also, immunoinformatics uses subcellular localization prediction in order to select proteins that are in contact with cells of immune system. The objective of this work is to select genes from the protozoan to vaccine targets development against CVL. For this, complete L. infantum predicted proteome was locally installed and used in all subsequent analyses. Epitope prediction was made with the following algorithms installed in local server: a) to MHC-I, NetCTL and NetMHC; b) to MHCII, NetMHCII; c) to B cells, BepiPred and BCPreds (BCPred12 and AAP12 models). Twelve human and 7 mouse MHC-I alleles were analyzed and, in the context of MHC-II, 14 human and 3 mouse alleles were analyzed. Subcellular localization prediction was made with WoLF PSORT. NetMHC predicted 277,398 epitopes and NetCTL totaled 1,700,109 predicted epitopes. In the context of MHCII, NetMHCII 5,859,600 epitopes were predicted. Concerning B-cell epitopes, BepiPred made 47,482 predictions, AAP12 made 2,361,313 predictions and BCPred12 made 957,493 predictions. Regarding subcellular localization, WoLF PSORT identified 2,020 proteins, either secreted or located on plasmatic membrane. To integrate the results of WoLF PSORT and epitope predictions, a relational database was created in a Management System Database, MySQL, in order to establish an environment which made it possible to accommodate the data from diverse approaches predictions, to manipulate and to extract the relationships between entity classes in order to define vaccine targets in the L. infantum predicted proteome. To select the potential vaccine targets, specific criteria defined by us were used. Finally, five proteins with more than 10 interactions in the protein-protein interaction network were selected, since this interactions identifies vital proteins to parasite biology. The resource developed represents an important tool that can be used to drive vaccine development against CVL. Supported by: CNPq, CAPES, FAPEMIG AND UFOP - GENOMICS, TRANSCRIPTOMICS, PROTEOMICS, METABOLOMICS [1072] P 243 - COMPARATIVE SECRETOME ANALYSIS OF LEISHMANIA (VIANNIA) BRAZILIENSIS STRAINS ISOLATED FROM PATIENTS WITH CUTANEOUS AND DISSEMINATED MANIFESTATIONS OF AMERICAN CUTANEOUS LEISHMANIASIS RODRIGUEZ VEGA, C.A.1; SABOIA-VAHIA, L.1; RODRIGUES, C.M.1; VALLEJO, A.2; DOMONT, G.3; CARVALHO, P.4; B. DE JESUS, J.5; CUERVO, P.1 1.IOC-FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 2.UNIVERSIDAD NACIONAL DE COLOMBIA, BOGOTA, COLOMBIA; 3.UFRJ, RIO DE JANEIRO, RJ, BRAZIL; 4.FIOCRUZ, CURITIBA, PA, BRAZIL; 5.UFSJ, MINAS GERAIS, MG, BRAZIL.
Keyword:secretome; leishmania (viannia) braziliensis; proteins
Abstract: Leishmania (Viannia) braziliensis is widely distributed throughout the American continent being responsible for a large number of cases of tegumentary leishmaniasis (TL). This species has been associated with a broad spectrum of clinical manifestations ranging from self-limiting cutaneous lesions to disseminated forms, with mucosal involvement. To date, the role of this parasite in the development of the different clinical forms remains unclear. Proteins secreted by Leishmania are key mediators of the host–parasite interaction so their identification could lead to understanding its survival mechanisms and the pathophysiology of leishmaniasis. We have previously identified several virulence factors in the secreted fraction of L. (V.) braziliensis; however, the function of most molecules secreted by this parasite is still unknown. The aim of this study is to compare the protein profiles secreted by two L. (V) braziliensis strains associated to single cutaneous leishmaniasis and disseminated leishmaniasis to ultimately identify proteins potentially related to each clinical manifestation of TL. Secreted proteins were obtained from parasite cultures in conditioned medium. Parasites integrity and viability were assessed by propidium iodide and FACS to ensure over 97% viability during secretion assays. Proteins in the supernatants were collected and trypsin digested. Derivatization of peptides with isotopically distinguishable labeling agents was used for unbiased, quantitative profiling of L. (V.) braziliensis secretome from the strains associated with different clinical manifestations of TL. Labeled peptides from each strain were mixed and subjected to nanoLC fractionation in line with a hybrid LTQ XL-Orbitrap mass spectrometer. Peptide identification was performed using the ProLuCID software and quantification with PatternLab’s SEProQ module. A total of 241 proteins were identified from 1358 peptides derived from the purged, combined MS/MS spectra of both L. (V) braziliensis strains secretomes of which 98 were considered reliable quantified based on the statistical analysis of peptide identifications (p<0.01, FDR<0.01, min 2 peptides) and reproducible presence in technical and biological replicates. Of these 98 proteins, 35 proteins were differentially expressed, 19 up-regulated and 16 down-regulated in the disseminated strain when compared to the cutaneous one. - GENETICS, EVOLUTION AND TAXONOMY [1076] P 244 - MICROSATELLITE MARKERS FOR THE CHARACTERIZATION LEISHMANIA INFANTUM ZYMODEME MON-1 ISOLATES SILVA, S.O.; CUNHA, J.L.R.; BARTHOLOMEU, D.C.; MELO, M.N. DEP. DE PARASITOLOGIA ICB - UFMG, BH, MG, BRAZIL.
Keyword:l. infantum; microsatellite; genetic diversity
Abstract: In Brazil Visceral Leishmaniasis (VL) constitutes a serious public health problem, due to its wide geographic distribution, the high number of cases and severity of this clinical form. Leishmania (Leishmania) infantum (syn. L. chagasi) zymodeme MON-1 is responsible for most cases of visceral leishmaniasis in the world. Several molecular markers have been developed to resolve differences within the Leishmania taxon and to try to elucidate genetic, epidemiological and population structure aspects of the disease. In this study, we used a panel of microsatellite markers described in the literature for L. infantum and another new one selected using bioinformatics approaches derived from different chromosomes of the parasite with different nucleotide compositions, namely: LinJ 05, LinJ 09, LinJ 10, LinJ 29, LinJ 33. These microsatellites were used to investigate, the level of diversity of the population of L. infantum strains isolated of humans and dogs cases from a geographical urban area of Brazil, compared with isolates from Portugal. The multilocus microsatellites typing showed that the technique was sensitive in the detection of different profiles within the population of L. infantum studied. Variable ranges of polymorphism were detected among the microsatellite loci analyzed. The polymorphism was low, as demonstrated for isolates of L. infantum MON-1. Population
structures were inferred by distance-based and Bayesian-based approaches. The samples were divided into two populations POP1 and POP2, and grouped into 17 different genotypes differing only in one or two loci. Despite being represented by a low number of isolates, the POP1 revealed some geographical association, grouping the reference strain WHO IPT1/Old World with strains from Europe. However, it was not possible to correlate the parasite genotype with the host. In this context, POP1 was also the group with the highest allelic variability between canine samples. In POP2 we found a prevalence of genotypes very similar between human and dogs but could not correlate them to a given geographic origin. In conclusion, our results demonstrate that the Brazilian L. infantum isolates are genetically homogeneous and the presence of similar genetic profiles in endemic areas is consistent with the zoonotic transmission of LV, since the domestic dog has been incriminated as the main reservoir of the disease in both studied urban area. Supported by CNPq and FAPEMIG
- OTHER SPECIAL TOPICS [1082] P 245 - SUSCEPTIBILITY TO CUTANEOUS AND MUCOSAL LEISHMANIASIS IN PERUVIANS IS ASSOCIATED TO SLC11A1 AND CXCR1 BARRETO, T.V.1; HERRERA-VELIT, P.1; LLANOS-CUENTAS, A.2; TAYPE, C.A.3; MIRANDAVERÁSTEGUI, C.2; LAZO-ESCALANTE, M.2; CRUZ, M.2; ESPINOZA, J.R.1
1.MOLECULAR BIOTECHNOLOGY UNIT, UNIVERSIDAD PERUANA CAYETANO HEREDIA, LIMA, PERU; 2.INSTITUTO DE MEDICINA TROPICAL “ALEXANDER VON HUMBOLDT, UNIVERSIDAD PERUANA CAYETANO HEREDIA, LIMA, PERU; 3.3PULMONARY AND CRITICAL CARE MEDICINE, UC SAN DIEGO, SCHOOL OF MEDICINE, CALIFORNIA, UNITED STATES.
Keyword:leishmaniasis; candidate susceptibility ; slc11a1, cxcr1, cxcr2, vil1
Abstract: The present work is a case/control study to test SLC11A1, VIL1, and CXCR1/CXCR2 for association with cutaneous and mucosal leishmaniasis in patients infected with Leishmania braziliensis species that acquired the infection in the Amazonian Region of Peru. Patients (47 cutaneous, CL, and 51 mucosal leishmaniasis, ML) and 102 healthy controls were evaluated by genotyping SLC11A1 (INT4, D543N, 3'UTR), CXCR1 (+2607 G/C) (IL8RA), CXCR2 (+ 785 C/T) (IL8RB) and VIL1 (LD4, rs733925). Case-control analysis suggested association to INT4 (469 + 14G/C) polymorphism to CL (P=0.020) and ATL (P=0.024) and were associated to INT4-D543N-3'UTR-LD4 haplotype distribution (P-values ranging 3.8 x 10-7 – 0.0007). The CG+G haplotype was associated with ML (P=0.0001, OR 4.69 CI95% 1.89-11.81), and ATL (P=5.7 x 10-6; OR, 5.02; 95%CI, 2.28-11.25), and CA–G haplotype was associated to ML (P=0.0002, OR 6.88 CI95% 2.06-24.23) and ATL (P= 0.0002, OR 5.97, CI95% 1.99-19.13), but no significant association was observed to CL. In addition CXCR1 appears associated to CL with CC genotype (P= 0.0043, OR 9.33 95% CI 1.60-70.48), to ML (P=0.0077, OR 8.0 95% CI 1.38-59.87) and to ATL (P=0.0014, OR 8.62 95% CI 1.74-57.62). Our results suggest that SLC11A1 and CXCR1 are genes that influence the susceptibility to cutaneous and mucosal leishmaniasis by Leishmania braziliensis species.
- CELLULAR AND MOLECULAR BIOLOGY [1087] P 246 - INVOLVEMENT OF IPLA2 IN ADHERENCE, INTERNALIZATION AND INFECTIVITY OF LEISHMANIA AMAZONENSIS IN PERITONEAL MACROPHAGES FERNANDES, A.C.S.; SOARES, D.C.; SARAIVA, E.M.; SOUTO-PADRON, T. UFRJ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:leishmania amazonensis; phospholipase a2; bromoenolactone
Abstract: Exposure of such important molecules on the surface of both cells or their secretion into the extracellular environment depend on the participation of endo/exocytic pathways where the fusion between distinct intracellular compartments are mediated by multiple factors among them the action of phospholipases. We have been investigating the participation of the Ca (2+)independent phospholipase A2 (iPLA2) in the fusion of endo/exocytic compartments of Leishmania amazonensis and its consequences in the process of interaction with mouse peritoneal macrophages. Metacyclic promastigotes of L. amazonensis grown in Schneider medium for 144h were incubated for 1 h with 2.5 µM of bromenolactone (BEL), an irreversible inhibitor of iPLA2. After treatment parasites were incubated with macrophage and the and adhesion to, internalization and survival of the parasite within the macrophage were analyzed for 48 hours. BEL reduced by approximately 80% the adhesion of promastigote to macrophage surface. Unlike the control, BEL treated parasites adhere to macrophages exclusively by the flagella. BEL also reduced the percentage of infected macrophage and the number of parasites within the parasitophorous vacuole (PV) in 80 and 90%, respectivily, in a period of 48 h post-infection. The formation of the PV in macrophages infected with BEL treated parasites showed significant differences compared to control. Microscopic analysis showed changes in the morphology of vacuoles, a delay in the differentiation of promastigote to amastigote forms and a reduction in the acidity of the PV. The results indicate that inhibition of iPLA2 in metacyclic promastigotes of L. amazonensis caused changes in parasite surface leading to reduced adhesion to and infection of the host cell, inhibition of parasite differentiation and changes in the morphology and functionality of the PV. Supported by CNPq, CAPES, FAPERJ, Pronex - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1092] P 247 - THE EFFECTS OF RECOMBINANT PROTEIN BASED ON TRYPANOSOMA CRUZI P21-HIS6 TREATMENT IN INFECTED MICE WITH LEISHMANIA AMAZONENSIS LUISI JUNIOR, C.M.; TEIXEIRA, T.L.; MACHADO, F.C.; QUINTAL, A.P.N.; DA SILVA, C.V. UNIVERSIDADE FEDERAL DE UBERLÂNDIA, UBERLÂNDIA, MG, BRAZIL.
Keyword:leishmania amazonensis; t. cruzi p21-his6 treatment; inflammatory infiltrates
Abstract: The cutaneous form of leishmaniasis is caused by the protozoan Leishmania amazonensis. This form cause a lesion in the dermis characterized by a large inflammatory infiltrates composed, among others cell types, by macrophages infected with amastigotes forms inside a parasitophorous vacuole. P21 protein is expressed in all forms of Trypanosoma cruzi. The recombinant protein based on the T. cruzi P21 (P21-His6) enhances phagocytosis on inflammatory macrophages. In this study we evaluate the effects of the P21-His6 on the site of lesion in Balb/c mice infected with L. amazonensis, and remove some aspects of the immune response of the hostage. The animals were divided in three groups: (1)Infected with L. amazonensis and not treated; (2)Infected with L. amazonensis and treated with 40µg of P21His6; and (3)Not infected and treated with P21-His6. The previous results showed an increase of the inflammatory infiltrates in the group (3), composed by different cell types. The group (2) showed an increase of the inflammatory infiltrates and a large number of parasites internalized
in comparison of group (1). The assays to measure the cytokines of the spleen, and the cell population residing in this tissue are being performed, to evaluate some aspects of the immune response against L. amazonensis in the presence of the P21-His6. Financial support: FAPEMIG/UFU/CNPq. - CELLULAR AND MOLECULAR BIOLOGY [1096] P 248 - CLONING AND CHARACTERIZATION OF SAND FLY AQUAPORINS ORTA, J.F.1; GONZALEZ, L.1; SHARMA, M.1; MANDAL, G.1; ALVES, C.R.2; KAMHAWI, S.3; MUKHOPADHYAY, R.1 1.FLORIDA INTERNATIONAL UNIVERSITY, MIAMI, UNITED STATES; 2.INSTITUTO OSWALDO CRUZ - FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 3.NATIONAL INSTITUTES OF HEALTH, ROCKVILLE, UNITED STATES.
Keyword:aquaporin; sand fly; transmission
Abstract: There are no reports of any aquaporins from sand flies. It was recently reported that the malaria vector Anopheles gambiae modulates its AQP expression during blood feeding and humidity adaptation. Since, sand flies are absolutely dependent on humidity and blood for their survival and oviposition, we propose that their AQP(s) will be intricately involved. It is established that there are intimate interactions between Leishmania promastigotes and vector (sand fly) gut epithelium. However, there is a huge knowledge gap as to how these interactions propel the parasites from hind or midgut to foregut and lead to transmission. Leishmania aquaglyceroporin AQP1 plays a major role in parasite survival and osmotaxis of promastigotes. Osmotaxis is a major propulsive force for directional movements of promastigotes. Therefore, we hypothesize that, identification of the interplay of parasite and vector AQPs will lead to major tools for transmission interventions. Transmission intervention is a highly unexplored area of research in the Leishmania biology. Accordingly, primary objectives of this study are cloning and initial characterization of the putative aquaporins from sand flies and gut epithelial cell lines. Characteristics of vector aquaporins will be discussed. - CELLULAR AND MOLECULAR BIOLOGY [1103] P 249 - CELULAR IMMUNE RESPONSE OF BALB/C MICE IMMUNIZED WITH LEISHMANIA CHAGASI A2 RECOMBINANT PROTEIN JUSI, M.M.G.1; OLIVEIRA, T.M.F.S.2; NAKAGHI, A.C.H.1; ANDRÉ, M.R.1; MACHADO, R.Z.1 1.FCAV-UNESP, JABOTICABAL, SP, BRAZIL; 2.USP, PIRASSUNUNGA, SP, BRAZIL.
Keyword:leishamania chagasi; a2 protein; flow cytometry
Abstract: Visceral leishmaniasis is a zoonotic ilness considered one among the six most important tropical diseases in developing countries. In Brazil, the etiological agent of this disease, Leishmania chagasi, is transmitted by Lutzomyia longipalpis, that acquires the parasite when accomplish hematophagism in infected animals. In domestic environment, dog is considered the main parasite reservoir and, in consequence, it is also the main target of campaigns aiming at disease control. “A2” and “K39” recombinant antigens have been assessed in an attempt to develop simpler, faster and more specific serological diagnosis and vaccines. In this aspect, A2 protein family is highlighted, since it is expressed exclusively in amastigote phase. Proteins from this family play a role in parasite survivor strategies into the host, once they are expressed under inhospitable environment conditions such as lower pH and higher temperature. In the present study, BALB/c mice were immunized with a recombinant protein expressed from Jaboticabal strain L. chagasi A2 gene. The isolated gene, which showed similarity to a L. chagasi-A2
family, was expressed using pET28a vector in a BL21 strain (DE3) Escherichia coli expression system. Forty mice, used as experimental models, were divided in 4 groups: G1, 10 animals inoculated with L. chagasi promastigotes; G2, 10 animals inoculated with A2 recombinant protein; G3, 10 animals immunized with A2 recombinant protein and challenged with L. chagasi promastigotes; G4, 10 animals inoculated with buffered saline (controls). Preliminary results, obtained from flow cytometry showed a considerable increase in the percentage of immunostained CD4 and CD8 lymphocyte found in spleen from groups 2 and 3, when compared to the total cell number. The above finding was also verified in the lymph nodes collected from animals belonging to groups 1 and 4. The presence of anti-A2 antibodies in immunized mice sera was assessed using amastigotes and promastigotes forms of L. chagasi in an Immuofluorescence Indirect Assay (IFA). Anti-A2 antibodies were detected only in slides containing amastigote forms, showing that A2 family protein are solely find in this phase. These results confirm the antigenic and immunogenic properties of the expressed protein. The process of characterization of this protein remains in ongoing studies. Financial support: FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo; Process #2011/17594-5 and #2011/01961-9)
- CLINICAL LEISHMANIASIS [1106] P 250 - PATIENTS INFECTED WITH LEISHMANIA CHAGASI NATURALLY RESISTANT TO NITRIC OXIDE (NO) HAVE HIGHER SERUM LEVELS OF CYTOKINES AND TRANSAMINASES THAN THE PATIENTS INFECTED WITH NO SUSCEPTIBLE ISOLATES DOS SANTOS, P.L.1; OLIVEIRA, F.A.1; SANTOS, M.L.B.1; VIEIRA, E.1; TELES, M.1; RANGEL, M.1; LEOPOLDO, P.T.G.1; DE MOURA, T.R.1; REED, S.G.2; DUTHIE, M.2; GUDERIAN, J.2; JESUS, A.R.1; ALMEIDA, R.P.1 1.UNIVERSIDADE FEDERAL DE SERGIPE, ARACAJU, SE, BRAZIL; 2.INFECTIOUS DISEASE RESEARCH INSTITUTE, WASHINGTON, UNITED STATES.
Keyword:nitric oxide; immunology; transaminase
Abstract: Introduction: Visceral leishmaniasis (VL), a systemic inflammatory disease, is the most severe form of leishmaniasis. Leishmania sp. naturally resistant to NO has been associated with more infectivity to macrophages in vitro and with disease severity. Aim: To evaluate the serum levels of cytokines and transaminases in patients infected with Leishmania chagasi naturally resistant or susceptible to NO. Methods and results: Leishmania chagasi obtained from bone marrow aspirates of visceral leishmaniasis patients were exposed to increasing concentrations of NaNO2 (0-16 mM) and viability was determined by the MTT colorimetric method. We considered resistant to NO the parasites that presented more than 70% survival at 16 mM NaNO2. Sera cytokines (IFN-g, IL-12p40, IL-10, TNF-a and transaminases (ALT and AST) were measured in seven patients infected with L. chagasi naturally resistant to NO and six with susceptible isolates before and during the Antimony treatment, the cytokines were measured using high sensitive Luminex technique. VL patients infected with NO-resistant parasite (RVL) showed higher levels of these cytokines (IFN-g 714 pg/mL, IL-12p40 305 pg/mL, IL-10 197 pg/mL) and transaminases (ALT 105 UI and AST 87 UI) than patients infected with NO susceptible isolates (SVL) (IFN-g 553 pg/mL, IL-12p40 65 pg/mL, and IL-10 132 pg/mL; ALT 43 UI and AST 50 UI p=0,02). However, we observed lower concentrations of TNF-a in the RVL patients (62 pg/mL), as compared to SVL (99 pg/mL). Along the treatment the levels of theses molecules decreased in both groups. Conclusion: Infection with NO-resistant L. chagasi induces increased
production of pro-inflammatory cytokines and consequently more hepatic damage, reflected by the release of transaminases. Financial Support: Fundação de Apoio à Pesquisa e à Inovação Tecnológica do Estado de Sergipe (FAPITEC)/ Brazilian Research National Council (CNPq) (PRONEX) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). - CLINICAL LEISHMANIASIS [1109] P 251 - HIGH PREVALENCE OF LIVER INJURY IN NO CHILDREN VISCERAL LEISHMANIASIS PATIENTS FRANCISCATO, C.; PONTES, E.R.J.C.; DORVAL, M.E.C.; PANIAGO, A.M.M.; MATOS, M.F.C. PPGDIP, CAMPO GRANDE, MS, BRAZIL.
Keyword:kala-azar; aspartate aminotransferase; alanine aminotransferase
Abstract: Liver involvement in human visceral leishmaniasis (VL) has been little studied, however mild to fulminant hepatitis are reported. Hepatomegaly occurs in 90,0% of the cases, with none or few alterations of hepatic enzymes levels, leading to no severe disturbances in the course of the disease. Studies about the function of the liver were performed in children. To assess the prevalence of hepatocytes injury in patients with visceral leishmaniasis and enlist epidemiologic data were reviewed medical records of 109 patients, totaling 110 episodes of the disease. All patients over age 13 treated at the University Hospital of the Federal University of Mato Grosso do Sul, Campo Grande, between 2005 and 2009, were included. Those with no record of examinations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were excluded. Fifty-two (47.3%) patients met the criteria for classification of severe disease and seven (6.4%) died. At the diagnosis, before the treatment, 50.5% of the cases showed elevated levels of ALT and AST of 74.5%, indicating a high prevalence of liver injury in the patients studied. About 50% of patients in this study presented hepatomegaly and 25% of patients with nausea and vomiting. Most patients were male (68.2%) and lived in Campo Grande (82.0%). Mean age was 36.9 years. Mean time elapsed from the onset of symptoms to diagnosis was 53 days. Comorbidities and/or other associated situations occurred in 40.0% of cases and about one fourth of the patients stated they were alcoholic, smokers, or chronic medicament users. The drug most often administered to treatment was Glucantime (meglumine antimoniate), followed by amphotericin B deoxycholate. Epidemiological and clinical laboratory findings were similar to those described in the literature. Although liver abnormalities have been described in humans, dogs and other models, the hepatic impairment in VL is poorly known. Further studies, especially prospective ones, are needed to ensure a better follow-up of patients by healthcare workers. These studies could be applied to the daily practice of the sufferers´ care. These alterations could determine the severity of clinical manifestations, the prognosis and progression factors of the disease, the severity criteria and a greater surveillance for these patients. - CELLULAR AND MOLECULAR BIOLOGY [1110] P 252 - AFAP1 IS RECRUITED DURING INVASION AND INTRACELLULAR TRAFFIC OF LEISHMANIA AMAZONENSIS PROMASTIGOTES. MACHADO, F.C.; DA SILVA, C.V. UNIVERSIDADE FEDERAL DE UBERLÂNDIA, UBERLÂNDIA, MG, BRAZIL.
Keyword:actin; kinetic assay; cytoskeleton
Abstract:
AFAP1 is the most known member of the actin-filament associated protein (AFAP) family. It has been previously demonstrated that AFAP1 has a strong affinity for actin filaments and c-Src (an enzyme that promotes changes in cellular adhesion in addition to the capacity of assemble, disassemble and remodeling the actin cytoskeleton). Engulfment and formation of the Leishmania phagolysosome is dependent on the host cytoskeleton, mainly with actin rearrangement. In order to verify if this protein accompanies the actin during intracellular traffic of Leishmania amazonensis, peritoneal macrophages from C57Bl/6 mice were harvested and incubated with metacyclics promastigotes forms in different time-points (0.25, 0.5, 1, 3, 6 and 12 hours). This kinetic assay was stained for immunofluorescence with phalloidin-TRITC, capable of binding to actin filaments, anti-AFAP1 monoclonal antibody and a secondary FITCconjugated. Images obtained showed a recruitment of AFAP1 to the invasion site, co-localizing with actin filaments during all time-points. These results indicates that AFAP1 co-localizes with actin in 30-70% of the cases. Financial support: UFU, CNPq, FAPEMIG. - VACCINE DEVELOPMENT AND TRIALS [1111] P 253 - SCREENING OF LEISHMANIA (LEISHMANIA) CHAGASI PUTATIVE T CELL ANTIGENS AS NEW VACCINE CANDIDATES NASCIMENTO, F.C.1; CANAVACI, A.M.C.1; TESTASICCA, M.C.S.1; MACHADO, L.F.M.1; SERUFO, A.V.1; COELHO, C.H.1; NASCIMENTO, M.S.L.2; KETELUT, N.2; DA SILVA, J.S.2; COELHO, E.A.F.3; ANTONELLI, L.R.D.V.4; FERNANDES, A.P.1 1.FACULDADE DE FARMﾃ，IA, UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL; 2.FACULDADE DE MEDICINA DE RIBEIRﾃグ PRETO - USP, Sﾃグ PAULO, SP, BRAZIL; 3.COLTEC - UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL; 4.CPQRR/FIOCRUZ, BELO HORIZONTE, MG, BRAZIL.
Keyword:leishmania, (leishmania), chagasi; t, cell, antigens; new, vaccine, candidates
Abstract: Introduction: Considering the ability of Leishmania to escape of host immune responses, the heterogeneity MHC molecules of the host populations and the limitations of current vaccines, our main goal is to identify and evaluate new Leishmania (Leishmania) chagasi antigens to develop multi-component vaccines. The A2 antigen was shown to induce protective responses against visceral leishmaniasis in different formulations. A2 is expressed only by visceral leishmaniasis species, it has potent CD8 epitopes and it is a stress protein associated linked to the ability of the parasite survive in high temperatures in visceral organs. Therefore, using A2 as model antigen we are searching for new molecules that could be associated in a multicomponent vaccine. Methods and Results: We selected molecules that were previously shown to enhance the ability of L. (L.) major parasites to migrate to liver and spleen or that were identified by immunoproteomic analysis using sera of dogs with visceral leishmaniasis, and submitted them to in silico analysis for searching of T cell epitopes. The peptides corresponding to the identified epitopes were synthesized. Lymph node cells or spleenocytes from chronnically infected or vaccinated mice were stimulated with pool of peptides and submitted to CSFE flow cytometry proliferation assays. Preliminary results indicated that 21 out of 63 peptides induced significant proliferative indexes. Conclusion: Several selected peptides were recognized by lymphocytes of infected mice and will be further characterized for their T cell phenotype and cytokine profile, aiming to select antigens candidate to vaccine development against leishmaniasis. Financial Support: INCTV, CNPq, FAPEMIG, CAPES - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1112] P 254 - IL-17 IN THE MOUSE MODEL OF LEISHMANIA INFANTUM RODRIGUEZ, O.L.; TOVAR, B.; DIAZ, N.L.; TAPIA, F.J.; SANCHEZ, M.A. INSTITUTO DE BIOMEDICINA, CARACAS, VENEZUELA.
Keyword:leishmania infantum; il-17; t lymphocytes
Abstract: Th17 cells produce IL-17 in response to IL-23 and are associated with inflammation. In visceral leishmaniasis (VL), the inflammatory processes are designed to eliminate parasites but if they are not regulated they may be involved in chronic infection or immunopathology. The aim of this study is to immunolocalize the cytokines associated with Th17, Th1, Th2 and regulatory T cells (Treg) in susceptible BALB/c mice during the course of infection with Leishmania infantum. For that purpose, we determined by immunocytochemistry the expression of IL-17 (Th17), IL-4 (Th2), IFN- (IL-1), IL-12 (Th1) and TGF- (Treg) and IL-10 (Treg) using frozen sections from isolated spleen sections taken at days 7, 14, 21, 28, 35, 42 post-infection (pi); and measuring parasite load on apposition smears from infected spleen stained with Giemsa. The results showed a gradually increased in parasite load until day 21 pi (359 ± 42.88), when commenced a gradual decrease. IL-17 increase was observed on day 21 pi (1083 cells/mm2), coinciding with the highest parasite load,), and maintaining about 600 cells/mm2 on days 28, 35 and 42 pi. In contrast, a decreased expression of Th1 cytokines was progressively observed until day 21 (IFN- : 28cells/mm2; IL-12: 203 cells/mm2) with its maximum value (p <0.001) on day 35 pi (740 cells /mm2 and 988 cells /mm2, respectively). In addition, IL-4 and IL-10 positive cells increased gradually during the course of infection with higher expression on day 42 pi (IL4: 294 cells/mm2, IL-10: 212 cells/mm2), while TGF- decreased on day 21 (50 cells/mm2) but increased on day 42 pi (263 cel/mm2), as compared with the early days of infection (day 9: 144 cells/mm2, day 14: 122 cells / mm2) (p <0.05). These results suggest that infection by Leishmania infantum promotes Th17 cells even in the presence of of a strong Th1 response and regulatory cytokines, suggesting that these cells may play a role in the immunopathology associated with experimental LV. Financed by FONACIT G-2005000375 (MS). - OTHER SPECIAL TOPICS [1114] P 255 - VITAMIN A MODULATION OF REGULATORY T CELLS IN HUMAN VISCERAL LEISHMANIASIS. MACIEL, B.L.1; VALVERDE, J.G.2; RODRIGUES NETO, J.F.2; NETO, F.F.2; DO NASCIMENTO, P.P.2; MONTEIRO, G.R.2; PONTES, N.N.2; KEESEN, T.2; JERONIMO, S.M.B.2 1.DEPARTAMENT OF NUTRITION, FEDERAL UNIVERSITY OF RIO GRANDE DO NORTE, NATAL, RN, BRAZIL; 2.DEPARTAMENT OF BIOCHEMISTRY, FEDERAL UNIVERSITY OF RIO GRANDE DO NORTE, NATAL, RN, BRAZIL.
Keyword:treg cells; vitamin a; visceral leishmaniasis
Abstract: Infection by Leishmania infantum is endemic in northeast Brazil and may cause a spectrum of clinical outcomes that ranges from asymptomatic self-healing infection to visceral leishmaniasis (VL). The natural history from infection to development of symptoms and disease are not clearly understood, but nutritional status may influence. People with poor nutritional conditions are at a greater risk of symptomatic VL in many endemic areas. Vitamin A supplementation has long been used in developing countries to prevent mortality by diarrheal and respiratory diseases, but there are no studies on the role of vitamin A in Leishmania infection, although we and others have found vitamin A deficiency in VL patients. Regulatory T (Treg) cells are induced in vitro by vitamin A metabolites and have recently been suggested as important cells implicated CD4+ suppression in human VL. This work analyzed the effect of retinoic acid (RA), an active vitamin A metabolite, in Treg cells isolated from children with VL. Peripheral mononuclear cells (PBMC) isolated from children with VL (n=7) were cultured with soluble L. infantum antigens (SLA, 10µg/mL) or RA (0.25nM) or SLA (10µg/mL) and RA (0.25nM). Cells were cultured for 20 hours and the production of IL-10 and TGF- was analyzed in Treg cells (CD4+CD25+highFOXP3+ cells) by flow cytometry. The results showed that RA increased the production of IL-10 in CD4+C25+ cells (6.2% vs 3.4% without any stimulation, p < 0.05). TGF- was also increased in CD4+CD25+ in RA cultures (2.7% vs 1.4% without any
stimulation, p < 0.05). CD4+CD25+highFOXP3+ cells were increased in RA cultures, especially in the ones with RA and SLA stimulates (32.0% vs 16.2% without any stimulation, p < 0.05). Data is consistent with immunomodulation by vitamin A improving regulatory mechanisms of the host. Further work to investigate whether these modulation actions benefit the outcome of Leishmania infection need to be done. Support: National Institutes of Health AI-30639 and CNPq - GENOMICS, TRANSCRIPTOMICS, PROTEOMICS, METABOLOMICS [1115] P 256 - IN SILICO PREDICTION OF MOLECULAR TARGETS FOR EXPERIMENTAL CHEMOTHERAPY OF SUSCEPTIBLE AND ANTIMONY RESISTANT LEISHMANIA BRAZILIENSIS. ROCHA, F.A.; LIARTE, D.B.; MURTA, M.F.; RUIZ, J.C.; BEVERLEY, S.M. UNIVERSIDADE FEDERAL DO PIAUĂ?, TERESINA, PI, BRAZIL; ; ; .
Keyword:chemoterapy; drug resistance; bioinformatic
Abstract: Introduction: Mechanisms of drug resistance in New World Leishmania species are poorly understood. Our group selected in vitro populations of Leishmania braziliensis, which are 20fold more resistant to potassium antimony tartrate (LbSbR) than its susceptible counterpart (LbWTS) (Liarte & Murta, 2010). Microarray analysis showed 252 genes and 957 transcripts up and down-regulated in LbSbR. In this study, we analyze that sequences in search for drug targets and metabolic pathways that contribute to Leishmania chemotherapy. Materials and Methods: Amino acid sequences from microarray studies (as previously described) and protein targets obtained from DrugBank 3.0 database were compared using BLAST algorithm. The results were classified according to microarray (into genomic or transcriptomic data, up or down-regulated) and the pharmacological nature of the target. These data were compiled in a new local database and used for drug target prediction. Results and Discussion: BLAST comparisons showed approximately 101 molecular targets into proteins associated to Leishmania antimony resistance. Most of them are common to several proteins involved in cell metabolism. The substances that interact with the targets were classified as follow: featured drugs so as toxico-pharmacological, compounds related to metabolism physiologic and other chemical compounds which interacts with amplified/overexpressed or deleted/down expressed proteins of LbSbR in the microarrays. Of these, 09 were single metabolites (amino acids, cofactors, co-called metabolites, among others); 26 were drugs commercially available and 141 were classified as other products. Highlighting 3 substances (1 single metabolites and 2 drugs) interact to proteins up or down regulated identified in both genomic and transcriptomic microarrays. These compounds refer to key targets related to the resistance phenotype, source of new research. Metabolic pathways and common side effects were analyzed to predict the efficacy of the use of the drugs and metabolites found. These products are under test to evaluate its contribution to resistance phenotype and chemotherapy of leishmaniasis.
- CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1120] P 257 - CYTOKINES AND T CELL ACTIVATION IN PERIPHERAL BLOOD FROM DOGS WITH VISCERAL LEISHMANIASIS FROM MARGARITA ISLAND ENDEMIC AREAS RODRIGUEZ, O.L.; GUERREO, R.; ALFONSO, E.; QUIJADA, J.D.; SANCHEZ, M.A. INSTITUTO DE BIOMEDICINA, CARACAS, VENEZUELA.
Keyword:canine visceral leishmaniasis; cytokines; t cell activation
Abstract: Canine Visceral Leishmaniaisis (VL) is a public health problem in Venezuela and particularly in Margarita Island, the control measure so far is based in the culling of infected dogs. Therefore, characterize immunological events associated with progression of the LVC is important to identify key molecules associated with immunoprophylactic immunity and long term disease control in therapeutic or vaccine trials. Objective: To evaluate the degree of activation and the expression of Th1, Th2 and Th17 circulating T cells in dogs with VL. Methods: We evaluated 30 dogs with VL randomly selected from endemic areas of Margarita Island, Nueva Esparta State. Based on the presence of none, few or several clinical signs were classified as asymptomatic-AS, oligosymptomatic-OS and symptomatic-S. Activation molecules (CD44, MHC-II, CD18) and intracellular cytokines: (IFN- , IL-4, IL-17) in CD4+ and CD8+ T cells of canines PBMC where determined by flow cytometry. Results: S-Dogs showed higher proportion of CD8+ IFN- + lymphocytes (p = 0.02) and a higher density of IL-4 expression in CD4+ and CD8+ (p <0.05), compared with AS and OS. Regarding activation molecules we found an increased expression of the adhesion molecule CD18 on lymphocytes from AS dogs when compared to OS-dogs (47.35 ± 9.8 vs 26.16 ± 9.3, respectively, p <0.05),although particularly on the CD8 T Cells the S-dogs show higher proportion of CD18+ when compared to OS-dogs (33.86% ± 15.8 vs23.15% ± 11.13, p <0.05). The MHC-II expression was highly increased in CD4+ and CD8+ lymphocytes of OS and S-dogs when compared to AS-dogs (CD4+: 44.6% ± 9.32, 42.2 % ± 19.2 vs 24.45% ± 8.8, CD8+: 24.76% ± 13.97, 28.03% ± 9.28 vs 15.8% ± 8.34, p <0.05). Conclusions: The symptomatic dogs have higher cell activation (MHC-II +) that may represent a condition of immune regulation by the cognate interaction between T cells, but also allows the progression of disease. Results also indicate that the role of CD8 + cells in the immunopathogenesis of LVC has to be further evaluated. Supported by grants: FONACIT2005000375, CDCH-PSU 09-7878/2009. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1122] P 258 - PROFILE OF INFLAMMATORY AND ANTI-INFLAMMATORY CYTOKINES IN PATIENTS WITH AMERICAN CUTANEOUS LEISHMANIASIS IN AMAZON-BRAZIL PINHEIRO, F.G.1; ESPIR, T.T.2; NAIFF, M.F.1; FIGUEIRA, L.P.1; DA COSTA, A.G.3; PIMENTEL, J.P.D.3; MALHEIRO, A.3; FRANCO, A.M.R.1 1.AINSTITUTO NACIONAL DE PESQUISAS DA AMAZÔNIA , AV.ANDRÉ ARAUJO, N/2936, 69080-971,, MANAUS, AM, BRAZIL; 2.UNIVERSIDADE FEDERAL DO AMAZONAS, MANAUS, AM, BRAZIL; 3.CFUNDAÇÃO DE HEMATOLOGIA E HEMOTERAPIA DO AMAZONAS, MANAUS, AM, BRAZIL.
Keyword:leishmania (viannia) guyanensis; american cutaneous leishmaniasis ; cytokines
Abstract: The northern region of Brazil has the highest incidence of American Cutaneous Leishmaniasis (ACL), estimated at more than 2,000 new cases per year only in the State of Amazonas. In general, the clinical manifestations are dependent on different Leishmania species and with the concentrations of pro-inflammatory and anti-inflammatory properties, which generally in high concentrations may contribute to immunopathology of the disease. The aim of this study was to quantify the inflammatory and anti-inflammatory cytokines in patients treated to ACL at the Hospital of Rio Preto da Eva-AM. This serial cases study was conducted from June 2009 to February 2012. Were analyzed patients aged between 21 and 58 years old, with infection duration about a month in 83% of cases, most activities in the forest area in 100% of cases. Concentrations of cytokines IL-2, IL-4, IL-6, IL-10, TNF- , IFN- and IL-17 were quantified in plasma from 48 patients (24 individuals infected by Leishmania (Viannia) guyanensis and the same number of uninfected was used as a control group) using the technique of CBA (Cytometric Bead Array) with the kit TH1, TH2 and TH17 (BD Biosciences). The reading of the samples was performed on FACSCanto II flow cytometer (BD) and analyzes of plasma
concentrations were conducted with FCAP-Array software (v 3.0). The results were analyzed with the GraphPad Prism software® (v 5.0), using a non-parametric test (Mann-Whitney). Levels of cytokines IL-2, IL-4, IL-6, IL-10, IL-17 and IFN- , TNF- were significantly higher among the infected group compared to non-infected individuals (p <0.0001, p <0.0001, p = 0.0329, p <0.0001, p = 0.0036, p = 0.0073 p <0.0001, respectively). Thus, there is a worsening in the immune response of ACL caused by L. (V.) guyanensis infection with simultaneous production of pro-inflammatory and anti-inflammatory cytokines and TH17profile. Support: FAPEAM, CAPES - OTHER SPECIAL TOPICS [1123] P 259 - MORPHOLOGICAL CHANGES INDUCED ON LEISHMANIA PROMASTIGOTES BY CERAGENINS, ULTRASTRUCTURAL ANALYSIS. CHIRINOS, Y.1; VELAZCO, E.2; MEJIA, E.2; SAVAGE, P.B.3; SANCHEZ, M.A.1 1.INSTITUTO DE BIOMEDICINA UCV-MPPS, CARACAS, VENEZUELA; 2.J. M. VARGAS MEDICAL SCHOOL UCV, CARACAS, VENEZUELA; 3.BRIGHAM YOUNG UNIVERSITY, UTAH, UNITED STATES.
Keyword:visceral leishmaniasis; ceragenins, leishmania promastigotes; ultraestructure
Abstract: Ceragenins, formerly known as Cationic steroid antibiotic (CSA) have proven antibacterial activity, We have previously demonstrated the leishmaniostátic effect of CSA-8 and CSA-13 as coadyuvant candidate in an alternative chemotherapeutic protocol, yet their mode of action and effect on the parasite surface remains undisclosed. This study aims to evaluate the ultrastructural changes in the cell membrane and organelles of Leishmania promastigotes after treatment with ceragenins. Methods: L. infantum (MCAN/VE/98/IBO-78) and L. mexícana (MHOM/BZ/82/BEL21) promastigotes (4x106 parasites) in log phase where respectively treated with CSA-8, and CSA-13 at concentrations below the LD 50 (5 ug / ml) samples were prepared and studied with conventional transmission electron microscopy. Results: Controls parasites, show fusiform morphology, cytoplasm rich in small vacuoles and electron dense. Mitochondria, rounded nuclei with prominent nucleoli and no alterations in the flagella and flagellar pocket. Both L. infantum and L mexicana promastigotes experiment profound morphological changes after treatment. Parasites exposed to CSA-8 exhibited prolonged forms mainly characterized by a content of foamy cytoplasm rich in large irregular vacuoles electron density variable; the rough endoplasmic reticulum (RER) is abundant in some cases show profuse mitochondria of different sizes and varying electron density. The dense nuclei are characterized by irregular contours with abundant chromatin in clumps or irregularly distributed. Some nuclei possess numerous clusters of degraded chromatin netlike. and evident nucleoli, the nuclear membrane shows small not prominent grooves and in some cases are very apparent. The normal appearance kinetoplast have shown flagella with flagellar pocket of increased in diameter. The plasma membrane also has an irregular contour after treatment with CSA-8. On the other hand the parasites treated with CSA-13 are characterized by having regularly contour shapes mainly distinguished by the presence of small irregular vacuoles with variable electron density in their cytoplasm, accompanied by the presence of small dense mitochondria. The nuclei of these promastigotes possess irregular contours due to the presence of deep grooves with apparent nucleoli, vacuolated euchromatin. The spaces between the flagellar pockets and their flagella are increased, the plasma membranes has irregular contours. Other results show residues of foamy cytoplasm where persist mitochondria of a different electronic density and large vacuoles with electron dense debris. Future analysis using scanning electron microscopy allow to elucidate the precise changes induced by ceragenins in the cytoplasmic membrane of Leishmania parasites Supported by grants: CDCH UCV PSU-09-7878-2009/1; FONACIT G-2005000375 MS
- GENOMICS, TRANSCRIPTOMICS, PROTEOMICS, METABOLOMICS [1124] P 260 - PROTEIN PROFILE OF THE MIDGUT OF FEMALE LUTZOMYIA (NYSSSOMYIA) UMBRATILIS PINHEIRO, F.G.; GOMES, L.H.M.; ROCHA, L.C.; FRANCO, A.M.R. INPA, MANAUS, AM, BRAZIL.
Keyword:lutzomyia umbratilis; peritrophic matrix; protein
Abstract: In the Amazon region Lutzomyia umbratilis is the main vector of Leishmania guyanensis causing the clinical form of American Cutaneous Leishmaniasis (ACL), transmitted to humans by the bite of infected sand flies. These parasites realize part of their cycle of development in the vector, which is considered a potential target for disease control. Leishmania must overcome physical and chemical barriers such as peritrophic matrix (PM) and midgut digestive enzymes (MI) for their establishment in these insects. The epidemiological study of the transmission dynamics of vector-borne infections is covered by interest in new techniques that can help in understanding the mechanism of parasite-vector interaction. Accordingly, such strategies require the characterization of proteins of MI and MP. This study aimed to identify proteins from MI and MP of adult females of L. umbratilis captured in nature. Sandflies collected in the field, receiving a diet of laboratory blood supplied through the blood meal in white rats adult Wistar and were after 24 and 72 hours dissected and identified according to the taxonomic key by Young and Duncan (1994). From two-dimensional gels and mass spectrometry analysis, the resulting spectra were processed using the Mascot software and compared with the protein database (NCBI, UniProtKB, FlyBast). Were used 80 MI / MP, dissected after blood feeding. 100 spots were evealed including 21 proteins identified by mass spectrometry and classified into five groups: (1) four glycoproteins, (2) three proteoglycans, (3) seven nucleoproteins, (4) a cromotoproteína and (5) five peptides with unknown function. The groups of proteins identified in the MI of L. umbratilis are related to MP formation, resistance, adhesion, structure, maintenance and degradation, and may participate in interaction with Leishmania parasites. Financila support: FAPEAM/INPA - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1129] P 261 - OXIDATIVE RESPONSES OF J774.16 MURINE MACROPHAGE CELL LINE INFECTED BY LEISHMANIA (L.) CHAGASI. CARVALHO, S.S.; DOS SANTOS, P.L.; BRAZ, J.M.; MONTEIRO, M.B.A.; BARBOSA DOS, M.L.; OLIVEIRA, F.A.; RIBEIRO, A.J.; DE MOURA, T.R.; DE ALMEIDA, R.P.; LEOPOLDO, P.T.G. UFS, ARACAJU, SE, BRAZIL.
Keyword: l. (l.) chagasi; superoxide anion; murine macrophages cell line j774.16
Abstract: (1) Universidade Federal de Sergipe – Aracaju, Brazil. Introduction. Leishmania spp. are protozoans that survive and replicate intracellularly in mammalian macrophages. Macrophages play crucial roles in leishmania infection, inducing synthesis of oxygen and nitrogen reactive intermediates, both of them, toxic agents to these parasites. Diethyldithiocarbamate (DETC), and inhibitor of superoxide dismutase 1 (SOD1) play a crucial role in Leishmania killing. In this study we evaluated the infective capacity of Leishmania (L.) chagasi isolates in a murine macrophages cell line (J774.16) and the production
of superoxide anions and nitric oxide in these cells. Methods and Results. Two isolates of L. (L.) chagasi promastigotes (previously characterized as resistant and susceptible to menadione, a anion superoxide donor) were used to infect j774.16 cells. J774.16 cells were incubated in 8well plates for 2hrs, then infected with L (L.) chagasi (in stationary phase of growth) at a cell ratio of 5:1 at 37 oC for 2 , 24, 48 and 72 hours. After each end infection time, the plates were stained with Giemsa to assess by light microscopy the percentage of amastigotes-loaded macrophages and the number of parasites per macrophages. Infection of J774.16 cells with menadione resistant L. (L.) chagasi, both in control conditions as stimulated with LPS and IFN(in any period of infection evaluated), presented a higher load than the menadione susceptible ones. In cultures of J774.16 stimulated with DETC, both susceptible and resistant L. (L.) chagasi presented low amastigotes-loaded macrophages after 48 hours of infection, correlating with increased superoxide anion production in these cultures. Conclusion These data suggest that amastigotes of L. (L.) chagasi are not resistant to leishmanicidal agents produced by macrophages, as was observed for their promastigotes forms exposed in vitro to increasing dose of menadione. Financial Support: Fundação de Apoio à Pesquisa e à Inovação Tecnológica do Estado de Sergipe (FAPITEC)/Brazilian Research National Council (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
- CELLULAR AND MOLECULAR BIOLOGY [1143] P 262 - IMPACT OF LEISHMANIA INFANTUM ON MACROPHAGE IRON METABOLISM PROTEINS TOMAS, A.M.1; CRUZ, T.1; PASSOS, J.1; LIMA, N.1; CASTRO, H.1; CANNONE-HERGAUX, F.2; PINTO, J.1 1.INSTITUTE FOR MOLECULAR AND CELL BIOLOGY, PORTO, PORTUGAL; 2.UNIVERSITÉ OF TOULOUSE, TOULOUSE, FRANCE.
Keyword:iron; macrophage; infection
Abstract: Macrophages, which are central components of both the innate and the adaptive immune system, are also the host cells for several of bacteria and protozoan parasites as is the case of Leishmania. The presence of microorganisms inside these phagocytic cells has been described to modify the expression of different proteins, including those involved in iron metabolism. Nevertheless, there is limited information regarding the expression of iron metabolism genes and proteins of the macrophage upon a Leishmania infection. The exception is transferrin receptor 1 (TfR1) which was reported to be up-regulated in Leishmania donovani-infected macrophages in vitro [1], possibly to allow the parasite to fulfill its nutritional requirements for this metal. Here we performed a series of in vitro infections, using peritoneal macrophages of BALB/c mice, to analyze the impact of Leishmania infantum on the expression of proteins involved in the macrophage iron metabolism. Our results indicate that: 1) There is a positive regulation of both hemic- and non-hemic acquisition pathways as well as of iron export in infected macrophages. Interestingly, the increased iron release observed in vitro might also occur in vivo as i) hepcidin (an hormone that abrogates iron efflux in macrophages) is not increased in BALB/c mice infected with L. infantum, and ii) these mice do not show signs of anaemia upon infection.
2) Infected macrophages have increased iron content. In these cells the metal locates mainly in the parasite phagolysosome and within the parasite itself. 3) Alongside with the changes observed in the macrophage iron metabolism, L. infantuminfected cells preferentially express a set of molecules characteristic of anti-inflammatory M2 sensus stricu (M2ss) macrophages. We propose that the alterations in iron homeostasis that accompany M2ss polarization in infected macrophages are one of the factors contributing to the increased permissiveness of these cells to L. infantum. They might allow the parasite a means to obtain iron while avoiding microbicidal host defense.
[1] Das N.K., Biswas S., Solanki S., Mukhopadhyay C.K., Leishmania donovani depletes labile iron pool to exploit iron uptake capacity of macrophage for its intracellular growth; Cell Microbiol. 2009 Jan;11 (1):83-94. Financed by FCT(Portugal)/FEDER(EU) through project PTDC/CVT/100090/2008.
- CELLULAR AND MOLECULAR BIOLOGY [1152] P 263 - THE SIALOGLYCOPEPTIDASE-LIKE PROTEIN OF LEISHMANIA MAJOR: POSSIBLE ROLE IN PARASITE GROWTH AND FLAGELLUM HOMEOSTASIS ROMAO, A.B.D.; DOS REIS, F.C.G.; COSTA, T.F.R.; PENHA, L.L.; LIMA, A.M.; LIMA, A.P.C.A. UFRJ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:metallopeptidase; leishmania; essential genes
Abstract: Leishmania major was predicted to contain about 154 peptidase genes representing 2% of the genome. It has a single copy gene with similarity to the sialoglyco metallopeptidase (OSGEP) of Pasteurella haemolytica. Bacterial OSGEP specifically hydrolyses peptide bonds between Oglycosylated and sialylated aminoacids and processes surface antigens of leukocytes. Other OSGEP-like proteins are found in bacteria and eukaryotes. Among those, the bacterial Kae-1 putative peptidase shares 34% sequence identity with OSGEP, but does not have proteolytic activity. It was reported that Kae-1 is an atypical DNA interacting protein that is important in the maintenance of genome integrity and transcription regulation. We set out to characterize L. major OSGEP. The L. major OSGEP gene shares 27% sequence identity to P. haemolytica OSEGP and 60% identity to bacterial Kae-1, revealing that Leishmania OSGEP does not encode a peptidase and is likely homologous to Kae-1. Importantly, the conserved Glu residue, proposed as the catalytic residue of bacterial OSGEP, is replaced by non-conservative Val in L. major OSGEP, suggesting that it does not encode a functional peptidase. Recombinant Histagged OSGEP was expressed in E. coli as inclusion bodies, purified and used to raise antiserum. The sub-cellular localization of the protein was assessed by the generation of a L. major line expressing OSGEP fused to GFP. Confocal microscopy revealed co-localization of the GFP fusion protein with the mitochondrion-selective probe, Mitotracker red. We generated L. major lines overexpressing OSGEP from an episomal vector, and confirmed increased OSGEP protein levels in parasite lysates by Western blot. Overexpressing lines have accelerated growth as promastigotes, reaching stationary phase earlier than WT parasites but have reduced infectivity to murine macrophages. A proportion of cultured promastigotes display morphological
alterations in the cell body and shorter flagella. In addition, we observed the secretion of amorphous material emerging from the flagellar tip. The mechanisms underlying the growth alterations of the mutants are under investigation. - CELLULAR AND MOLECULAR BIOLOGY [1155] P 264 - FUNCTIONAL STUDIES TO EVALUATE CYTOKINE REGULATION IN SUBJECTS WITH THE POLYMORPHISMS AT -176 G/C IL6 AND -2518G/A MCP1 GENES MAGALHﾃウS, A.S.1; OLIVEIRA, J.1; CASTELLUCCI, L.1; RAMASAWMY, R.1; WILSON, M.2; JAMIESON, S.3; BLACKWELL, J.3; CARVALHO, E.1; JESUS, A.R.4 1.UFBA, SALVADOR, BA, BRAZIL; 2.UNIVERSITY OF IOWA, IOWA, UNITED STATES; 3.UWA, PERTH, AUSTRALIA; 4.UFSE, ARACAJU, SE, BRAZIL.
Keyword:functional studies; immune response; leishmaniasis
Abstract: Leishmaniasis presents a wide clinical spectrum that is related to host innate and adaptative immune responses, and genetic factors of the parasite and human host. It has been reported several associations of leishmaniasis with some polymorphisms in genes that affect the immune response. Our group has also documented these associations in individuals of cutaneous (CL) and mucosal (ML) leishmaniasis in an endemic area of Corte de Pedra, Bahia, Brazil. The data presented in this study show the importance of functional studies to prove the influence of genetic polymorphisms in multifactorial diseases and the regulation of certain cytokines that are involved in its pathogenesis. This study provides support to genetic association between the C allele (low producer) and the G allele (high producer) of the IL6 SNP -174 G/C and -2518 G/A CCL2/MCP-1, respectively, and susceptibility to mucosal leishmaniasis (ML) after infection with L. braziliensis in the endemic area individuals under study. This study demonstrate an low basal concentrations of IL-6 individuals carrying the CC genotype of the polymorphism -174 G/C IL6 and high basal concentrations of MCP-1 for individuals who carry GG genotype polymorphism -2518 G/A CCL2/MCP1, which is also associated with higher TNF- production may contribute to the exaggerated pro-inflammatory response and the severe mucosal disease in L. braziliensis infection. Our study adds to the growing importance of the genetic regulation of pro and anti-inflammatory have in determining disease susceptibility to ML after infection with L. braziliensis. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1157] P 265 - GENE EXPRESSION FOR INTERFERON-GAMMA (IFN- ) IN PATIENTS WITH ACTIVE LEISHMANIASIS TEGUMENTARY ALMEIDA, T.M.; SOUZA, M.A.; DE CASTRO, M.C.A.B.; DE ALMEIDA, A.F.; DE ALMEIDA, A.P.; DE OLIVEIRA, B.C.; PEREIRA, V.R.A. CENTRO DE PESQUISAS AGGEU MAGALHﾃウS-FIOCRUZ, RECIFE, PE, BRAZIL.
Keyword: american tegumentary leishnmaniasis; cytokines; qpcr.
Abstract: American tegumentary leishmaniasis (ATL) is an infectious disease caused by several species of protozoa from the order Kinetoplastida, family Trypanosomatidae and genus Leishmania. In Brazil, Leishmania (Viannia) Braziliensis is the main causative specie of ATL. It is known that in all forms of the disease, the immune response is dependent of T cells and that the difference between resistance and susceptibility to infection is associated with the expansion of Th1 and Th2 cytokine producing T cells. Interferon-gamma (INF- ), a major Th1 cytokine, plays an essencial role in control of leishmania infection by mediating macrophage killing of intracelular
parasites. Thus, this study aimed to quantify mRNA expression of INF- using peripheral blood mononuclear cells (PBMC) of patients with active leishmaniasis. Cells from twenty-one patients and six controls were incubated for 24 hours in the presence of the concanavalin A (2.5 mg / ml), phytohemaglutinin (5.0 ug / ml) and L. (V.) braziliensis soluble (AgSol, 1,25µg/ml) or insoluble (AgIns, 2,5µg/ml) antigens. The expression of INF- was evaluated using real time quantitative PCR, and the results were obtained through the comparative Ct method. Data were considered significant when p < 0,05 (Wilcoxon and Mann-Whitney tests). Under the mitogen stimulation, INF- was significantly expressed in patients and controls. In response to AgSol, INF- expression was 24,80 fold higher in patients ( CT=14,93±2,652) in comparison to healthy individuals ( CT=19,56±1,580) (P=0,0028). In patients ( CT=13,21 ±2,829), this cytokine was 65,09 fold more expressed in cells stimulated with AgIns when compared to control( CT= 19,23±1,584) (P=0,006). A significant mRNA production 0.3 fold higher for INFin PBMC of patients was promoted by AgIns in relation to AgSol (P = 0,0006). Some studies has been demonstrated that cytokine secreted during leishmania infection are important to determine progression or control of the disease. Our data suggestion that the increase of expression of mRNA for INF- by lymphocytes demonstrated that these patients can mouting an effective Type-1 immune response capable of killing intracellular Leishmania. Thus, this study may help to better understand the role of IFN- in the process of healing of the disease. - OTHER SPECIAL TOPICS [1159] P 266 - PANCYTOPENIA AND IGF-I MRNA EXPRESSION IN BONE MARROW IN VISCERAL LEISHMANIASIS IN HAMSTER TORRES, A.R.A.1; CARVALHO-DANTAS, I.M.1; PINHO, F.A.1; GOTO, H.1; MIYASHIRO, S.I.2; HAGIWARA, M.K.2 1.INSTITUTO DE MEDICINA TROPICAL DE SÃO PAULO, SÃO PAULO, SP, BRAZIL; 2.FACULDADE DE MEDICINA VETERINÁRIA E ZOOTECNIA DA UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL.
Keyword:leishmaniasis; pancytopenia; hamster
Abstract: Active visceral leishmaniasis (VL) is characterized by fever, hepatosplenomegaly, pancytopenia, hypergammaglobulinemia and cachexia. Pancytopenia is a frequent manifestation in VL and the genesis of the alterations in the red or white blood cell series is often related to marrow dysfunction but the pathogenesis is still not clear. Insulin-like growth factor-I (IGF-I) plays a role in the development of the infection by Leishmania but it is also an important promoter of the growth of hematopoietic cells in vitro. Since we have observed low level of serum IGF-I in human and canine VL we considered that it may be related to the alterations in the hematopoiesis in VL. The aim of this study was to characterize the establishment of pancytopenia and to evaluate the expression of IGF-I mRNA in the bone marrow of hamsters intraperitoneally infected by 20 million Leishmania (Leishmania) infantum amastigotes. We evaluated 10 infected and 14 control hamsters. We followed for 90 days, the peripheral blood was collected every 30 days and processed in an automated veterinary hematology ABC VET analyzer for differential blood cell counts, and bone marrow was collected for IGF-I mRNA evaluation. IGF-I expression was quantified by Real Time PCR Quantification relative method. Infected animals presented progressive hematological alterations during follow up. Ninety days post-infection all infected hamsters presented hepatosplenomegaly and hematological alterations. The leucocyte count was 8.11 ± 0.99 (mean + standard deviation) x103/mm3 in the infected and 12.20 ± 1.73 x103/mm3 in non infected animals (p = 0.0079, Mann-Whitney test). The red blood cell count was 7.4 ± 0.24 x106/mm3 in the infected and 7.8 ± 0.38 x106/mm3 in non infected animals. The platelet count was 451.14 ± 13.02 x103/mm3 in the infected and 527.4 ± 81.88 x103/mm3 in non infected animals. Analysis of mRNA expression of IGF-I in bone marrow at 90 days post-infection revealed 2.7 times lower expression in the infected than in control animals. These results suggest that the L. (L.) infantum-infected hamster is a good model
to study the pathogenesis of pancytopenia in VL since it presents progressive hematological alterations similar to human VL. Low IGF-I mRNA expression was observed which may be related to the alterations in the hematopoiesis in the bone marrow during disease development. - OTHER SPECIAL TOPICS [1160] P 267 - LIPID ALTERATIONS IN LEISHMANIA (LEISHMANIA) CHAGASI-INFECTED HAMSTER AND PARASITE LOAD UNDER EFFECT OF CIPROFIBRATE. CARVALHO-DANTAS, I.M.; TORRES, A.R.A.; PINHO, F.A.; GOTO, H. INSTITUTO DE MEDICINA TROPICAL, SÃO PAULO, SP, BRAZIL.
Keyword:visceral leishmaniasis; hamster; ciprofibrate
Abstract: In the active phase of infection by the Leishmania (Leishmania) chagasi lipoprotein changes occur with reduction in HDL and increase in triglyceride (TG) levels. It is also shown that fatty acids are important as energy source for Leishmania amastigotes thus it is likely that the reduction in lipid levels using drugs would interfere in the parasite growth. The drug ciprofibrate is known to reduce plasma lipoprotein levels. Aim: To evaluate the plasma lipoprotein fraction changes and the effect of the drug ciprofibrate on the parasite load in hamster model of visceral leishmaniasis. Material and Methods: Hamsters were infected with 2x107 purified L. (L.) chagasi amastigotes. After 60 days post-infection, they were treated for 15 days with 0.5 mL of ciprofibrate (7.6mg/mL in sorbitol). The 40 animals were divided in non-infected, infected, treated and non-treated groups (5-9 hamsters/group). Lipoprotein fractions were measured by enzymatic colorimetric’s method and the parasite load evaluated in the liver and spleen by optic microscopy. Further we evaluated higher doses, 15 mg, 50 mg and 100 mg of ciprofibrate in 1 mL (3 animals/ dose) and compared to 14 controls since the effect of the drug was not as expected. Results: We observed an increase in the TG levels (median = 242.5 mg/dL) in infected and non-treated animals when compared with non-infected and non-treated animals (median = 186.5 mg/dL), while the median of cholesterol levels was similar between both groups (92 mg/dL and 86 mg/dL, respectively). In the infected and non-treated animals the median of HDL levels was 33.5 mg/dL and in the non-infected and non-treated was 44.0 mg/dL. Upon treatment the parasite load did not change significantly in relation to non-treated controls. Increasing the dose, however, treatment with 15 mg induced significant reduction in the TG levels (median of 89 mg/dL versus 174 mg/dL of control) and of cholesterol levels (median of 63 mg/dL versus 82.5 mg/dL of control) (p=0.0098). In non-treated animals the parasite load reaches 3.6 (median x 106) in the spleen and 87.8 in the liver. In those treated with 15 mg we did not detect any amastigotes. Conclusion: In visceral leishmaniasis in hamster lipoprotein levels were altered, and the treatment with ciprofibrate normalized these alterations and reduced the parasite load. Supported by FAPESP 2011/03768-1, CNPq, LIM-38 (HC-FMUSP) ivemcd@usp.br
- OTHER SPECIAL TOPICS [1166] P 268 - OBSERVATION OF MICROSCOPIC CHANGES AND FIBROSIS IN THE LIVER OF DOGS WITH VISCERAL LEISHMANIASIS IN THE CITY OF ARAÇATUBA – SP. COSTA, L.D.; SILVEIRA, T.L.; VASCONCELOS, R.O.; ROZZA, D.B. UNESP, ARAÇATUBA, SP, BRAZIL.
Keyword:histological; fibrosis; leishmaniasis
Abstract: In canine leishmaniasis (CL) there are reports of liver disease associated with connective tissue proliferation. This study aimed to observe the microscopic changes and fibrosis in the liver of dogs with visceral leishmaniasis in the city of Araçatuba SP. We used 25 naturally infected dogs from an endemic area for CL, ranked in the symptomatic group (S), asymptomatic (A) and control (C). Immunohistochemistry was used to detect L. chagasi in the liver and Tricômio Masson staining with aniline blue (Kit EasyPath) and Picrosirius Red (Kit EasyPath) in the detection of fibrosis. Histopathologically, 3 patterns were described. The pattern A, presence of inflammatory infiltrate composed of lymphocytes, plasma cells and some macrophages mainly in the sinusoids, mild to moderate, and discreetly in the central vein and in the portal space. Pattern B, inflammatory infiltrate evident in central vein and in particular in the portal space, moderate or severe, being composed mainly of lymphocytes, plasma cells and macrophages and a mild to moderate amount of epithelioid macrophages. Parenchyma, aggregates of lymphocytes, plasma cells and macrophages now in greater quantities and discrete epithelioid macrophages to moderate granulomas characterizing diffuse and varied sizes, absence of giant cells. In some cases there was a slight amount of polymorphonuclear cells (neutrophils) around the vessels and parenchyma. The pattern C, similar to the pattern B, but with marked chronic inflammatory infiltrate in the portal space and centrolobular vein and multiple granulomas in the parenchyma diffuse and varied size, the presence of epithelioid macrophages is much more evident, moderate to severe, with no giant cells. Lack of increase in fibrous connective tissue. In this study we observed a histological pattern in the livers of dogs with L. chagasi and no proliferation of fibrous connective tissue. Financial support: FUNDUNESP (Foundation for the development of UNESP), Case No. Proc.: 00965/11-DFP.
- CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1168] P 269 - THE DURATION OF MUCOSAL DISEASE AND THE TIME OF CLINICAL CURE INFLUENCES THE IN VITRO CYTOKINE PRODUCTION BY PERIPHERAL MONONUCLEAR CELLS FROM LONG TERM HEALED MUCOSAL LEISHMANIASIS PATIENTS NOGUEIRA, R.S.1; GOMES-SILVA, A.1; BITTAR, R.C.1; MENDONÇA, D.S.1; AMATO, V.S.2; MATTOS, M.S.3; OLIVEIRA-NETO, M.P.4; COUTINHO, S.G.1; DA-CRUZ, A.M.1 1.LABORATÓRIO INTERDISCIPLINAR DE PESQUISAS MÉDICAS/IOC/FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 2.SERVIÇO DE DOENÇAS INFECCIOSAS E PARASITÁRIAS, HOSPITAL DAS CLÍNICAS, FACULDADE DE MEDICINA, USP, SÃO PAULO, SP, BRAZIL; 3.INSTITUTO DE BIOFÍSICA CARLOS CHAGAS FILHO, UNIVERSIDADE FEDERAL DO RIO DE JANEIRO, RIO DE JANEIRO, RJ, BRAZIL; 4.INSTITUTO DE PESQUISA CLÍNICA EVANDRO CHAGAS/FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:mucosal leishmaniasis; clinical cure; cytokines
Abstract: Introduction: Several reports have unequivocally shown that an exacerbated type 1 immune response to leishmanial antigens is the basis of severe tissue destruction observed in active mucosal leishmaniasis (ML) patients. Furthermore, persistent production of high levels of inflammatory cytokines post therapy can confer a bad prognosis. The present study investigated whether the conditions defined during active disease of ML could influence the magnitude of anti-Leishmania immune response. Material and Methods: Twenty-one clinical cured ML patients were studied. Peripheral blood mononuclear cells (PBMC) were cultured with Leishmania braziliensis antigens (Lb-Ag), Toxoplasma gondii antigens (Tg-Ag), Concanavalin A (Con-A) or medium alone, and measured the lymphocyte proliferative response (LPR) and cytokine secretion by ELISA (IFN-gama, TNF, IL-18, IL-10 and IL-5). Retrospectively, medical records of the patients were reviewed for clinical and laboratorial parameters as: delayed type-hypersensitivity response during diagnosis (Montenegro skin test, MST), duration
of clinical cure after treatment or duration of ML disease. Results: Trend of positive correlation between duration of ML disease and MST induration (r=0.46, p=0.06, n=16) showed us an evidence that duration of ML illness could influence the immunopathogenesis. The IFN-gama production in response to Lb-Ag was positively correlated with duration of illness (r=0.55; p=0.02; n=17). In contrast, no association was observed when PBMC of the same patients were stimulated with Tg-Ag or Con-A. Also we observed a negative correlation between the duration of ML disease and production levels of IL-10 (r=-0.77, p=0.04, n=7). It is interesting to note that the magnitude of MST was negatively correlated with the IL-5 production after clinical cure (r=0.76, p=0.01, n =10). The IFN-gama/IL-10 ratio was negatively correlated with the duration of clinical cure (r=-0.78; p=0.02; n=8). Conclusions: Together, these results suggest that the prognosis of patients seems to be determined, at least in part, by the duration of ML disease. Also, the time of clinical cure can contribute to the better modulation of immune response essential to the control of parasite replication without triggering tissue damage. Support: CNPq, FAPERJ and IOC/FIOCRUZ. - CELLULAR AND MOLECULAR BIOLOGY [1172] P 270 - A COMPLEX PATTERN OF POST-TRANSLATION MODIFICATIONS ACTS UPON A SINGLE HOMOLOGUE OF THE TRANSLATION INITIATION FACTOR EIF4E FROM DIFFERENT LEISHMANIA SPECIES PEREIRA, M.M.C.; NASCIMENTO, L.M.D.; DE MELO NETO, O.P. CPQAM/FIOCRUZ, RECIFE, PE, BRAZIL.
Keyword:translation initiation; eif4f; post-translation modification
Abstract: Protein biosynthesis is a fundamental mechanism for the survival of all organisms and its regulation, mainly at the translation initiation stage, is likely to be influenced by many factors through different regulatory mechanisms. The assembly of the eIF4F complex (formed by the eIF4A, eIF4E and eIF4G subunits) is critical for mRNA recognition, via eIF4E binding to its capped 5’ end, and ribosome assembly. In Leishmania sp., several eIF4E and eIF4G homologues were identified, with two eIF4F complexes described so far (based upon the EIF4E3/EIF4G4 and EIF4E4/EIF4G3 subunits). These multiple eIF4F complexes might be related to the parasite’s complex life cycle, a possibility reinforced by the finding that, in L. amazonensis, both EIF4E3 and 4 were found to be targeted by post-translational modifications generating multiple isoforms. In L. infantum the EIF4E4 expression pattern is conserved, however the high molecular weight isoform of EIF4E3, typical of stressed cells, is missing. Here the genes encoding the wild type EIF4E3 genes from L. infantum and L. amazonensis were cloned and expressed in transfected cells fusioned to an N-terminal HA tag, accessible to detection with an anti-HA antibody. In both species the expression of the L. amazonensis EIF4E3 gene led to the high molecular weight isoform missing from its L. infantum orthologue, nevertheless when both genes were transcribed and translated in vitro the protein shifted to a smaller size, compatible with its predicted molecular weight. These results are consistent with differences in primary amino acid sequence between the two EIF4E3 orthologues being associated with unique sites for post-translation modification of the L. amazonensis protein. Sequence alignment comparing both genes revealed candidate serine and threonine residues found within the L. amazonensis sequence, but missing from the L. infantum EIF4E3. Mutation of the serine residue specifically eliminated the high molecular weight isoform in transfected cells. 2D analysis of the mutated and wild type proteins revealed that the changes in isoform profile are not associated with changes in pI, implying that post-translation modifications other than phosphorylation might be associated with this specific isoform. The 2D analysis, however, uncovered other isoforms, likely associated with phosphorylation events, which are common to the EIF4E3 orthologues from both Leishmania species investigated.
- CELLULAR AND MOLECULAR BIOLOGY [1175] P 271 - CHARACTERIZATION OF NEW LEISHMANIA CHAGASI/INFANTUM PROTEINS WITH POTENTIAL FOR USE IN THE DIAGNOSIS AND IMMUNIZATION AGAINST VISCERAL LEISHMANIASIS DO NASCIMENTO, M.B.1; DE CASTRO NETO, A.L.1; DE CARVALHO, L.C.P.2; OLIVEIRA, G.G.S.2; MAGALHﾃウS, F.B.3; DE MELO NETO, O.P.1
1.CPQAM-FIOCRUZ, RECIFE, PE, BRAZIL; 2.CPQGM-FIOCRUZ, RECIFE, PE, BRAZIL; 3.FACULDADE ASCES, CARUARU, PE, BRAZIL.
Keyword:leishmania; antigens; molecular biology
Abstract: The identification and characterization of novel antigens which can be evaluated as tools for the diagnosis of visceral leishmaniasis (VL), as well as for inducing protective immunity, is still a valid strategy considering the reduced number of recombinant polypeptides available for diagnostic purposes and vaccine development against this disease. Our group has previously identified new L. chagasi/infantum antigens through serological screenings of genomic and cDNA libraries using sera from humans and dogs. A total of 13 recombinant polypeptides derived from these antigens were evaluated and displayed sensitivities for the serological diagnosis of VL ranging from 34% to 93%. This work aimed to better characterize some of these proteins, all hypothetical and in general being rich in repetitive motifs, investigating aspects of their localization and conservation in different species of trypanosomatids. The selected proteins, named Lci5, Lci9, Lci10, Lci11 and Lci12, were analyzed through Western blots, immunofluorescence and immunocytochemistry, using rabbit polyclonal sera produced against each recombinant antigen. These were used to investigate various properties of the targeted proteins in both forms of the Leishmania life cycle, promastigote and amastigote, as well as their conservation in different species of the parasite. The proteins were further analyzed in cellular extracts made by cell fractionation using gradients of digitonine and freeze/thawing. The various proteins had distinct patterns of subcellular localization with a few localized to specific spots within the cytoplasm (Lci10 and Lci12) or flagellum (Lci5), while others were dispersed throughout the cytoplasm (Lci9 and Lci11). Four of them, Lci9, Lci10, Lci11 and Lci12, were found to be constitutively expressed in growth curves of both promastigotes and amastigotes of L. chagasi/infantum and were detected only in samples from Leishmania species. Lci10 and Lci11 were identified as phosphorylated proteins, indicative of being targeted by post-translational modifications. Further studies are necessary to better clarify the functional role of these proteins and in order to understand the reasons responsible for their immunogenicity and their promising properties for potential use in both diagnosis and vaccine against VL. - CLINICAL LEISHMANIASIS [1193] P 272 - VISCERAL LEISHMANIASIS: A RARELY DIAGNOSED ADULT CASE IN THE THRACE REGION, TURKEY* SAKRU, N.1; ATA, A.1; VELET, M.1; DEMIR, M.1; RAVEL, C.2; PRATLONG, F.2 1.TRAKYA UNI, EDIRNE, TURKEY; 2.UNIVERSITY OF MONTPELLIER 1, MONTPELLIER, FRANCE.
Keyword:visceral ; leihmaniasis; thrace, turkey
Abstract: In Turkey, Leishmania infantum is responsible for human visceral leishmaniasis, which is endemic along the Aegean and Mediterranean coasts and occurs sporadically in other regions.
We report a case of visceral leishmaniasis in a 65-year-old female, a resident of the Thrace Region of Turkey, which is non-endemic zone for leishmaniasis. She had been referred to our hospital for evaluation of hepato-splenomegaly. Routine blood investigations showed pancytopenia and increased gamma-globulin serum level with a high polyclonal peak. Diagnosis of leishmaniasis was established by bone marrow examination and culture. Leishmania strain isolated from patient was identified as L.infantum MON-1 by isoenzyme analysis at the French Reference Centre for Leishmaniasis. The patient was treated by meglumine antimonite for 28 days. All symptoms were disappeared within 10 days and there were no adverse events depending on Glucantime. Patient was cured and no relapsed within two years. *part of the study presented in 16th National Parasitology Congress, Adana, Turkey, 2009 - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1196] P 273 - ACTIVATION AND CYTOKINES PROFILE OF NKT CELLS IN HUMAN VISCERAL LEISHMANIASIS KEESEN, T.S.L.; VALVERDE, J.G.; MONTEIRO, G.R.; PONTES, N.N.; JERONIMO, S.B. UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE, NATAL, RN, BRAZIL.
Keyword:human visceral leishmaniasis; nkt cells; inflammatory cytokines
Abstract: Leishmaniasis is a disease caused by different species of Leishmania and affects millions of individuals worldwide. Visceral leishmaniasis (VL) is a chronic and fatal disease in humans if untreated. NKT cells are among the cell types that have been postulated to link the two arms of the immune system and are candidates for being able to respond rapidly and subsequently to activate other cell types. Moreover, NKT cells are known to be effectors and share features with other conventional T lymphocytes and they are unique in their rapid, concomitant cytokine production. Although NKT cells are a small subpopulation in the peripheral blood, they play crucial roles in early stage of infection with various pathogens including protozoa. Our goal was determine de activation status and cytokine profile of NKT cells in visceral leishmaniasis (VL) patients comparing to endemic (EC) or non endemic controls (NEC). To reach this aim, we evaluated VL patients (n=9) at diagnosis and compare to EC (n=9) and NEC (n=7). Whole blood assay was used and cells were labeled to determine the level of NKT cells (CD3+6B11+) and the intracellular level of IFN- , IL-17 and IL-10 using flow cytometry. The striking findings were that the total frequency of NKT cells did not change comparing VL versus EC. However, the NEC presented a lower frequency of NKT cells compared with VL and EC. Moreover, in VL patients the NKT cells have a hyperactivated profile compared with both controls. Interestingly, we found that the NKT cells from VL patients have a higher pro-inflammatory cytokines production (IFN-gamma and IL-17) than endemic control. As regards the IL-10 production, the NKT cells did show difference in the expression of IL-10 to compare VL versus controls. These findings suggest that NKT cells may be an important source of proinflammatory immune response in VL patients and that the great activated/inflammatory profile may direct the initial immune response in this disease. Support: National Institutes of Health AI-30639 and CNPq - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1199] P 274 - LOCAL IMMUNE RESPONSE PATTERN IN ACTIVE LESIONS AND IN SCARS OF CUTANEOUS LEISHMANIASIS DUE TO LEISHMANIA (VIANNIA) BRAZILIENSIS OLIVEIRA, M.P.; OLIVEIRA-NETO, M.P.; PIRMEZ, C. FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:foxp3; ifng; inflammatory infiltrate
Abstract: In cutaneous leishmaniasis (CL), local immune response is determinant not only in the pathogenesis but also in the control of the infection. The aim of this study was to investigate the inflammatory patterns in patients exhibiting active CL and in individuals with clinical cure. Skin biopsies from patients with active CL and cured individuals, all of them living in endemic areas of Leishmania (Viannia) braziliensis infection in the southern of Brazil, were enrolled. Inflammatory infiltrate was characterized by detection of T cells (CD4+, CD8+ and FoxP3+ cells/Tregulatory cells) and cytokines (IL-4, IL-10 and IFN ) in 4µm frozen sections using immunoperoxidase assays. While the frequency of CD4+ T cells was similar between groups, in CL lesions the frequency of CD8+T cells was elevated, when compared to scars samples (p<0.001). In scars samples, the CD4+/CD8+ T-cell ratio was significantly higher when compared to active CL lesions (p<0.001). FoxP3+ cells were observed in all samples tested, with a significantly higher frequency in scar samples than in CL active lesions (p<0.001). Also, when comparing to normal skin tissue, scars samples displayed a higher accumulation of T regulatory cells (p<0.05). Cells producing IL-4 were detected in 31/43 (72.1%) of CL samples but only in 6/29 (20%) of scar samples tested (p<0.001). All CL tissues were positive for IFN , IL-10. IFN- + cells were observed in 22/29 and IL-10 was detected in 16/22 (72.7%) of scar samples tested. In situ levels of IFN + (p<0.001) as well as IL-10+ cells (p<0.05) were increased in scars, when compared to active CL. Increased of CD4+/CD8+ T-cell ratio, in combination with an overproduction of IFN- and a local accumulation of T regulatory cells might be requirements for the control of skin infection caused by L. (V.) braziliensis. Corresponding author: pirmez@fiocruz.br Supported by POM/Fiocruz and CNpq
- CLINICAL LEISHMANIASIS [1200] P 275 - ASSOCIATION OF PARASITEMIA BY LEISHMANIA INFANTUM INFECTION IN HUMANS WITH BLOOD CELLULAR AND BIOCHEMICAL ABNORMALITIES COSTA, C.H.N.1; ZACARIAS, D.A.2; SENE, I.S.1; DA SILVA, J.C.2; SILVA, F.O.2; COSTA, D.L.1; ISHIKAWA, E.A.Y.3 1.FEDERAL UNIVERSITY OF PIAUÍ, TERESINA, PI, BRAZIL; 2.INSTITUTE OF TROPICAL DISEASES NATAN PORTELLA, TERESINA, PI, BRAZIL; 3.FEDERAL UNIVERSITY OF PARA, BELEM, PA, BRAZIL.
Keyword:cala-azar; parasitemia; pathogenesis
Abstract: Background: Since parasitemia is related to severe kala-azar, it may induce laboratory alterations related to the pathogenesis. Therefore, we decided to evaluate the relationship of blood tests with kala-azar severity. Methods: Six hundreds and ninety six patients with kala-azar were studied. All patients underwent routine laboratory tests.From whole blood, DNA isolation was performed with QIAmp DNA Blod Mini Kit (Qiagen, Germany), according to manufacturer’s instructions. Real-time PCR (qPCR) was based on TaqMan probe for accurate quantification of the target sequence. Specific primers based on kinetoplast minicircle DNA (kDNA) (Applied Biosystems, USA) were used. A standard curve was constructed using 10-fold serially diluted L. infantum parasite DNA corresponding to 104 to 1 parasite per reaction. Amplification and detection was performed in StepOn™ Real-Time PCR System (Applied Biosystems, USA). Cycling parameters were 50 ºC for 2 min, 95 ºC for 10 min, and 40 cycles at 95 ºC for 15 s and 60 ºC for
1 min. Standards, samples and negative controls were analyzed in triplicates. The threshold cycle (Ct) value was plotting by the standard curve. Results: Hemoglobin decreases significantly with increasing parasitemia (r = - 0,10; p= 0,008), as well as the number of circulating leukocytes (r= - 0,17; p < 0,001) and platelets (r= - 0,23; p < 0,001). The erythrocyte sedimentation rate (ESR) in the first hour increased with parasitemia (r= 0,11; p = 0,03). Conclusion: The results suggest that changes in these tests may be associated with signs, symptoms and complications such as anemia, bacterial infections, bleeding, as well as systemic inflammation.
- GENOMICS, TRANSCRIPTOMICS, PROTEOMICS, METABOLOMICS [1202] P 276 - NAGT IS NOT A VIRULENCE FACTOR FOR KALA-AZAR CAUSED BY LEISHMANIA INFANTUM. COSTA, C.H.N.1; AGUIAR, B.G.1; GAIDO, S.B.1; FIGUEIREDO, L.C.2
1.INSTITUTO DE DOENÇAS TROPICAIS NATAN PORTELLA, UNIVERSIDADE FEDERAL DO PIAUÍ, TERESINA, PI, BRAZIL; 2.UNIVERSIDADE FEDERAL DA PARAÍBA, BANANEIRAS, PB, BRAZIL.
Keyword:polymorphisms; visceral leishmaniasis; genomics
Abstract: Background: Most patients with visceral leishmaniasis present the classical clinical picture, and are successfully treated without complications. However, some develop complications such as bleeding and bacterial infections. This, like other studies have seek to look at the association of genetic variations in the genome of the parasite with the clinical manifestations. Methods: To check parasite genotype in patients with kala-azar from North-Northeast Brazil, the gene N-acetylglucosamine-1-phosphate transferase - NAGT of Leishmania infantum from 68 patients was sequenced. Patients belonged to two distinct clinical manifestations – 35 subject with (a) classic kala-azar without complications and 33 with complications, such as bacterial infections, bleeding and death. The gene sequence of NAGT was amplified using primers (L0 and L5) designed and optimized for the external regions of the gene NAGT generating a fragment of approximately (~) 1706 bp. After amplification by PCR (polymerase chain reaction), the fragments were sequenced (in duplicate) with the addition of two internal primers (L2B and L3B), so the occurrence of overlapping reads was possible. The assembly of reads to form contig for each sample was performed by scripts using the CAP3, followed by analysis for identifying polymorphisms through the tool Nucmer using the Mummer program. With the action strategy used, the NAGT gene sequencing was well done and the assembly of contigs with an efficiency of ~ 1630 bp was easily possible. Results: The analysis of the presence of genetic polymorphisms, showed absence of any SNPs (single-nucleotide polymorphism) or gaps. All 68 contigs, from 544 reads, analyzed presented 100% homology with each other and with the reference sequence regardless of clinical manifestation presented. Conclusion: With completely identical sequences, NAGT is a gene that has a high sequence similarity to L. infantum JPCM5 (NCBI) used for comparison and is not involved with the severity of kala-azar. It is highly conserved among different isolates of the same species and may be used in phylogenetic inferences or, eventually, for the diagnosis.
- CELLULAR AND MOLECULAR BIOLOGY [1207] P 277 - DEVELOPMENT OF AN RNAI TOOLKIT FOR FUNCTIONAL GENETIC ANALYSIS OF LEISHMANIA BRAZILIENSIS AND ITS APPLICATION TO FLAGELLAR GENES LYE, L.; OWENS, K.; NOTTON, T.; BRETTMAN, E.; MARCUS, J.; BEVERLEY, S.M. WASHINGTON UNIVERSITY SCHOOL OF MEDICINE, ST. LOUIS, UNITED STATES.
Keyword:axenic amastigote; flagellum; rna interference
Abstract: RNA interference (RNAi) is a powerful tool for genetic analysis. While many Leishmania species lost the RNAi pathway deep in evolution, species within the Leishmania subgenus Viannia retained RNAi pathway genes. Correspondingly, introduction of constructs expressing stem-loop constructs generating dsRNA in L. (V.) braziliensis resulted in substantial down regulation of target mRNAs and/or proteins (Lye et al PLoS Pathogens 2010). We are exploring a number of variables in order to optimize and/or increase the efficiency of RNAi. We have developed new vectors allowing rapid generation of stem-loop (StL) constructs using Gateway (Invitrogen速) technology. We are also exploring the development and use of axenic amastigote lines of L. braziliensis. The remaining task is to develop an inducible RNAi system. Amongst the many interesting targets evident in the genome, we have focused some effort on the Leishmania flagellum, which plays important roles in cell division and flagellar pocket formation, promastigote motility, and potentially amastigote virulence. Previously we showed that RNAi targeting the two major paraflagellar rod proteins PFR1 or PFR2 yielded parasites lacking the PFR and exhibiting motility defects. The phenotypes were very similar to those seen in PFR knockout parasites of L. mexicana previously. We probed the role of the intraflagellar transport pathway (IFT) by targeting four genes implicated in anterograde or retrograde trafficking. We were unable to recover transfectants bearing the respective StL constructs. As a control, the same StL constructs were transfected into an Argonaute null mutant which lacks a functional RNAi pathway; these transfections were successful These data suggest that the IFT pathway is essential for the viability of L. braziliensis promastigotes. We are now extending these studies to other flagellar genes.
- CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1210] P 278 - GBV-C INFLUENCES DELAYED TYPE HYPERSENSITIVITY RESPONSES TO LEISHMANIA INFANTUM PONTES, N.N.1; LIMA, J.R.1; LEUNG, D.T.; QUEIROZ, J.W.1; FERNANDES, M.Z.2; GOMES, C.E.M.1; MARTINS, D.R.1; MARINHO, P.1; MCLIDEN, J.H.3; PEARSON, R.D.4; WILSON, M.E.3; STAPLETON, J.T.3; JERONIMO, S.M.B.1
1.DEPARTAMENTS OF BIOCHEMISTRY AND DEPARTAMENT OF CELL BIOLOGY AND GENETICS, BIOSCIENCE CENTER, UFRN, NATAL, RN, BRAZIL; 2.DEPARTAMENT OF INTERNAL MEDICINE, UFRN, NATAL, RN, BRAZIL; 3.DEPARTAMENT OF MICROBIOLOGY AND DIVISION OF INFECTIOUS DISEASES, UNIVERITY OF IOWA, IOWA CITY, UNITED STATES; 4.DIVISION OF INFECTIOUS DISEASES, DEPARTAMENTS OF INTERNAL MEDICINE AND PATHOLOGY, UVA, CHARLOTTESVILLE, UNITED STATES.
Keyword:gbv-c; leishmania infantum infection; type 1 response
Abstract: GB virus C (GBV-C) infection promotes a type 1 immune response associated with prolonged survival in HIV infection. Leishmania infantum infection leads to either asymptomatic infection or symptomatic visceral leishmaniasis (VL). We characterized the GBV-C status of subjects living in a region of Brazil endemic for L. infantum. The peak age of active GBV-C viremia in both urban blood donors (16%) and families of VL patients (7.0%) was young adulthood. The incidence of positive anti-E2 serology increased with age (p=0.0121). No difference in ongoing GBV-C viremia was discerned between subjects with VL versus subjects with asymptomatic infection (p=0.269). Nonetheless, VL were more likely to have had prior cleared GBV-C infection than asymptomatic subjects (p=0.0012). Furthermore, the mean size of Leishmania delayed type hypersensitivity response (DTH) was larger in E2 antibody negative versus positive subjects (7.1 mm and 4.5 mm respectively; p=0.0023). This suggests that clearance of GBV-C infection is associated with a diminished type 1 response and could alter the immune response to Leishmania infantum infection. Support: National Institutes of Health AI-30639 and CNPq - CLINICAL LEISHMANIASIS [1212] P 279 - THE SPLEEN AND THE LIVER IN VISCERAL LEISHMANIASIS. COSTA, C.H.N.1; ROCHA, R.L.2; COSTA, D.L.1
1.FEDERAL UNIVERSITY OF PIAUĂ?, TERESINA, PI, BRAZIL; 2.FEDERAL UNIVERSITY OF MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL.
Keyword:spleen; liver; pathogenisis
Abstract: Backgound: The clinical features of visceral leishmaniasis (VL) are highly variable. Fever, anemia, hepatosplenomegaly and pancytopenia are present in most patients. These data are not very specific in determining the severity of disease. Patients and Methods: A prospective, hospital-based study was conducted in the northeast of Brazil, from September 2005 to August 2008. The cohort was composed of symptomatic VL patients of both sexes and of all ages, untreated and without a history of leishmaniasis. Statistical analysis was performed by Stata / SE 10.0. Results: The risk of death was higher among patients who had hepatomegaly (RR=1.8, 95% CI 1.0 to 3.2) compared to patients with normal liver size, but was much lower among patients with splenomegaly (RR=0.01, 95% CI from 0.0 to 0.01) compared to those who did not have an enlarged spleen. A positive correlation between edema and hepatomegaly (r=0.15, p <0.001), edema and splenomegaly (r=0.07, p=0.04), edema and death (r=0.27, p<0.001) and edema and bleeding (r=0.32, p<0.001). No association was observed between the presence of hepatomegaly or splenomegaly and death. It was observed that the patients had comparable mean the extent of spleen and liver, independent of height, weight or age of the patient. As the extent and volume of an organ has direct relation to weight and height, the size of the spleen and liver were corrected by its proportionality to body surface area. When the variables corresponding to the extension of these organs corrected for body surface were analyzed, there was a strong negative correlation with age, weight and stature of the individual. The extent of the spleen was negatively correlated with the duration of disease (r= -0.15, p<0.001), platelet count (r= -0.55, p<0.001) the number of neutrophils (r= -0,15, p<0.001), hemoglobin level (r = 0.20, p<0.001), serum globulin (r= -0.11, p=0.001) and the presence of epistaxis (r= -0.12 , p<0.001). A positive correlation with the extent of spleen was found with leukocytes (r=0.22, p<0.001), lymphocytes (r=0.47, p<0.001), liver enzyme AST (r=0.12, p<0.001), the occurrence of edema (r= 0.22, p<0.001), bacterial infection (r= 0.10, p= 0.002) and renal failure (r=0.21, p<0.001). The extent of the liver was positively correlated with the occurrence of gastrointestinal bleeding (r= 0.06, p=0.05), bacterial infection (r = 0.09, p=0.009) and negatively
with the levels of globulin (r= -0.11, p=0.002). Conclusion: Hepatosplenomegaly is much more than a diagnostic sign in patients with LV. These organs actively participate in the individual's response to infection and its role in the pathogenesis of the disease needs to be assessed. The negative correlation with the extent of spleen hematological parameters reinforces the hypothesis that splenic uptake of red blood cells, platelets and macrophages opsonized have an important role in defining the pancytopenia. - CLINICAL LEISHMANIASIS [1215] P 280 - BACTERIAL INFECTIONS AND BLEEDING DISORDERS IN PATIENTS WITH VISCERAL LEISHMANIASIS. COSTA, C.H.N.1; ROCHA, R.L.2; COSTA, D.L.1 1.FEDERAL UNIVERSITY OF PIAUĂ?, TERESINA, PI, BRAZIL; 2.FEDERAL UNIVERSITY OF MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL.
Keyword:bacterial infection; bleeding ; sepsis
Abstract: Backgound: Bacterial infections and blood clotting disorders are the most often complications associated with death in patients with visceral leishmaniasis. The frequency of bacterial infections in LV ranges from 11% to 60% and they may be the first manifestation of LV. Several pathogens and several presentations of these infections have been described, but respiratory and urinary infections seem to be the most important. Patients and Methods: A prospective, hospital-based study was conducted in the northeast of Brazil, from September 2005 to August 2008. A total of 883 symptomatic patients with visceral leishmaniasis of both sexes and of all ages untreated and without a history of VL were followed from admission to till discharge from hospital. Statistical analysis was performed by Stata / SE 10.0. Results: Bacterial infections were documented in 147 (16.6%) patients and were significantly associated with mortality (RR = 2.3, 95% CI 1.4 to 3.8) (Table 05A). Sepsis was diagnosed in 59 (40.1%) patients, infection of the skin or subcutaneous tissue in 33 (22.4%), pneumonia in 32 (21.8%), urinary tract infection in 29 (19.7 %). Mixed infections were identified in 37 (25.2%) patients. Cultures of usual secretions, blood or urine were negatives for bacteria in 54 (36.7) patients. Two children had proctitis pseudomonas without the classic signs of VL. Bleeding and concomitant bacterial infection was reported in 46 (5.2%) patients. Hospital infections, defined by the onset of clinical manifestations after three days of admission, occurred in 37 (4.2%) patients, and of these, 25 (67.6%) were infections of the skin or subcutaneous tissue. As the information was collected only during the period of hospitalization, it was not possible to rule out the possibility of hospital infection after discharge. Blood clotting disorders associated with bacterial infections were present in 49 (74.2%) of 66 patients with lethal outcome. Some variables were simultaneously associated with the risk of the patients present with bleeding and bacterial infection (vomiting, diarrhea, dyspnea, cough, rales, jaundice, edema, sepsis, pneumonia, HIV / AIDS, low platelets count, elevated aminotransferases level and renal failure). Among the 226 patients with bleeding disorders, 48 (21.2%) had normal number of platelets in the peripheral blood. Conclusion: Bacterial infections in patients with VL are associated with coagulation disorders, kidney failure, thrombocytopenia, elevated aminotransferases levels and other signs and symptoms such as vomiting, diarrhea, dyspnea, cough, edema, jaundice. Blood clotting disorders may result from systemic inflammatory response due to bacterial infections. - CLINICAL LEISHMANIASIS [1216] P 281 - RESPIRATORY DISTRESS IN PATIENTS WITH VISCERAL LEISHMANIASIS. COSTA, C.H.N.1; ROCHA, R.L.2; COSTA, D.L.1
1.FEDERAL UNIVERSITY OF PIAUÍ, TERESINA, PI, BRAZIL; 2.FEDERAL UNIVERSITY OF MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL.
Keyword:pneumonia; bacterial infection; respiratory syndrome
Abstract: Backgound: Respiratory symptoms are common in patients with visceral leishmaniasis (VL) and are often attributed to bacterial infections of the respiratory tract. Patients and Methods: A prospective, hospital-based study was conducted in the northeast of Brazil, from September 2005 to August 2008. A total of 883 symptomatic patients with VL were followed from admission to discharge from hospital and information on respiratory tract symptoms were assessed daily. Statistical analysis was performed by Stata / SE 10.0. Results: Chest radiography was performed in 860 (97.4%) patients. The clinical syndrome of pneumonia characterized by fever and cough associated with dyspnea, rales or runs, was present in 128 patients, 126 of whom underwent chest radiography. Alveolar opacities were observed in 18 (14.3%) of patients with respiratory symptoms and interstitial opacities in 8 (6.3%) patients. On the other hand radiographs suggestive of infection were seen in 46 patients without respiratory symptoms. Of all patients radiographed, which included symptomatic and asymptomatic patients, 51 (5.9%) had alveolar opacities and 24 (2.8%) had interstitial opacities. There were five deaths (9.8%) among patients with alveolar opacities and 10 deaths (38.1%) among patients with interstitial opacities (p<0,001). The chance of death in patients presenting alveolar opacities compared to patients with normal radiographs was 1.6 (p>0,05), whereas the relative risk of patients with interstitial opacities type evolve to death was 8.9 (p < 0.001). When this association was controlled by the presence of HIV infection, the risk of patients with interstitial opacities progression to death was 5.2 (95% CI 1.7 to 16.2) compared to patients with normal radiographs (p < 0.001). Eight of the patients with interstitial opacity were HIV positive (p <0.001) and six of them subsequently died. Interstitial opacities were also associated with malnutrition (p <0.001), cough (p = 0.04), dyspnea (p <0.001), cyanosis (p<0.001), and rales (p <0.001). Blood cultures for bacteria were performed in 34 (45.3%) of respiratory symptomatic patients and were positive in 6/20 (30.0%) patients with pulmonary alveolar opacity, but in none of the 14 patients with interstitial opacity. Conclusions: Pathogenic mechanisms of alveolar and interstitial involvement in patients with VL may be different. Alveolar infiltrates were probably related to bacterial pneumonia its prognosis may be improved by the early antibiotic therapy. Interstitial infiltrates unresponsive to antibiotics support the hypothesis of Pneumocystis jiroveci pneumonia. There are some similarities between respiratory distress in VL patients with acute lung injury (ALI) and its most severe form acute respiratory distress syndrome (ARDS), although the syndrome has not been described in patients with LV.
- GENOMICS, TRANSCRIPTOMICS, PROTEOMICS, METABOLOMICS [1217] P 282 - IDENTIFICATION OF LEISHMANIA AMAZONENSIS MOLECULES THAT ARE CANDIDATES FOR MEDIATING A POTENTIATING EFFECT ON LEISHMANIA BRAZILIENSIS INFECTION IN BALB/C MICE ARAÚJO, C.F.1; DA SILVA, V.M.G.2; MACHINI, F.K.3; DA CUNHA, J.P.4; DOS-SANTOS, W.L.1; DE CARVALHO, L.C.P.1 1.CENTRO DE PESQUISA GONÇALO MONIZ (IGM-FIOCRUZ), SALVADOR, BA, BRAZIL; 2.UNIVERSIDADE ESTADUAL DO SUDOESTE DA BAHIA, JEQUIÉ, BA, BRAZIL; 3.INSTITUTO CARLOS CHAGAS (FIOCRUZ-PR), CURITIBA, PR, BRAZIL; 4.INSTITUTO BUTANTAN, SÃO PAULO, SP, BRAZIL.
Keyword:leishmania; molecules; identification
Abstract: Introduction: Parasites of the genus Leishmania secrete molecules that modulate the host immune system and thus contribute to the establishment of the infection. Identification and functional characterization of these parasite molecules may clarify the pathogenesis of leishmaniasis and contribute to the development of prophylactic and therapeutic methods. Objectives: This study aimed at identifying biologically active molecules in aqueous extracts of Leishmania amazonensis amastigotes. Methods: L. amazonensis extract (LaE; Infect. Immun. 79:1236-1243, 2011)) was subjected to non-denaturing gel electrophoresis in 12% polyacrylamide gel, according to a previously described protocol (PLOS One 4:e5820, 2009). The intradermal injection of molecules eluted from gel fragments containing bands with apparent molecular weights (MW) around 68, 45, 36, and 28 kDa significantly increased the lesion caused by the inoculation of Leishmania braziliensis in the foot pad of Balb/c mice. The proteins from these bands were digested in gel (Anal. Chem. 85: 907 914, 2013), and the generated peptides were extracted and analyzed by mass spectrometry. Results: The molecules with apparent MW around 68 kDa were identified, through comparison with sequences deposited in gene banks and molecular weight congruity, as being heat shock protein 70 (HSP70) and succinate dehydrogenase flavoprotein. Discussion and conclusion: HSP-70 has been shown to be released by Leishmania in the cytoplasm of infected macrophages and in the intracellular medium. L. amazonensis HSP-70 could, therefore, by interfering with the nature of the macrophage activation, down-modulate an effective immune response, leading to disease aggravation. No similar information on succinate dehydrogenase flavoprotein, to the best of our knowledge, has been reported in the literature. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1223] P 283 - HUMAN EXPOSURE TO SALIVA OF LUTZOMYIA LONGIPALPIS AND THE PARASITE LEISHMANIA CHAGASI IN ENDEMIC AREA OF CANINE LEISHMANIASIS FRAGA, T.L.1; FERNANDES, M.F.2; OLIVEIRA NASCIMENTO, J.M.1; CASTRO, L.1; NEGRÃO, F.2; BARRAL, A.3; CRISTAL, J.R.3; DORVAL, M.E.C.1 1.UNIVERSIDADE FEDERAL DE MATO GROSSO DO SUL, CIDADE UNIVERSITÁRIA, 79070-900, CAMPO GRANDE, MS, BRAZIL; 2.UNIVERSIDADE FEDERAL DA GRANDE DOURADOS, DOURADOS, MS, BRAZIL; 3.CENTRO DE PESQUISA GONÇALO MUNIZ - FUNDAÇÃO OSWALDO CRUZ, SALVADOR, BA, BRAZIL.
Keyword:leishmaniasis; sand flies; salivary
Abstract: Some studies show that saliva from sand flies performs an important role as an anticoagulant, besides having the most potent vasodilator known, the maxidilan that inhibits the action of macrophages, increases blood flow and thus accelerates and optimizes blood repast. Besides maxidilan, we know of the existence of salivary proteins with skills that include the inhibition of hemostasis, vasoconstriction, inflammation and immune response, and that proved to be immunogenic for humans, dogs and rats. Mato Grosso do Sul, the LV is endemic and in spite of wide distribution and the growing number of cases, little is known about several epidemiological, biological and clinical characteristics. The features of introduction and spread of the disease in the State have virtually the same epidemiological characteristic, with the disease in dogs by preceding the human cases. In the city of Dourados-MS, cases of canine LV have been identified by the Center for Zoonosis Control since 2003, and today the figure as an important disease further among the animals being cause for concern for both clinicians and the local control authority. This work intends to assess human exposure to saliva of Lutzomyia longipalpis and the parasite Leishmania chagasi in the population of Dourados, area not endemic to human visceral leishmaniasis, contributing to the understanding of the interactions between host-vector in this interlocutory appeal of great importance in public health. So far, 47 individuals were evaluated with no history of leishmaniasis, from different areas of the city,
between the ages of two and 76 years, average of 39 years. Of these, eight were children below twelve years (three boys and four girls), 15 men and 14 women. ELISA reagents for antisaliva were (17) 38 and anti-Leishmania (21) 44 suggesting a significant exposure to saliva of the insect vector and the protozoan Leishmania chagasi. - CELLULAR AND MOLECULAR BIOLOGY [1224] P 284 - AN ACTIN-BINDING PROTEIN, TWINFILIN, LOCALIZES IN THE NUCLEUS AND REGULATES LEISHMANIA CELL DIVISION SAHASRABUDDHE, A.A.; KUMAR, G. CENTRAL DRUG RESEARCH INSTITUTE, LUCKNOW, INDIA.
Keyword:twinfilin; chromatin remodeling; nuclear spindle
Abstract: Leishmania constitute a clinically important group of protozoan parasites that are responsible for a range of disease manifestation in human. The current regime of treatment for leishmaniasis is undoubtedly torturous and mostly suffers from drug-resistance, thus necessitating identification of newer and potential drug targets. In this direction, the most important cellular process of therapeutic interest has been the duplication and expansion of these parasites in both insect vector and human host. Recent advances in the understanding of eukaryotic cell division processes have implicated indispensable functions of actin-network in the various nuclear processes such as chromatin remodeling, mRNA transport and karyokinesis. However, a highly conserved nature of actin has always distracted interests of researchers in exploring it as a therapeutic candidate in the parasitic infections. Interestingly, Leishmania-actin (LdACT) has shown a marked divergence in its structure and function making it an attractive drug target in Leishmania parasites. It has been shown that LdACT besides being distributed throughout the cell also co-localizes with the nucleus and kinetoplast DNA. Recent, biochemical analysis has shown that LdACT directly binds and creates nicks in the double stranded DNA molecule with special reference to the kinetoplast-DNA suggesting an importance of actin-network in the chromatin remodeling process. Here we show that an actin-binding protein, twinfilin, localizes in the nucleus and redistributes specifically on the intranuclear spindle during cell division in Leishmania. We found that this protein establishes a tripartite interaction with chromatin (DNA), intranuclear spindles and LdACT in the nucleus indicating its plausible role in the Leishmania cell division. By generating gene knockout mutants of the twinfilin gene we further show that ablation of its expression up to ~50% affects spindle formation and partially arrests the cells in ‘S’ phase of the cell division. This inidicated that twinfilin might be complecting the events associated with spindle formation and nuclear DNA synthesis. In addition, all the attempts to obtain null mutants failed categorically while only aneuploid population survived, which suggesed a vital requirement of twinfilin for the survival of these parasites. The present study reveals an essential physiological function of twinfilin and invites therapeutic intervention towards the treatment of leishmaniasis. - GENETICS, EVOLUTION AND TAXONOMY [1226] P 285 - ANALYSIS OF GENETIC VARIABILITY OF CIRCULATING STRAINS OF LEISHMANIA FROM HUMANS AND DOGS IN RIO GRANDE DO NORTE. SILVA, V.P.M.1; MARTINS, D.R.A.1; MONTEIRO, G.R.G.1; LACERDA, H.G.1; NUNES, J.1; QUEIROZ, P.V.S.2; TORRES, T.M.1; LIMA, J.P.M.S.1; WILSON, M.E.3; JERÔNIMO, S.M.B.1; XIMENES, M.F.F.M.1 1.UFRN, NATAL, RN, BRAZIL; 2.UERN, MOSSORÓ, RN, BRAZIL; 3.NIH, IOWA, UNITED STATES.
Keyword:leishmania braziliensis; leishmania infantum; pcr
Abstract: Infection with Leishmania spp is spectral, varying from both asymptomatic and symptomatic cutaneous and visceral leishmaniasis (VL). Genetic polymorphisms in Leishmania may influence the natural history following infection. The objective of this study was to evaluate whether genetic polymorphisms of Leishmania were associated in part with the clinical outcomes. We genotyped and sequenced Leishmania isolates obtained from humans with skin lesions suggestive of cutaneous leishamaniasis due Leishmania braziliensis, and patient with symptomatic VL and people with asymptomatic Leishmania infantum infections and isolates from dogs with VL from different areas from the State of Rio Grande do Norte. Samples were genotyped for HSP 70 and ITS1, followed by sequencing the PCR products. The sequences were analyzed and the reads were mounted using Phred/Phrap package (phred score > 20), edited by using Consed and the alignment with reference samples using MAFFT program with defaults, thereafter the best-fit nucleotide substitution model was estimated using the jModelTest tool. The relationships between strains were estimates based under a Bayesian coalescent model using BEAST package, and the final dendrograms were visualized in FigTree tool. We obtained 119 sequences for L. infantum strains (54 for ITS1 and 65 for HSP70) and 34 for L. braziliensis strains (12 for ITS1 and 22 for HSP70). One nucleotide transition among L. infantum strains for HSP70 were identified when compared to control samples, whereas one transition on three L. braziliensis sequences were found. More genotypic variation was observed for canine L. infantum isolates (5 transversions and 3 transitions) in HSP70 sequence. The HKY substitution model was consistent for all groups of data and the ln of Likelihood is -2092.4776 and -688.4843 for HSP70 and ITS1, respectively. These might indicate similar selective pressures for both Leishmania species. The dendrograms analysis showed two groups for each marker, and these groups were correlated with geographic regions. Our data suggest that the presence of local selection pressures that promote prevalence of a few strains in a specific district. Resampling are in progress to elucidate unsolved questions. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1228] P 286 - INCREASED FREQUENCY OF ACTIVATED CD8 T CELLS PRODUCING IFN- IN THE HEALING RESPONSE DURING LEISHMANIA INFANTUM INFECTION. VALVERDE, J.G.; KEESEN, T.L.; MONTEIRO, G.G.; PONTES, N.N.; NETO, F.F.; JERÔNIMO, S.B. UFRN, NATAL, RN, BRAZIL.
Keyword:leishmania infatum; flow cytometry; gamma interferon
Abstract: Visceral Leishmaniasis (VL) caused by Leishmania infantum is characterized by lack of activation of peripheral blood mononuclear cells (PBMC) and inability to produce IFN- in response to Leishmania antigen, which is restored after clinical cure. However, recent studies of symptomatic VL patients from Indian showed IFN- and IL-10 production by peripheral blood cells in response Leishmania antigens. Our objective was to determine the role of activated CD4 and CD8 T cells in the production of IFN- and IL-10 in VL. To reach this aim, we followed, prospectively, VL patients (n=16) at diagnosis and after successful treatment (at 4 months and 14 months) and endemic controls (n=15) (EC). Parasite load in the blood was assessed by qPCR, to estimate the level of circulating Leishmania in the study subjects. Whole blood assay was used and cells were labeled to determine the level of late activation (CD25) and the intracellular level of IFN- and IL-10 on CD4 and CD8 cells using flow cytometry. VL patients had parasitemia at diagnosis (Mean: 329.8 ± 459.1), and four months post-treatment (Mean: 801.7 ± 1491.7); but cleared after a year post-treatment (Mean:0.035 ± 0.0495). In addition, 20% of endemic control individuals had positive parasitemia (Mean: 7,98 ± 22,44). A decrease in activated CD8+CD25+ T cells “ex vivo” was observed 4 months post-treatment, (p=0.0121)
however, TCD4+CD25+ T cells remained without changes (p>0.05). The frequency of TCD8+CD25+ expressing IFN- did not vary after SLA stimulation in symptomatic VL (p>0.05) however, those cells increased after 4 months (p<0.05), and after 14 months post treatment (p<0.05) as well as in EC (p<0.05). On the other hand the frequency of TCD8+CD25+ expressing IL-10 decrease after SLA stimulation in symptomatic VL (p<0.05) and no change, stimulus specific, was observed in this cells after treatment. Surprisingly EC present an increased frequency after SLA stimulation. Otherwise no significant change in the frequency of TCD4+CD25+ expressing IFN- or IL-10 was observed. We conclude that despite the decrease in levels of activated CD8 T cells after treatment, these cells remain as an important source of IFN- after cure and no IL-10 is observed after recover. Thus CD8 Tcells could be involved in the control of parasitemia and clinical recovery observed in visceral leishmaniasis. Support: National Institutes of Health AI-30639 and CAPES
- CLINICAL LEISHMANIASIS [1235] P 287 - COOCCURANCE OF VISCERAL LEISHMANIASIS AND LYMPHOMA: CASE REPORT ERDEM, H.A.1; ISIKGOZ TASBAKAN, M.1; PULLUKCU, H.1; OZENSOY TOZ, S.2; SIPAHI, O.R.1; YAMAZHAN, T.1; TURGAY, N.2 1.EGE UNIVERSITY MEDICAL SCHOOL DEPARTMENT OF INFECTIOUS DISEASES AND CLINICAL MICROBIOLOGY, IZMIR, TURKEY; 2.EGE UNIVERSITY MEDICAL SCHOOL DEPARTMENT OF PARASITOLOGY, IZMIR, TURKEY.
Keyword:leishmaniasis; lymphoma; coinfection
Abstract: Clinicians have been usually considered malignancies during follow up of the patients who have infectious diseases as pre-diagnosis. However, malignancy and an infection disease are seen together more rarely with the exception of immunosuppressed patients. This presentation is a case report followed up for fever of unknown origin. The patient was first diagnosed parasitologically and treated for visceral leishmaniasis (VL) using Liposomal Amphotericin B but had diagnosed as diffuse B cell lymphoma in the investigation of bone marrow aspiration samples applied for the control of Leishmania amastigotes to evaluate the success of the treatment. Case report: A 59 year old female patient admitted to the outpatient clinic of internal medicine with the symptoms as fever, weight loss, abdominal pain and weakness during last 4-5 months. The malignancy was first considered and only hypercellular bone marrow was observed in bone marrow biopsy sample, and the patient was transferred to department of infectious diseases for investigation of fever of unknown origin. The laboratory test results were as follows; leucocyte: 5880 x103/mm3 (neutrophil: %42, monocyte: % 41), Hg: 7 g/dl, sedimentation: 68 mm/h and CRP: 1.41 mg/dl. Protein electrophoresis was revealed polyclonal hyperglobulinemia (kappa light chain: 495 mg/dl, lambda light chain: 248 mg/dl). No other pathology except splenomegaly was detected by total body computed tomography. After evaluating of laboratory tests and clinical signs, the patient had diagnosed as visceral leishmaniasis clinically and bone marrow aspiration was applied. Amastigotes were seen in giemsa stained smears and the parasite was isolated in NNN medium. Patient was treated with Liposomal Amphotericin B as 3 mg/kg/day for five days, and repeated in 10th and 28th days. Erythrocytes and plasma replacements were also applied but the abdominal pain was continued and general situation was not improved. Abdominal computed tomography was revealed hepatosplenomegaly, bilateral pleural effusion and minimal intraabdominal free fluid. Fever could not be solved and consecutive blood cultures were resulted as negative. Because of no response to leishmaniasis treatment, the examination for malignancy
was continued. There was no atypical cell or blast in peripheral blood smear. At the 28th day of VL treatment, bone marrow aspiration and biopsy was repeated for checking the amastigotes and no amastigotes were seen but diffuse B cell lymphoma infiltration was detected and the patient was transferred to clinic of hematological diseases. Visceral leishmaniasis is one of the important parasitological diseases mimicking malignancies. VL can also be accompanied to malign diseases as in the case of our patient. In these situations, the diagnosis can be difficult and very late. Therefore, in endemic areas, especially in immunosuppressive group of patients co-infections of VL should be taken into account by the clinicians. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1242] P 288 - THE CYTOKINE PROFILES OF SUDANESE PATIENTS PRESENTED WITH DIFFERENT CLINICAL FORMS OF LEISHMANIASIS MUKHTAR, M.M.; ABDEL HALEEM, A.A.; BABIKER, D.E.; BAKHIET, S.M. INSTITUTE OF ENDEMIC DISEASES, UNIVERSITY OF KHARTOUM, KHARTOUM, SUDAN.
Keyword:leishmaniasis, sudan; clinical forms; cytokines
Abstract: Leishmania donovani causes different clinical forms in Sudan including Visceral Leishmaniasis (VL), Post-Kala Azar Dermal Leishmaniasis (PKDL), Mucosal Leishmaniasis (ML) and cutaneous Leishmaniasis (CL). Genomic analysis of the parasite isolates from the different clinical forms showed no major diversity between the isolates. Cytokine analysis of whole blood samples from patients stimulated with live Leishmania donovani, showed a mixed TH1/ Th2 responses with a dominant inflammatory response in dermal and mucosal leishmaniasis patients and a dominant Th2 response in VL patients. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1244] P 289 - SEX AND AGE ARE ASSOCIATED WITH DIFFERENCES IN IL-10 PRODUCTION AFTER LEISHMANIA INFANTUM INFECTION AND ITS CLINICAL OUTCOME. MONTEIRO, G.R.G.1; LOVELL, A.M.2; NASCIMENTO, E.T.1; QUEIROZ, J.W.1; NASCIMENTO, P.P.1; PEARSON, R.D.3; WILSON, M.E.2; JERONIMO, S.B.1 1.UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE, NATAL, RN, BRAZIL; 2.UNIVERSITY OF IOWA, IOWA CITY, UNITED STATES; 3.UNIVERSITY OF VIRGINIA, CHARLOTTESVILLE, UNITED STATES.
Keyword:visceral leishmaniasis; interleukin-10; males
Abstract: Visceral leishmaniasis (VL) due to Leishmania infantum is endemic in Rio Grande do Norte, Brazil. L. infantum induces an altered immune response leading to distinct clinical outcomes in humans, which varies from asymptomatic self-healing infections to severe, progressive VL. A positive delayed type hypersensitivity skin test for Leishmania antigen (DTH) develops after resolution of symptomatic or asymptomatic infection. Males exposed to L. infantum are at increased risk of developing VL after puberty, although the sex ratio is equal in younger children. Interleukin 10 (IL-10) is an immune modulatory cytokine that inhibits macrophage activation and suppresses type 1 cellular responses. We hypothesized that the greater male risk of developing VL rather than asymptomatic infection is due to enhanced production of IL-10. We determined IL-10 by ELISA in sera of VL patients and healthy endemic controls. All subjects underwent a physical examination, delayed type hypersensitivity testing (DTH) for Leishmania antigen, and Leishmania serology (Ab). Based on these evaluations, subjects were grouped in 5 categories: symptomatic visceral leishmaniasis (VL), recent infection (DTH-Ab+),
asymptomatic seronegative (DTH+Ab-), asymptomatic seropositive (DTH+Ab+), and presumed exposed with negative DTH and Ab (DTH-Ab-). The levels of IL-10 in the sera of persons in these groups were, respectively, 95.7±51.3 pg/mL (VL), 32.4±4.7 pg/mL (DTH-Ab+), 20.8±3.9 pg/mL (DTH+Ab-), 10.8±1,6 pg/mL (DTH+Ab+) and 8.3±2.9 pg/mL (DTH-Ab-). IL10 was higher during symptomatic VL (88.4 pg/mL) than after treatment (9.3 pg/ml), (p <0.0001). Individuals with recent infection (DTH-Ab+) had higher levels of IL-10 than the other groups with resolved asymptomatic infections (DTH+Ab+, DTH+Ab) or presumed exposed with no apparent infection (DTH-Ab-), (p<0.05, ANOVA). A significant difference in the level of IL-10 during symptomatic VL (n=89) was found in males, 104.8 ±60.8 pg/mL, compared to females (n=39) 74.46±39.4 pg/mL (p<0.05). In (DTH-Ab+), levels of IL-10 in males (n=18) and females (n=26) were respectively 35.8±5.3 pg/mL and 30.1±2.4pg/mL p<0.05). No significance difference in IL-10 was found by gender in the (DTH+Ab-), (DTH+Ab+) and (DTH-Ab-) groups. In summary, IL-10 levels are associated with distinct clinical outcomes of L. infantum infections and could explain the greater risk of males to developing symptomatic VL in Brazil. - CLINICAL LEISHMANIASIS [1248] P 290 - ASSOCIATION TO CLINICAL STATUS OF THE VISCERAL LEISHMANIASIS WITH PARASITE LOAD AND PARASITEMIA BY REAL-TIME PCR COSTA, C.H.N.; SENE, I.S.; ZACARIAS, D.A.; COSTA, D.L.; SILVA, J.C. UNIVERSIDADE FEDERAL DO PIAUI, TERESINA, PI, BRAZIL.
Keyword:qpcr; parasite load; parasitemia
Abstract: Objectives: Visceral leishmaniasis (VL) is lethal but the pathogenesis of severe disease is poorly understood. Little is known about the contribution of the amount of parasites to mortality. Since real-time PCR (qPCR) has been applied successfully to estimate parasitemia and parasite load, this study was conceived to evaluate the effect of parasite load and parasitemia, and their correlation between themselves. Methods: The study population was comprised by 146 hospitalized patients from the city of Teresina, Brazil. A single experienced physician prospectively and carefully examined each patient. They were classified according to the presence of symptoms and signs such as, hemorrhage, fever, abdominal pain, edema, epistaxis, lung x-ray, dyspnea, bacterial infection and others. Bone marrow and blood DNA isolation was performed by QIAmp DNA Blood Mini Kit (Qiagen, Germany) according to manufacturer’s instructions. Real-time PCR was based on TaqMan probe for accurate quantification of the target sequence of the genes. Specific primers based on mini-circle kinetoplast DNA (kDNA) and albumin (Applied Biosystems, USA) were used. A standard curve was constructed using 10-fold serially diluted L. infantum parasite DNA corresponding to 104 to 1 parasite per reaction and human cells serially diluted 2 x 104 to 2 cells per reaction (housekeeping gene). Amplification and detection was performed in StepOne™ Real-Time PCR System (Applied Biosystems, USA). Cycling parameters were 50 oC for 2 min, 95 oC for 10 min, and 40 cycles at 95 oC for 15 s and 60 oC for 1 min. Standards, samples and negative controls were analyzed in duplicates. The threshold cycle (Ct) value was plotting by the standard curve. Statistical analysis was performed by Mann-Whitney test to analyzed clinical status. Correlation was evaluated using Spearman/Pearson correlation, been statistically significant at p < 0.05.
Results: The following associations with clinical data were found: a) parasitemia: bleeding, and gender. b) parasite load: lung x-ray, lung rules, edema, and epistaxis. c) correlation between parasitemia and parasite load (r = 0.2564; p-value = 0.001).
Conclusions: Parasite load and parasitemia are slightly correlated and contribute differently to the signs of VL severity.
- CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1250] P 291 - INOS EXPRESSION IN THE SKIN LESIONS OF CEBUS APELLA PRIMATE EXPERIMENTALLY INFECTED WITH L. (L.) AMAZONENSIS AND L. (V.) BRAZILIENSIS LAURENTI, M.D.1; SILVEIRA, F.T.2; FRANCESQUINI, F.1; TOMOKANE, T.Y.1; SANCHES, F.1; PASSERO, L.F.D.1; GOMES, C.M.C.1; CORBETT, C.E.P.1 1.MEDICAL SCHOOL-USP, SÃO PAULO, SP, BRAZIL; 2.PARASITOLOGY-IEC, BELÉM, PR, BRAZIL.
Keyword:experimental cutaneous leishmaniasis; nitric oxide; monkey
Abstract: Background: Clinical and immunopathological findings concerning the evolution of the experimental Leishmania-infection in Cebus apella primate have confirmed its susceptibility and support previous indications that this primate may be useful as an experimental model for studying cutaneous leishmaniasis. In order to evaluate the role of nitric oxide on the tissue parasitism, a correlation between amastigotes and iNOS+ cells density was studied in C. apella. Methods: C. apella was inoculated intradermally in six points at the dorsal face of the tail with 3x106 promastigotes of L. (L.) amazonensis and L. (V.) braziliensis. Skin biopsies were collected at 30, 60, 90, 120, 150 and 180 days PI. The skin sections were processed by immunohistochemistry using mouse anti-Leishmania (produced in our laboratory) and rabbit anti-human NOS2 (Santa Cruz) as primaries antibodies. Novolink Max Polymer (NovoCastra) was used for amplification and visualization the reaction. The immunostained cells were counting in 10 different microscope fields (40x objective) for estimating the cellular density, using an image analysis system (Karl-Zeiss). Results: Both L. (L.) amazonensis and L. (V.) braziliensis showed an increase on the skin parasite burden till 60 days PI, with decrease since 90 days PI. There were no visible amatigotes in the tissue since 150 days PI. In spite of the same profile in the tissue parasitism, the parasite burden was higher in L. (L.) amazonensis than in L. (V.) braziliensis infection. Concerning the iNOS expression, higher number of positive cells was observed in the first days PI (30 and 60) in L. (L.) amazonensis infection (p<0.05) followed by decrease since 90 days PI. However, higher number of iNOS+ positive cells was observed in L. (V.) braziliensis since 90 days PI (p<0.05). Conclusion: These results showed a direct correlation between the parasite burden and iNOS expression in the skin lesions of Cebus apella primate experimentally infected with L. (L.) amazonensis and L. (V.) braziliensis, suggesting an increase on the microbicidal mechanisms related to nitric oxide production in the outcome of experimental Leishmania-infection in Cebus apella model. Supported by FAPESP, CNPq and LIM50 HC-FMUSP. - VACCINE DEVELOPMENT AND TRIALS [1251] P 292 - EVALUATION OF CYTOKINE PROFILE IN DOGS AT DIFFERENT TIMES POST VACCINATION AGAINST LEISHMANIASIS AFTER LEUKOCYTES CULTIVATION
UNDER STIMULATION OF SOLUBLE ANTIGEN OF LEISHMANIA CHAGASI PEREIRA, C.C.1; MOREIRA, M.L.1; DIAS, M.H.F.1; MARTELETO, B.1; ALVES, M.L.1; RIBEIRO, V.M.2; VIANA, K.F.3; GIUNCHETTI, R.C.3; FILHO, O.A.M.1; ARAÚJO, M.S.S.1 1.FIOCRUZ-MG, BELO HORIZONTE, MG, BRAZIL; 2.CLÍNICA VETERINÁRIA SANTO AGOSTINHO, BELO HORIZONTE, MG, BRAZIL; 3.UFMG, BELO HORIZONTE, MG, BRAZIL.
Keyword:canine visceral leishmaniasis; vaccine; cytokines
Abstract: Visceral leishmaniasis is a high growing disease around the world, especially in Brazil. The vertebrate hosts recognized as reservoirs are limited to mammals. The large number of cases of Canine Visceral Leishmaniasis (CVL) and also the skin parasitism became the main source of sand fly vectors infections and therefore of humans, with a profile of antropozoonotic disease. The current strategy for the control of this disease is based on the elimination of seropositive dogs, systematic human cases treatment and vector control. Although dog euthanasia would be the main strategy to control the disease, it is not widely accepted, especially by the owners. However, most therapeutic strategies studied on dogs have failed to achieve consistent parasitological cure at CVL. In this context, the development of a protective vaccine against CVL could be a promising tool for control of CVL and consecutively human visceral Leishmaniasis. Usually there is a consensus that the cytokine microenvironment plays a central role in the pathogenesis result Leishmaniasis. The major effector mechanism involved in protective immune response in dogs infected with Leishmania is the activation of macrophages by INF- in order to destroy intracellular parasites amastigote form mediated by the production of nitric oxide. In this context, immunobiological based Leishmania antigens can be used to induce a protective immune response on the type 1, which is a prerequisite for creating a successful vaccine CVL. The aim of our study was to evaluate of cytokine profile in dogs at different times post vaccination with Leishmune® against leishmaniasis after cultivation of leukocyte under stimulation with soluble antigen of Leishmania chagasi. We employed flow cytometry and ELISA to analysis the production of cytokines. Our results showed that Leishmune® was able to induce an increased the level of production of IL-8 and INF- and decrease of IL-10 at the sixth month after a vaccination and theses cytokine the levels retuned at the normal values after one year, these results are suggesting that immune responses remains in effect during six months been necessary booster dose vaccination at one year. Financial support: CNPq, CAPES, FAPEMIG and FIOCRUZ - CLINICAL LEISHMANIASIS [1253] P 293 - COINFECTION AMERICAN CUTANEOUS LEISHMANIASIS-HIV IN ELDERLY PATIENT: A CASE REPORT BRITO, M.E.F.1; ARAUJO, A.I.F.1; RODRIGUES, E.H.G.1; ALMEIDA, E.L.1; VIANA, J.B.M.1; ALEXANDRE, J.L.A.1; WERKHAUSER, R.P.1; BRANDÃO-FILHO, S.P.1; RESENDE, M.O.2; CAVALCANTI, S.M.M.2; VIDAL, M.L.2; HOROWITZ, M.R.2; ANDRADE, M.S.2; MEDEIROS, A.C.R.2 1.CENTRO DE PESQUISAS AGGEU MAGALHÃES, RECIFE, PE, BRAZIL; 2.HOSPITAL UNIVERSITÁRIO OSWALDO CRUZ, RECIFE, PE, BRAZIL.
Keyword:american cutaneous leishmaniasis; co-infection leishmaniasis/ hiv; pernambuco
Abstract: Coinfection leishmaniasis-HIV is considered emerging disease with highly severe in many regions of the world. It has been observed in patients co-infected an important clinical diversity, with more severe and spread refractory to conventional treatment. Male, 71 years old, farmer, coming from Amaraji, Pernambuco, referring appearance of lesions on the left leg which after 3
months evolved as confluent ulcers whose greatest diameter was about 10 x 6 cm2, associated with erythema and edema. Diagnosis of american cutaneous leishmaniasis (ACL) by PCR was carried and the treatment with N-methyl-glucamine (15mg/kg/day) for 20 days was done. It was observed posterior clinical cure. After five months, the patient was admitted at the hospital with loss of appetite, significant weight loss (20 kg), asthenia and persistent diarrhea. Besides, he had infiltrated erythematous plaques on both legs that stretched to the distal third of the left thigh and reported a history of leprosy treated 10 years ago. It was performed histological exam from biopsy of the plate on the left thigh by clinical suspicion, where was observed structures suggestive of ACL. Given the clinical systemic associated with the appearance of atypical skin lesions, immunosuppression was investigated and, thus, was found the positive serology to HIV. Was prescribed antiretroviral drugs and amphotericin B, what provided the regression of lesions. The Brazilian population growth, rapid urbanization, anthropic environmental change and massive migration have contributed to the spread of LTA in new areas. Also the epidemiology of HIV has undergone major changes, with an increase in cases among women, heterosexuals, internalization and persons with less education. It was also observed the increase of the disease in the elderly, which can be attributed to technological advances that have prolonged they sexual life, as well as its resistance to condom use. It is important to investigate immunodeficiencies in patients with atypical evolutions of leishmaniasis and alert to the changes in epidemiological patterns of seropositive patients. Financial support: CNPq, FACEPE, FIOCRUZ. - CELLULAR AND MOLECULAR BIOLOGY [1264] P 294 - THE CONTRIBUTION OF INFLAMMATORY CD14+CD16+ MONOCYTES IN THE PATHOLOGY OF LEISHMANIA BRAZILIENSIS INFECTION DINIZ, A.G.; BONFIM, T.D.; VENDRAME, C.V.; CARVALHO, L.P.; CARVALHO, E.M.; BACELLAR, O.A. HUPES, SALVADOR, BA, BRAZIL.
Keyword:inflammatory monocytes; subclinical infection; leishmania braziliensis
Abstract: Introduction: In cutaneous leishmaniasis (CL) caused by Leishmania braziliensis, the pathology is associated with exaggerated production of pro-inflammatory cytokines. In contrast individuals who are exposure to L. braziliensis infection but do not develop disease (subclinical L. braziliensis infection) have a lower T cell activation and their macrophages produce less inflammatory cytokines as compared with CL patients. In contrast to the wide knowledge of T cell in human leishmaniasis, very little is known about the role of monocytes/macrophages in human L. braziliensis infection. Objective: The aim of the present study was to determine the frequency of inflammatory monocytes (CD14+CD16+) in CL patients and SC individuals, and characterize phenotypically their functions in tegumentary leishmaniasis (TL). Methods: The expression of CD14, CD16, HLA-DR, CD80 and CD86 in monocytes was assessed by flow cytometry. The analyses were performed with cells cultured in the absence of any stimulus to determine the ex-vivo expression of these molecules. To determinate the intracellular source of TNF in monocyte subpopulations, PBMCs were incubated with soluble leishmania antigen (SLA) and with LPS and the percentage of cytokine producing cell was determined in the CD14+CD16+ and CD14+CD16- cells. Results: The frequency of CD14+CD16+ monocytes was higher in CL patients, median was 25% (range 14-47%) compared with SC individuals 15% (8.4-33%) and the HS group 12% (5.5-37%), (*p<0.01; **P<0.001). The expression of HLA-DR, CD80 and CD86 were significantly higher in the CD14+CD16+ monocytes compared with CD14+CD16- cells in both groups (*p<0.05; **p<0.01; *** p<0.001). TNF expression was clearly higher in the CD14+CD16+ monocytes than in CD14+CD16- monocytes from both CL patients and SC individuals with SLA stimulation. Conclusion: These data suggest that the high frequency of CD16+ monocytes and the high activation status of these cells detected by
increased expression of HLA-DR, CD80, CD86 and TNF production may play an important role in the pathogenesis of L. braziliensis - CLINICAL LEISHMANIASIS [1268] P 295 - TRIPLE CO- INFECTION LEISHMANIASIS, BRUCELLOSIS AND TYPHOID FEVER SHKURTI, K.1; KRAJA, D.1; BINO, S.1; KOKICI, M.2; KIKA, B.3; DIKOLLI, E.4; BEGO, A.3 1.DEPT OF INFECTIOUS DISEASES UHC “MOTHER TERESA” ALBANIA, TIRANA,, ALBANIA; 2.DEPT OF LABORATORY UHC “MOTHER TERESA” ALBANIA, TIRANA,, ALBANIA; 3.LABORATORY OF NATIONAL PUBLIC HEALTH, TIRANA,, ALBANIA; 4.LBORATORY OF NATIONAL PUBLIC HEALTH, TIRANA,, ALBANIA.
Keyword:leishmaniasis; brucellosis; typhoid fever
Abstract: Author’s aim is to present a rare case with bacterial and parasite co-infection in an immunocompetent subject. A 24 years female was hospitalized in the Department of Infectious Diseases in UHC “Mother Teresa” in Tirana with fever of unknown origin. She lived in a rural zone in the north of Albania and was treated in the regional hospital for Leishmaniasis resistant to antimony. She had four weeks history of high fever, headache, malaise, prostration and pain in articulations. Objective examination showed pallor and toxic face, enlargement of liver and spleen. She had no meningeal signs, rales or abnormal cardiac murmur. Laboratory findings were: RBC 3560000, Hb 11 g%, PLT 150000, WBC 2800, absolute lymphocytosis. X rays and cardiac sonography didn’t show any pathology. Culture of the blood or urine didn’t find any pathogen. Serology exams were: HIV negative, Leishmania positive, Rose Bengal positive, Wright 1:1280, Widal TO 1:160, TH 1:320. Microscopic examination of marrow aspiration showed leishmania amastigote. She started treatment with antimony (glucantime) 5mg/kg, Trimetoprim+ Sulphometoxasol 960 mg x 2 and Rimphampicin 300 mg x2 and after ten days the patient had no more fever. Widal examination after a week treatment was TO 1:320, TH 1:320. The antibiotic treatment course finished after 42 days and antimony drug after 28 days. Laboratory findings after treatment were: Widal TO 1:40, TH 1:80, Rose Bengal positive, Wright B. melitensis 1:160. WBC 7100, RBC 3990000, Hb 11.7g%, PLT 290000. Microscopic marrow aspiration didn’t show more leishmania parasites. Serologic tests after six months were: Widal TO=0, TH 1:40, Wright 1:40. One year latter serology for leishmania and Wright were negative and Widal TO=0, TH 1:40. Conclusion: in developing countries, co-infection possibilities are to be checked in fever of unknown origin. Author’s aim is to present a rare case with bacterial and parasite co-infection in an immunocompetent subject. A 24 years female was hospitalized in the Department of Infectious Diseases in UHC “Mother Teresa” in Tirana with fever of unknown origin. She lived in a rural zone in the north of Albania
and was treated in the regional hospital for Leishmaniasis resistant to antimony. She had four weeks history of high fever, headache, malaise, prostration and pain in articulations. Objective examination showed pallor and toxic face, enlargement of liver and spleen. She had no meningeal signs, rales or abnormal cardiac murmur. Laboratory findings were: RBC 3560000, Hb 11 g%, PLT 150000, WBC 2800, absolute lymphocytosis. X rays and cardiac sonography didn’t show any pathology. Culture of the blood or urine didn’t find any pathogen. Serology exams were: HIV negative, Leishmania positive, Rose Bengal positive, Wright 1:1280, Widal TO 1:160, TH 1:320. Microscopic examination of marrow aspiration showed leishmania amastigote. She started - CLINICAL LEISHMANIASIS [1282] P 296 - UNCOMMON MUCOSAL LEISHMANIASIS IN CHILD: CASE REPORT MEDEIROS, A.C.R.1; ROCHA, M.A.W.1; RODRIGUES, E.H.G.2; ALEXANDRE, J.L.A.2; ARAUJO, A.I.F.2; VIANA, J.B.M.2; SILVA, C.J.3; ALMEIDA, E.L.2; WERKHAUSER, R.P.2; BRANDÃOFILHO, S.P.2; ALMEIDA, A.P.2; BRITO, M.E.F.2 1.HOSPITAL UNIVERSITÁRIO OSWALDO CRUZ, RECIFE, PE, BRAZIL; 2.CENTRO DE PESQUISAS AGGEU MAGALHÃES, RECIFE, PE, BRAZIL; 3.NUCLEO DE VIGILÂNCIA EM SAÚDE E AMBIENTE, MORENO, PE, BRAZIL.
Keyword:mucosal leishmaniasis; diagnosis; pernambuco
Abstract: American cutaneous leishmaniasis (ACL) is an infectious parasitic disease that predominates in young adults, but in recent years there has been a change in the epidemiological profile of the disease with involvement of children and adolescents. In Pernambuco State, Brazil, the number of reported cases of ACL is increasing, especially in the Rain Forest area, which is responsible for 64.2% of total cases recorded in the whole State. A 6-years-old, male, coming from Pinto Sugar mill, a locality in the city of Moreno, metropolitan area of Recife, Pernambuco, which is an endemic area for ACL. He was admitted at the Clinic of Infectious Diseases, Hospital Oswaldo Cruz, with exulcerated crusted lesions on the lower and upper lip accompanied by significant edema, and increase during one month. Treatment with Acyclovir was implemented for eight days by clinical suspicion of herpes, but there was no improvement in clinical status. A new anamnesis was done and in the histopathologic exam from biopsy of the lesion were viewed Leishmania forms. Collections of cutaneous exudate were made by bacteriological swabs from both crusted lesions and salivary fluids in the region below the tongue and oral mucosa, and were processed by polimerase chain reaction with B1/B2 primes specific for the subgenus Viannia at the Serviço de Referência em Leishmanioses do Centro de Pesquisas Aggeu Magalhães - Fiocruz - PE. The results of PCR from swabs were consistent with ACL. According to the researcher responsible for the study area, the child was treated in 2010 by present at that moment two cutaneous lesions undergoing the left foot, with regression of lesions. The treatment was implemented with N-methyl glucamine 20mg/kg/day. After eight days there was a reduction of edema and bilateral lesion regression. The diagnosis of ACL has been associated with clinical, epidemiological and laboratory aspects. Although the mucosal form is less common in children, the diagnosis should be recommended when there is origin from an endemic area. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1288] P 297 - EVALUATION OF PARASITIC LOAD AND PROFILE OF TH1 AND TH2 CYTOKINES IN MICE INFECTED WITH LEISHMANIA BRAZILIENSIS AND
SUBMITTED TO PDT-ALA MEDIATED ALVES, P.M.1; SILVA, M.L.F.2; SOUZA, D.M.2; CARLOS, S.M.1; SILVA, M.V.2; RODRIGUES, D.B.1; PAULINO, T.P.1; RODRIGUES, V.2 1.UNIVERSIDADE FEDERAL DO TRIÂNGULO MINEIRO / CEFORES, UBERABA, MG, BRAZIL; 2.UNIVERSIDADE FEDERAL DO TRIÂNGULO MINEIRO / DPTO CIÊNCIAS BIOLÓGICAS / LABORATÓRIO DE IMUNOLOGIA, UBERABA, MG, BRAZIL.
Keyword:leishmania braziliensis; photodynamic therapy; cytokines
Abstract: Leishmaniasis is a parasitic disease caused by protozoan flagellates that belong to the leishmania genus. Cutaneous diseases range from mild skin ulcers to severe mucocutaneous destruction. The outcome of infection depends mainly on the strain of Leishmania and on the host immunological response. Leishmanias develop inside the macrophage and IFN-gamma and others Th1 cytokines are essential in protecting against all Leishmania infections due to NO production. Conversely, the induction of a Th2 cytokine response is associated with susceptibility to infection. Photodynamic Therapy (PDT) utilizes an otherwise nontoxic chemical to create oxidative damage in cells when exposed to light. In this study, we analyzed the local and systemic expression of Th1 and Th2 cytokines in BALB/c mice experimentally infected with L. braziliensis after PDT-ALA mediated (Aminolevulinic Acid). Skin foot pad and spleens from infected animal, submitted or not to 1, 2 or 3 PDT sections were homogenized (Ultra-Turrax®) in PBS buffer containing surfactant and a protease inhibitor. Homogenates were centrifuged and supernatants were used for cytokines titration by CBA (Cytometric Beads Array). Cytokines levels are expressed in pg/mL of total tissue weight. Parasite burden were evaluated in skin imprints. We observed that IFN-gamma and IL-4 levels were higher in skin lesions than in spleens whereas levels of TNF-alpha, IL-2 and IL-5 were similar. Also, after a single PDT an increase of IL-4 levels and a decrease of IFN levels were observed only on skin samples. After the second and the third sections, levels of these cytokines were restored. Levels of TNF-alpha, IL-2 and IL-5 were not affected by PDT in any therapeutical approach used in this study. Moreover the parasites number decreased after a first PDT section and maintaining reduced during consecutive sections (about 4 times). This data suggested that a single PDT section improve Th2 cytokines and decrease Th1 cytokines, probably due to a direct effect on T lymphocyte or an indirect effect on Leishmania lyses, mediated by reactive species (oxigen and/or nitrogen) yielded after therapy. Supported by FAPEMIG, CNPq and Cefores. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1294] P 298 - INCREASE OF CD56BRIGHT NATURAL KILLER CELLS IN PATIENTS WITH LOCALIZED CUTANEOUS LEISHMANIASIS: A ROLE IN THE INDUCTION OF PROTECTIVE TH1 RESPONSE? CABRERA, M.1; CUENCA, M.2; RODRÍQUEZ, O.L.2; LUGO, D.3; ZERPA, O.2; SCOTT-ALGARA, D.4 1.INSTITUTO DE BIOMEDICINA, UNIVERSIDAD CENTRAL DE VENEZUELA, CARACAS, VENEZUELA; 2.INSTITUTO DE BIOMEDICINA, UNIVERSIDAD CENTRAL DE VENEZUELA, MPPS, CARACAS, VENEZUELA; 3.INSTITUTO DE BIOMEDICINA, UNIVERSIDAD CENTRAL DE VENEZUELA/MPPS, CARACAS, VENEZUELA; 4.LABORATORY OF RETROVIRAL INFECTIONS RÉGULATION DES INSTITUT PASTEUR, PARIS, FRANCE.
Keyword:nk cells; nk receptors; human cutaneous leishmaniasis
Abstract: Natural killer cells (NK) are an important component in early resistance against intracellular pathogens such as Leishmania spp. They are cytotoxic and potent cytokine producer, particularly interferon- (IFN- ) and tumor necrosis factor- (TNF- ), allowing the induction of Th1-like response, important in the resolution of leishmaniasis. NK cells can be subdivided into two subsets: CD56bright NK cells and CD56dim NK cells. Both populations define NK functions, which are governed by a fine tuned balance of signaling from inhibitory and activating receptors
expressed on these cells. To date, these aspects have not been investigated in human cutaneous leishmaniasis. In this study we evaluated peripheral NK cell subpopulations associated with different clinical forms of American cutaneous Leishmaniasis (ACL). Ten localized cutaneous leishmaniasis patients (LCL), ten mucocutaneous (MCL), four intermediate or cutaneous chronic leishmaniasis (ICL) and fifteen healthy volunteers (controls) were studied. NK cells phenotypes (CD56+CD16+, CD56bright, CD56dim,NKp80,NKp46, NKG2A, NKG2D, CD69) and NKT cells were assessed in peripheral blood by flow cytometry. LCL patients showed a decreased percentage of NKT cells compared to ICL patients and healthy volunteers (p = 0.02). Interestingly, the CD56bright NK cells were significantly increased in patients with LCL compared to the other groups (p <0.001). In contrast, patients with intermediate forms of Leishmaniasis (MCL and ICL) showed an increase in CD56dim NK cells (p <0.001) than LCL patients and controls. A higher percentage of CD56bright NK cells activated (CD69) was observed in LCL and ICL patients compared to controls and MCL patients. (p <0.001). Additionally, CD56dim NK cells have shown a higher expression of CD69 in LCL patients compared to the control group (p = 0.018). Finally, it was observed an increased percentage of CD56 bright NK cells expressing NKp46 (associated with cytotoxicity) in LCL patients compared to other groups (p <0.0001). Overall results suggest that CD56bright NK subpopulation is an important cellular effector in the localized form of ACL. Indeed, these cells might play a role in the induction of the adaptive immune response, particularly in LCL and ICL patients promoting Th1 responses. Supported by UCV CDCH-PG-09-7429-2008/2 and BNP Paribas France.
- CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1297] P 299 - IL-23 AND IL-27 LEVELS IN MACROPHAGES COLLECTED FROM PERIPHERAL BLOOD OF PATIENTS WITH HEALING VS NON-HEALING FORM OF CUTANEOUS LEISHMANIASIS HEJAZI, S.H.H.1; TOLOUEI, S.1; KHAMESIPOUR, A.2; GHAEDI, K.3; HASHEMINIA, S.J.4; JAVADI, A.5 1.DEPARTMENT OF PARASITOLOGY AND MYCOLOGY, SCHOOL OF MEDICINE, ISFAHAN UNIVERSITY OF MEDICAL SCIENCES, ISFAHAN, IRAN, ISLAMIC REPUBLIC OF; 2.CENTER FOR RESEARCH AND TRAINING IN SKIN DISEASES AND LEPROSY, TEHRAN UNIVERSITY OF MEDICAL SCIENCES, TEHRAN, IRAN, ISLAMIC REPUBLIC OF; 3.DEPARTMENT OF BIOLOGY, SCHOOL OF SCIENCES, UNIVERSITY OF ISFAHAN, ISFAHAN, IRAN, ISLAMIC REPUBLIC OF; 4.DEPARTMENT OF IMMUNOLOGY, SCHOOL OF MEDICINE, ISFAHAN UNIVERSITY OF MEDICAL SCIENCES, ISFAHAN, IRAN, ISLAMIC REPUBLIC OF; 5.DEPARTMENT OF SOCIAL SCIENCES, SCHOOL OF MEDICINE, QAZVIN UNIVERSITY OF MEDICAL SCIENCES, QAZVIN, IRAN, ISLAMIC REPUBLIC OF.
Keyword:il-23, il-27; macrophages; cutaneous leishmaniasis
Abstract: In this study the level of IL-23 and IL-27 produced by macrophages derived from peripheral blood mononuclear cell culture collected from patients with healing or non-healing form of cutaneous leishmaniasis lesion were compared before and after treatment with live Leishmania to explore whether IL-23 or IL-27 plays any role in healing process of cutaneous lesions induced by L. major. Twenty patients resident in Isfahan Province, with healing or non-healing form of cutaneous leishmaniasis lesion caused by Leishmania major participated in this study. In vitro productions of IL-23 and IL-27 by peripheral blood derived macrophages, before and after stimulation with live L. major (MRHO/IR/75/ER) promastigotes were evaluated using ELISA method. Patient with healing form of lesion received no treatment and patient with non-healing form of lesion received at least 2 courses of glucantime.The mean production of IL-23 and IL-27 from macrophages of patients with healing form of lesion was significantly higher than patients with
non-healing form of lesion. The levels of IL-23 and IL-27 in culture supernatants before and after stimulation in healing form of CL was significantly higher than non- healing form of CL (P < 0.001).It seems that IL-23 and IL-27 might play a role in human leishmaniasis and further studies are needed to understand the role of IL-23 and IL-27 in leishmaniasis. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1299] P 300 - TLR2 AND TLR4 IN CUTANEOUS LEISHMANIASIS CAUSED BY L. MAJOR TOLOUEI, S.1; HEJAZI, S.H.1; KHAMESIPOUR, A.2; GHAEDI, K.3; HASHEMINIA, S.J.4; JAVADI, A.5 1.DEPARTMENT OF PARASITOLOGY AND MYCOLOGY, SCHOOL OF MEDICINE, ISFAHAN UNIVERSITY OF MEDICAL SCIENCES, ISFAHAN, IRAN, ISLAMIC REPUBLIC OF; 2.CENTER FOR RESEARCH AND TRAINING IN SKIN DISEASES AND LEPROSY, TEHRAN UNIVERSITY OF MEDICAL SCIENCES, TEHRAN, IRAN, ISLAMIC REPUBLIC OF; 3.DEPARTMENT OF BIOLOGY, SCHOOL OF SCIENCES, UNIVERSITY OF ISFAHAN, ISFAHAN, IRAN, ISLAMIC REPUBLIC OF; 4.DEPARTMENT OF IMMUNOLOGY, SCHOOL OF MEDICINE, ISFAHAN UNIVERSITY OF MEDICAL SCIENCES, ISFAHAN, IRAN, ISLAMIC REPUBLIC OF; 5.DEPARTMENT OF SOCIAL SCIENCES, SCHOOL OF MEDICINE, QAZVIN UNIVERSITY OF MEDICAL SCIENCES, QAZVIN, IRAN, ISLAMIC REPUBLIC OF.
Keyword:tlrs; cutaneous leishmaniasis; l.major
Abstract: Cutaneous leishmaniasis (CL) is a self-healing skin disease; however rarely for unknown reason(s) the lesion develops to a non-healing form. It seems that the initial contact of Leishmania parasites with the host innate immune system is an important step in the outcome of the disease. Recent studies suggested that TLRs play a role in Leishmania recognition. In the present study the level of TLR2 and TLR4 were checked in patients with healing form of lesion and compared with that of patients with non-healing form of lesion caused by L. major infections. Gene expression of TLR2, TLR4 in peripheral blood derived macrophages, before and after stimulation with live L.major (MRHO/IR/75/ER) promastigotes were evaluated using quantitative real-time reverse transcription PCR and flow cytometry. The results showed that mean relative gene expression and difference membrane expression of TLR2 in macrophages of patients with healing form of lesion was significantly higher than patients with non-healing form of lesion (p<0.0001 and p=0.0034), respectively. The results also showed that mean relative gene expression and difference of protein expression of TLR4 in macrophages of patients with healing form of lesion was significantly higher than that of patients with non-healing form of lesion (p=0.021 and p=0.002).Our data indicated a possible role for TLR2 and TLR4 in the outcome of the lesion. Further studies are needed to understand more about the role of these immune factors in leishmaniasis. - VACCINE DEVELOPMENT AND TRIALS [1300] P 301 - LOW DOSES OF VIABLE LEISHMANIA MAJOR PLUS BCG AS A SPECIFIC IMMUNE STIMULATOR FOR IMMUNOTHERAPY OF CUTANEOUS LEISHMANIASIS ARJMAND, R.; HEJAZI, H.; SABERI, S.; KALANTARI, M.
PARASITOLOGY AND MYCOLOGY DEPARTMENT, SCHOOL OF MEDICINE, ISFAHAN UNIVERSITY OF MEDICAL SCIENCE, ISFAHAN, IRAN, ISLAMIC REPUBLIC OF.
Keyword:l. major, bcg; lst; immunotherapy
Abstract: This study aims to find out the efficacy of Bacillus Calmette-Guerin (BCG) bacterial system as a non specific cell immune stimulator in animal model of viable Leishmania major immunotherapy. In this study BCG was used as a cell immune stimulator with different doses of live L. major in Balb/c mice. L. major (MRHO/IR/75/ER) was cultured in N.N.N medium and produced in large scale using RPMI 1640. 8 groups of Balb/c mice were inoculated with
different doses of harvested L. major in stationary phase. For each dose two groups of ten mice were selected (One group received parasites with BCG, and the second received parasites without BCG). Leishmanin Skin Test (LST), clinical examinations and ulcer measurement were shown that immunoprophylaxis with low doses of parasite (120 and 1200 promastigote per mouse) with or without BCG could stimulate cell immunity to produce protection against lethal visceral leishmaniasis of Balb/c model. Moderate doses of parasite (12000 promastigote per mouse) produce partial protection. There was no protection with high doses of parasite (200000 promastigote per mouse).Using Low doses of live parasites plus BCG is suggested to apply in a human trial for prophylactic purposes. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1303] P 302 - CYTOKINES ASSOCIATED WITH TH17 RESPONSE IN SEVERE FORMS OF AMERICAN CUTANEOUS LEISHMANIASIS CABRERA, M.; LUGO, D.; CUENCA, M.; RODRĂ?GUEZ, O.L.; ZERPA, O. INSTITUTO DE BIOMEDICINA, UNIVERSIDAD CENTRAL DE VENEZUELA, MPPS, CARACAS, VENEZUELA.
Keyword:th17 cytokines; cutaneous leishmaniasis; human
Abstract: Th17 response is associated with the development of harmful autoimmune and inflammatory conditions and with protection against some infectious agents. Th17 cells produce IL-17, IL-22 among other cytokines, being important the IL-23 in the development and maintenance of this response. Previous studies have shown that the Th17 response is involved in the pathogenesis of the inflammatory reaction in Leishmaniasis, particularly in mucocutaneous Leishmaniasis patients (MCL). However, other severe forms of cutaneous Leishmaniasis (intermediate, ICL and diffuse DCL) have not been evaluated. In order to investigate that, we determined Th17 cytokines (IL-17, IL-22 and IL-23) in different clinical forms of American cutaneous Leishmaniasis (ACL). A total of 41 ACL patients and 18 healthy volunteers were studied. All patients were diagnosed using established clinical, epidemiological and histopathological criteria as either localized cutaneous (LCL=16), intermediated or cutaneous chronic (ICL n=14), mucocutaneous (MCL=13) and diffuse cutaneous leishmaniasis (DCL=8). IL-17, IL-22 and IL23 levels were detected by ELISA in serum samples and in supernatants from peripheral blood mononuclear cells (PBMC) stimulated with PHA-M and crude antigens of L.(L) mexicana (MHOM/BZ/82/BEL21) and Leishmania (V) braziliensis, (MHOM/BR/75/M2903). High serum IL-17 and IL-23 levels were observed in samples from DCL patients (0.0035 in vitro stimulation with L (V) braziliensis antigen (39.9 pg/mL) than other groups. In contrast, to than observed for IL-22 in supernatants from PMNC cultures from the same patient group; increased levels of IL-22 was produced in response to L (L) mexicana (50.4 pg/mL) and L (V) braziliensis (41.5 pg/mL) (MCL vs LCL, ICL, DCL and controls, p<0.05). Taken together, these results suggest the presence of Th17 response in LCL patients that may contribute to the control of Leishmania spp infection. Additionally, at the severe manifestation of the disease such as MCL and DCL, the presence of IL17 with low production of IL-22 may suggest the contribution of other cellular groups in the pathology observed in these susceptible forms of ACL. Research founded by CDCH-UCV No. PG-09-7429-2008-2.
- GENOMICS, TRANSCRIPTOMICS, PROTEOMICS, METABOLOMICS [1304] P 303 - KNOWLEDGE MANAGEMENT FOR IN SILICO MAPPING OF METABOLIC TARGETS TO DRUG CONTROL OF LEISHMANIA BRASILIENSIS SILVA, L.A.; BEZERRA, C.B.; CRAVO, P. UNIVERSIDADE FEDERAL DE GOIĂ S, GOIĂ‚NIA, GO, BRAZIL.
Keyword:leishmaniasis; metabolism; bioinformatic
Abstract: Introduction Leishmaniasis is a serious public health problem affecting 12 million people around the world. Although some of the anti-leishmania drugs used are partly effective, drug resistance has evolved and become a persistent problem. Therefore, research efforts are being directed towards discovering new drugs with different mechanisms of action, low cost and high efficacy. To this purpose, new strategies have been adopted in drug research, such as the use of genomics, proteomics and metabolomics. In this study, we have carried out an in silico analysis of the Leishmania braziliensis energy metabolism in order to identify the genes and their respective predicted enzymes participating in glycolysis and in the Krebs cycle. We then searched drug databases for potential inhibitors of these enzymes. Materials and Methods Information about the genome of L. brasiliensis was obtained from the GeneDB TritrypDB databases. In GeneDB, each gene was first identified by the nomenclature of the enzyme. Once the identity of the gene was obtained, TritrypDB was then interrogated for the corresponding predicted peptide sequence. This sequence was then used to search for target similarity in TTD and DrugBank, two freely available databases that contain information about known targets and their matching validated drugs. Each of the enzymes of the energy metabolism was treated as a potential therapeutic target. All drugs with an output expectation value (E-value) lower than 1e5 were listed. The compiled data were then compared with the target-drug data of L. braziliensis available in the TDR Targets database, and all data were integrated in light of the knowledge available about metabolic targets. Results We identify drugs which are predicted to interact with 14 out of 36 (39%) enzymes of the L. braziliensis energy metabolism. Out of these 14 enzymes, 5 and 9 participate in glycolysis or the Krebs cycle, respectively. Most of the drugs (64%) against the 14 targets identified are already approved for treatment of bacterial diseases, nutritional deficiency, parasitic infections and cancers, among others. Additionally, some of the compounds have been tested against parasites of medical interest. Conclusion We use bioinformatics and knowledge management as tools to search and analyze information available in genome and drug databases. The large numbers of data retrieved and information mismatches between different databases have made the task intricate. Still, it was possible to identify many potential drugs against L. braziliensis that interact with specific enzymes of the energy metabolism. Most of these drugs are approved for clinical use in humans, but have never been tested against Leishmania parasites. We now pretend to carry out in vitro tests with some of these drugs, which may pave the way for their use alone or in combination with drugs currently used in the treatment of leishmaniasis.
- VACCINE DEVELOPMENT AND TRIALS [1317] P 304 - INTRANASAL IMMUNIZATION WITH LACK-DNA PROMOTES PROTECTIVE IMMUNITY IN HAMSTERS CHALLENGED WITH LEISHMANIA CHAGASI GOMES, D.1; COSTA, B.L.2; GUEDES, H.L.2; LOPES, U.G.2; BERGMANN, B.R.2
1.UNIVERSIDADE FEDERAL DO ESPÍRITO SANTO, VITÓRIA, ES, BRAZIL; 2.UNIVERSIDADE FEDERAL DO RIO DE JANEIRO, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:leishmania chagasi; hamster; mucosal vaccine
Abstract: LACK(Leishmania analogue of the receptor kinase C) is a conserved protein in protozoans of the genus Leishmania which is associated with the immunopathogenesis and susceptibility of BALB/c mice to L. major infection. Previously, we demonstrated that intranasal immunization with a plasmid carrying the LACKgene of Leishmania infantum (LACK-DNA) promotes protective immunity in BALB/c mice against Leishmania amazonensis and Leishmania chagasi. In the present study, we investigated the protective immunity achieved in hamsters intranasally vaccinated with 2 doses of LACK-DNA (30 g). Compared with controls (PBS and pCI-neo plasmid), animals vaccinated with LACKDNA showed significant reduction in parasite loads in the spleen and liver, increased lymphoproliferative response and increased nitric oxide (NO) production by parasite antigen-stimulated splenocytes. Furthermore, hamsters vaccinated with LACK-DNA presented high IgG and IgG2a serum levels when compared to control animals. Our results showed that intranasal vaccination with LACK-DNA promotes protective immune responses in hamsters and demonstrated the broad spectrum of intranasal LACK-DNA efficacy in different host species, confirming previous results in murine cutaneous and visceral leishmaniasis. - GENETICS, EVOLUTION AND TAXONOMY [1326] P 305 - GENETICS AND EVOLUTION OF LEISHMANIA MAJOR STRAINS TWENTY YEARS LATER IN THE CENTER OF TUNISIA: INFERENCE FROM MICROSATELLITE DATA HARRABI, M.1; BETTAIEB, J.1; TOUMI, A.1; CHLIF, S.1; BAÑULS, A.L.2; SALAH, A.B.1 1.DEPARTMENT OF MEDICAL EPIDEMIOLOGY- (LR 11 IPT 02). INSTITUT PASTEUR, TUNIS, TUNISIA; 2.UMR MIVEGEC (IRD 224-CNRS 5290-UNIVERSITÉS MONTPELLIER 1 ET 2), CENTRE IRD, MONTPELLIER, FRANCE.
Keyword:microsatellite; leishmania major; genetics and evolution
Abstract: In Tunisia, the transmission of Zoonotic Cutaneous Leishmaniasis, caused by Leishmania (L.) major appeared for the first time in Kairouan in 1982 and spread to Sidi Bouzid where it emerged as an epidemics in1991. Since then, the disease is maintained in these areas and expanded to other governorates in the center and the south. To assess the evolution of the strains over twenty years, ten microsatellites were used to characterize 108 strains of L. major collected from patients in Kairouan and Sidi Bouzid: 35 Old Strains (Population 1 isolated between 1991 and 1992) and 73 new strains (Population 2 isolated between 2009 and 2011). A total of 35 alleles were detected. In historical population, two of the microsatellites were monomorphic, revealing 26 genotypes out of 35 strains, whereas, all the ten microsatellites were all polymorphic in contemporary samples exhibiting eleven more genotypes out of 73 strains. This shows that both genotypic (0.74 versus 0.5) and genetic diversity (0.28 versus 0.12) were much higher in the old samples. Observed heterozygosity ranged from zero to 0.4, which was considerably lower than the mean expected. Inbreeding coefficients Fis ranged from 0.514 in old strains to 0.799 in new samples indicating a degree of homozygosity more important in the recent populations. The phenetic tree revealed no strict partition between the two populations with the presence of overlapping clusters. Nevertheless, we obtained a moderate but significant genetic differentiation (Fst= 0.1672, p-value= 0.050). These results suggest the existence of high genetic connectivity over time. Microsatellite data showed that the turnover of the L. major population after 20 years since the emergence of the first epidemics led to some changes in genetic patterns. More specifically, we observed a decrease in genotypic and genetic diversity. This can be due to a better management of the disease in humans and thus an elimination of a part of the Leishmania population. Nevertheless, the low but significant genetic differentiation suggests a low turnover of the L. major population and a circulation of the parasites at a low level. The inclusion of strains isolated from vectors and reservoirs but also the seroprevalence study in the healthy inhabitants could give precious information in order to better understand the L. major transmission.
- GENETICS, EVOLUTION AND TAXONOMY [1327] P 306 - LEISHMANIA MAJOR IN TUNISIA: GENETIC STRUCTURE OF POPULATIONS ACCORDING TO THE GEOGRAPHICAL, ECOLOGICAL AND TEMPORAL CHARACTERISTICS. HARRABI, M.1; BETTAIEB, J.1; TOUMI, A.1; CHLIF, S.1; BAÑULS, A.L.2; SALAH, A.B.1 1.DEPARTMENT OF MEDICAL EPIDEMIOLOGY- (LR 11 IPT 02). INSTITUT PASTEUR, TUNIS, TUNISIA; 2.UMR MIVEGEC (IRD 224-CNRS 5290-UNIVERSITÉS MONTPELLIER 1 ET 2), CENTRE IRD, MONTPELLIER, FRANCE.
Keyword:leishmania major; tunisia; genetic structure
Abstract: The molecular epidemiology studies show that L. major is spreading from the South West to new areas in the center of Tunisia. This study had for objectives to investigate the population dynamics of L. major in order to explain how the parasites are organized according to the geographical distribution, the ecology and the chronological emergence of Leishmaniasis in humans. We have thus studied the population structure of 138 L. major human cases isolated from different endemic areas in the South West and in the Center of Tunisia. Genetic polymorphism was studied using 10 microsatellite markers. We estimated different genetic variability indices using the computer program FSTAT to characterize the population. Overall, inbreeding coefficient (Fis) was high in all the populations suggesting a deficit in
heterozygosity. As expected, observed heterozygosity among microsatellites was substantially low compared with the expected value. Our results support the recent postulate assuming that these parasites follow a mix-mating model. The genetic differentiation index, Fst showed a slight and marginal significant genetic differentiation between south and center populations, with higher allelic richness in the south. These results are in agreement with the chronology of Leishmaniasis emergence in humans (oldest in the south). Nevertheless, the important migration rate suggests the frequent or recent circulation of parasites between these populations and thus explains the low genetic differentiation (Fst). The different ecosystems do not seem to influence the population structure of L. major. The analyses, focused on two areas in the center with a different history of Leishmaniasis emergence in humans, showed that genetic diversity was faintly higher in the emerging focus even though the genetic differentiation was almost absent. This suggests a similar temporal origin of the two foci, but an emergence or detection in humans at different time. Nevertheless, to assess undoubtedly the population structure of L. major in Tunisia, it would be essential to perform a global genetic study integrating the sympatric strains of all the compartments of the cycle, i.e. strains from vectors and reservoirs. - GENETICS, EVOLUTION AND TAXONOMY [1336] P 307 - GENETIC ASSOCIATION OF INTERLEUKIN-1 (-511C/T) WITH SUSCEPTIBILITY TO CUTANEOUS LEISHMANIASIS CAUSED BY LEISHMANIA GUYANENSIS IN THE AMAZONAS STATE OF BRAZIL SABINO, K.C.1; SOUZA, L.M.1; SOUSA, P.B.1; TALHARI, A.C.1; GUERRA, J.A.1; TALHARI, S.2; RAMASAWMY, R.3
1.FUNDAÇÃO DE MEDICINA TROPICAL DR HEITOR VIEIRA DOURADO, MANAUS, AM, BRAZIL; 2.UNIVERSIDADE NILTON LINS, MANAUS, AM, BRAZIL; 3.FUNDAÇÃO DE MEDICINA TROPICAL DR HEITOR VIEIRA DOURADO; UNIVERSIDADE NILTON LINS, MANAUS, AM, BRAZIL.
Keyword:l. guyanensis; susceptibility; interleukin1
Abstract: Mostly Leishmania guyanensis cause cutaneous (CL) and mucosal leishmaniasis (ML) in the Amazonas state. It is not understood why only a proportion of individuals infected with L. guyanensis develop CL or ML. Notably, the development of CL or ML in only a proportion of individuals infected with L. guyanensis and the tendency for its familial aggregation suggest that genetic factors may be involved in such susceptibility. High levels of proinflammatory cytokines are observed in patients with CL or ML. IL-1 is a proinflammatory cytokine playing important role in immunity against Leishmania infection and the outcome of the disease and present functional polymorphisms in the promoter region of its gene IL-1B. Methods. We assessed, by polymerase chain reaction restriction fragment–length polymorphism analysis, IL-1B variant in the promoter region at position -511C/T (rs16944) in 150 cases of CL and 150 unrelated controls from the Amazonas state of Brazil. Chi-square analysis was performed to compare distribution of genotypes and alleles frequencies between CL and controls. Results. Case control analysis showed a trend of association between the C allele (OR 1.36; 95% CI 0.93-2.00; P = 0.031) and CL. Distribution of the genotypes were in Hardy-Weinberg equilibrium in both the CL and control groups. The distribution of genotypes frequencies among the CL group and the control group was (TT 29%; CT 50%; CC 21%) and (TT 37%; CT 50%; CC 13%) respectively. The CC genotype showed an increased frequency among the CL patients versus the controls (OR 1.96; 95% CI 0.93-3.93; P = 0.027). Conclusion. The functional variant in the promoter region of IL-1B in our study seems preliminarily to be a genetic factor contributing to the susceptibility to CL. However, further
studies are needed with greater sample size to confirm whether this variant is associated with susceptibility to CL. Acknowledgement: This study was financed by FAPEAM and CNPq.
- GENETICS, EVOLUTION AND TAXONOMY [1338] P 308 - HIGH RESOLUTION MULTILOCUS MICROSATELLITE TYPING (MLMT) AND MULTILOCUS SEQUENCE TYPING (MLST) OF NATURAL ETHIOPAN LEISHMANIA DONOVANI ISOLATES REVEAL PARENTAL DONORS AND GENETIC HYBRIDS, ORIGINATING FROM AT LEAST TWO INDEPENDENT HYBRIDISATION EVENTS. YEO, M.1; GELANEW, T.2; HAILU, A.2; MILES, M.A.1 1.LSHTM, LONDON, UNITED KINGDOM; 2.ADDIS ABABA UNIVERSITY, ADDIS ABABA, ETHIOPIA.
Keyword:multilocus; microsatellite; mlst
Abstract: Protozoan parasites of the genus Leishmania (Kinetoplastida:Trypanosomatidae) cause widespread and devastating human diseases. Visceral leishmaniasis is endemic in Ethiopia where it has also been responsible for fatal epidemics. It is postulated that genetic exchange in Leishmania has implications for heterosis, spread of virulent strains, resistance to chemotherapeutics, and exploitation of different hosts and vectors. In the current work over 90 biological clones of Leishmania donovani derived from human isolates from Ethiopia were characterised by high resolution Multilocus Microsatellite typing (MLMT) and Multilocus Sequence typing (MLST). Results showed that four isolates (and associated biological clones) are clearly genetic hybrids, each possessing allelic profiles from two parental donors in the Ethiopian panel. Outputs from both MLMT and MLST were congruent. Parental alleles situated on different chromosomes were inherited by the hybrid progeny. Differential allelic profiles of the hybrids suggest that a minimum of two separate recombination events have occurred among the panel of strains studied. FACS analysis demonstrated that all hybrids and associated clones were minimally diploid. Mitochondrial sequences show uniparental inheritance for all hybrids, each possessing a single mitochondrial sequence type. Two of the four hybrid isolates were from patients co-infected with HIV. Together the data suggest a meiotic mode of hybridisation with substantial phyloepidemiological implications. - CELLULAR AND MOLECULAR BIOLOGY [1344] P 309 - HISTOPATHOLOGCAL AND IMMUNOLOGICAL STUDIES OF THE GASTROINTESTINAL TRACT OF DOGS NATURALLY INFECTED WITH LEISHMANIA (LEISHMANIA) INFANTUM CHAGASI TAFURI, W.; FIGUEIREDO, M.; AMORIM, I.; JUNIO, A.; PINHEIRO, L.; BARBOSA, V.; SILVA, S.; DEOTI, B.; FARIA, A. UFMG, BELO HORIZONTE, MG, BRAZIL.
Keyword:canine visceral leishmaniasis ; gastrointestinal tract ; immunohistopathology
Abstract: The aim of this work was to better characterize the involvment of the gastrointestinal tract (GIT) in canine visceral leishmaniasis using histological, parasitological and immunological studies associated to the clinical status of the animals. Forty-four mongrel dogs naturally infected with Leishmania (Leishmania) infantum chagasi and obtained from the Control Zoonosis Center of
the Municipality of Ribeir達o das Neves, Belo Horizonte Metropolitan area, Minas Gerais (MG) state, Brazil, were analyzed. Other six uninfected dogs were used as controls. After necropsy, gross analysis was done without noticing any lesions or ulcers. Under histological analysis, all infected dogs showed an increased number of cells (mainly macrophages, plasma cells and lymphocytes) in lamina propria (LP) expect in esophagus in comparison to controls. Besides parasite distribution in the GIT was evident in all intestinal segments irrespective of the clinical status of the dogs, but the parasite load was statistically high in the caecum and colon. Some cellular antigens were analyses from cells obtained from LP of jejune and colon. In general, all studied receptors are high expressed in infected animals. We observed that expression of TLR2+ and CD11c+ as well as CD14+ Mannose+ was more frequent in the colon compared to the jejune. Rather, TLR9+ was more frequent in the jejunum compared to the colon. T CD4+ e TCD4+/FOXP3+ was more frequent in jejune than colon. Infected animals revealed increase CD4+ and Foxp3+ and decrease CD8+ frequencies. MFI (mean fluorescence intensity) was also carried out in cells of LP. There was difference between TLR9+, CD4+, CD8+, CD4+, FOXP3+ and CD11b+ whereas MFI was more intense in jejune. In contrast CD14+ and TLR2+ expression were more intense in colon. CD11c+ and Mannose+ did not present any MFI difference between jejune and colon. These results show that infected dogs have more CD4+ and Foxp3+, and less CD8+ antigen expression than uninfected dogs. We also found IL-10 and TNF- in tissue where they are more intense in jejune than colon. Moreover, the cytokine IL-4 were mainly found in colon. Thus, the TGI segment with lower parasite load showed high frequency and expression of receptors CD4+, Foxp3+, CD11b+, TLR9+, besides the presence of IL-10 and TNF- . In contrast, the TGI segment with higher parasite load showed also concomitant high frequency of CD14, TLR2, CD11c and IL-4. We have concluded that in CVL there is a high Foxp3+ in the LP of the jejune. Probably the normal intestinal mucosa has an IL-10 dependent immunological mechanism where Foxp3+ cells are able to produce this IL-10. As Foxp3+ was also found in colon in normal conditions it suggests a local role of these cells in the intestinal homeostasis. - CELLULAR AND MOLECULAR BIOLOGY [1345] P 310 - A POTENTIAL LINK BETWEEN, TRACE ELEMENTS, ANTIOXIDANT ENZYMES AND HISTOPATHOLOGY IN CANINE VISCERAL LEISHMANIASIS TAFURI, W.1; SOUZA, C.1; BARRETO, T.1; MADEIRA, I.1; AMORIM, I.1; JUNIO, A.1; CRUZ, J.1; ROCHA, O.2; CANGUSSU, S.3 1.UFMG, BELO HORIZONTE, MG, BRAZIL; 2.CETEC, BELO HORIZONTE, MG, BRAZIL; 3.UFOP, BELO HORIZONTE, MG, BRAZIL.
Keyword:canine visceral leishmanaisis ; trace elements ; histopathology
Abstract: Dogs are the main domestic reservoirs of Leishmania infantum in canine visceral leishmaniosis. Thirty mixed-breed dogs categorized as naturally infected with Leishmania infantum, symptomatic (n = 19) and asymptomatic (n = 11) were used. Nine uninfected controls were also included in the study. Serum levels of trace elements (copper, iron, zinc, selenium) and nitric oxide were measured. Blood and tissue levels of antioxidant enzymes of Cu-Zn superoxide dismutase, catalase and glutathione peroxidase were also measured. Otherwise, lipid peroxidation was measured in plasma. Histological and morphometric analyses of lesions were done in liver, spleen and lymph nodes. Our results showed alterations in all infected dogs, mainly in the symptomatic. Iron, zinc, selenium, catalase, glutathione peroxidase and oxide nitric were lower than controls. In contrast, cupper, superoxide dismutase and lipid peroxidation were higher than controls. A chronic inflammatory reaction, congestion, hemosiderin pigment deposition (iron deposition) and fibrosis was observed in all studied organs of all infected dogs, with symptomatic ones presenting severe lesions associated with a high parasite load. In parallel, a lower tissue antioxidant enzymes activity was also observed in infected dogs than controls. Tissue alterations as inflammation, hemosiderin pigment deposition and fibrosis were
negatively correlated with lower tissue antioxidant enzymes activity and positively correlated with iron tissue deposition. We confirmed oxidative stress in tissues of CVL, indicated by enhanced lipid peroxidation and altered status of trace elements. We have shown evidences that the enhanced of LPO, Fe tissue deposition and decrease antioxidant enzymes activity are linked to tissue damage and may provide an explanation for severe tissue lesions during the evolution of canine visceral. - CELLULAR AND MOLECULAR BIOLOGY [1346] P 311 - CANINE VISCERAL LEISHMANIASIS AS A FIBROTIC DISEASE TAFURI, W.; SILVA, L.; CASTRO, R.; FIGUEIREDO, M.; MICHALICK, M.; TAFURI, W. UFMG, BELO HORIZONTE, MG, BRAZIL.
Keyword:canine visceral leishmanaisis ; fibrosis ; histopathology
Abstract: We have proposed that canine visceral leishmaniasis (CVL) is a fibrotic disease, based on a systemic fibrotic picture found in the chronic condition in which the inflammatory cells apparently direct fibrosis formation. Twenty-four cases (symp- tomatic dogs) were selective from a total of one hundred and five cases naturally infected with Leishmania chagasi identified during an epidemiological survey of CVL carried out by the metropolitan area of the municipality of Belo Horizonte, MG, Brazil. The histological criterion was intralobular liver fibrosis as previously described in dogs with visceral leishmaniasis. In addition to liver, we describe and quantify a conspicuous and systemic deposition of collagen in other organs, including spleen, cervical lymph nodes, lung and kidney of all the infected symptomatic dogs. We have found a systematic fibrotic picture where inflammatory cells appear to direct fibrosis in all studied organs. We propose CVL as a fibrotic disease. - CELLULAR AND MOLECULAR BIOLOGY [1352] P 312 - ALTERED EXPRESSION OF RBP-ASSOCIATED ARGININE METHYLTRANSFERASE 7 IN LEISHMANIA MAJOR AFFECTS PARASITE INFECTIVITY FERREIRA, T.R.1; ALVES FERREIRA, E.V.C.1; PAPADOPOULOU, B.2; CRUZ, A.K.1 1.FACULDADE DE MEDICINA DE RIBEIRÃO PRETO - USP, RIBEIRÃO PRETO, SP, BRAZIL; 2.CENTRE DE RECHERCHE EN INFECTIOLOGIE - CRICHUL, QUÉBEC, CANADA.
Keyword:methyltransferase; virulence; rbp
Abstract: Protein arginine methylation is a post-translational modification conserved across several eukaryotic taxa. It is widely known to modulate protein-protein and protein-RNA interactions via methylase activity of enzymes termed protein arginine methyltransferases (PRMTs) on targeted arginine residues. Trypanosomatids are ancient eukaryotes that carry five PRMT homologs in their genomes, including a PRMT7 homolog, not found in any other unicellular eukaryote genome. However their functional potential in these parasitic protozoa are still largely under-explored. In this study, we have generated Leishmania major transfectants with completely disrupted or increased LmjPRMT7 expression from wild-type levels. Based on in vitro macrophage and in vivo mice infections we concluded that LmjPRMT7 expression restrains parasite virulence within mammalian host. The knockout mutant displayed an increased infectivity of macrophages while the overexpressor transfectant barely induced any lesion in susceptible mice. On the other hand, neither procyclic promastigotes nor metacyclogenesis seemed to be affected by the altered levels of PRMT7 expression. Interestingly, wild-type LmjPRMT7 expression levels peak at early-logarithmic stage and
decreases as the growth curve progresses into late-log and late-stationary phases. Furthermore, we identified putative LmjPRMT7 substrates and interacting partners by immunoprecipitation experiments using a HA-tagged version of this protein. This analysis revealed at least eleven RNA-binding proteins (RBPs) associated to this methyltransferase that may act as posttranscriptional regulators in the parasite. Since RBPs are well-known mammalian PRMT substrates, we believe that interactions established between the identified proteins and their RNA targets may be modulated by arginine methylation, which still needs further investigation. This work consists of the first study to describe a role of a unicellular parasite PRMT7 homolog in parasite-host interaction. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1356] P 313 - THE INFECTION SITE OF LEISHMANIA MAJOR PARASITES INFLUENCES THE PERFORMANCE OF LEISH-111F INTRANASAL IMMUNIZATION MATSUMOTO, Y.1; ISOMURA, K.1; TSUNASHIMA, R.1; GEN, F.1; MATSUMOTO, Y.2; REED, S.G.3 1.LAB. GLOBAL ANIM. RES. SCI., GRAD. SCH. AGRICULT. LIFE SCI, THE UNIV. TOKYO, TOKYO, JAPAN; 2.LAB. MOL. IMMUNOL., GRAD. SCH. AGRICULT. LIFE SCI, THE UNIV. TOKYO, TOKYO, JAPAN; 3.IDRI, SEATTLE, WA, UNITED STATES.
Keyword:intranasal immunization; leish-111f; infection site
Abstract: Mucosal immunization is a pain-less alternative strategy for delivering vaccine antigens. Recently, we demonstrated that BALB/c mice intranasally immunized with Leish-111f plus cholera toxin (CT) adjuvant control lesion development in Leishmania major infection at tail base. However, lesion development may vary according to the site of infection as demonstrated in na誰ve mice infected with L. major. In this study, the protective effect of Leish-111f intranasal immunization was evaluated in L. major footpad infection model. BALB/c mice immunized intranasally with Leish-111f plus CT or non-immunized were infected with L. major (MHOM/UZ/91/PM2 or MHOM/IL/80/Friedlin) intradermally at tail base or subcutaneously at footpad. Disease progression was monitored by measuring lesion size weekly. Parasite burden in the skin lesion of the infected mice were measured at 2, 3, and 4 weeks post infection (wpi). Splenocytes were collected after infection and the recall productions of IFNand IL-4 were measured. In non-immunized mice infected at tail base or at footpad, progressive lesion development with the increase in parasite burden was observed from 1 wpi. In immunized mice infected at tail base, immunoreactive swelling without increase in parasite number was temporally observed at 1-5 wpi, however, the lesion development was completely controlled. In immunized mice infected at footpad, immunoreactive swelling was also observed from 1 wpi, which gradually become smaller in some mice. However, the rest of mice showed similar lesion development as that of non-immunized mice. Surprisingly, even the controlled footpads started to develop the lesion with the increase in parasite burden at 6 wpi or later. In immunized and footpad-infected mice, strong Th1-polarized cytokine production demonstrated in immunized and tail baseinfected mice were not clearly observed, suggesting non-healing Th2-type immunoreactions were evoked and overcame the Th1-type reaction. Although under the experimental conditions, the impact of intranasal immunization seems to be different depending on the infection sites.
- CELLULAR AND MOLECULAR BIOLOGY [1361] P 314 - DIFFERENTIAL ROLE OF RAB5 ISOFORMS IN ENDOCYTOSIS IN LEISHMANIA RASTOGI, R.; MUKHOPADHYAY, A. NATIONAL INSTITUTE OF IMMUNOLOGY, NEW DELHI, INDIA.
Keyword:rab5; endocytosis; isoforms
Abstract: The early endocytic compartment is also known as the sorting compartment, however, the mechanism of sorting of endocytic ligands is not well defined. Interestingly, three isoforms of Rab5 are present on the early endosome in mammalian cells. It is worthwhile to determine the role of Rab5 isoforms in the sorting of endocytic ligands. In the present study, we have elucidated the functions of different isoforms of Rab5 in the various modes of endocytosis in Leishmania. Genome analysis reveals that Leishmania has two isoforms of Rab5. Subsequently, we have identified, cloned and characterized putative Rab5a and Rab5b like sequences from L. donovani. To determine the function of Rab5 isoforms in various modes of endocytosis in Leishmania, we have overexpressed these proteins in promastigotes and follow the kinetics of receptor-mediated endocytosis of hemoglobin (Hb) (J. Biol. Chem. 1999; EMBO J. 2003; J. Biol. Chem. 2005; Proc. Natl. Acad. Sci.USA 2008) and fluid-phase uptake of horse-radish peroxidase (HRP) in the parasites. We have found that the overexpression of LdRab5b specifically stimulate the receptor-mediated endocytosis of 125I-Hb in Leishmania whereas no significant change in Hb endocytosis is observed with overexpression of LdRab5a. Furthermore, we have found that overexpression of dominant negative mutant of LdRab5b (LdRab5b:N133I) specifically inhibits Hb-endocytosis. This observation is further supported by the fact that overexpression of LdRab5b in the parasite induces the transport of internalized Alexa 594-Hb to the lysosomes with faster kinetics than control and LdRab5a overexpressed cells. In contrast, LdRab5a and its dominant active mutant (LdRab5a:Q93L) specifically upregulates fluid-phase uptake of HRP whereas no change in HRP uptake is detected in LdRab5b overexpressed cells. This observation is further confirmed by the fact that overexpression of LdRab5a:N146I, the dominant negative mutant of LdRab5a, significantly inhibits the uptake of HRP. In addition, we have also found that overexpression of LdRab5a specifically accelerates the transport of dextran-TexasRed, another fluid-phase endocytic marker, to the lysosomes in comparison to control and LdRab5b overexpressed cells. Taken together, our results demonstrate that LdRab5a specifically regulates fluid-phase endocytosis whereas LdRab5b is involved in the regulation of receptor-mediated endocytosis in Leishmania.
- CELLULAR AND MOLECULAR BIOLOGY [1362] P 315 - CLATHRIN-MEDIATED HEMOGLOBIN ENDOCYTOSIS IS ESSENTIAL FOR SURVIVAL OF LEISHMANIA GUPTA, D.; MUKHOPADHYAY, A. NATIONAL INSTITUTE OF IMMUNOLOGY, NEW DELHI, INDIA.
Keyword:clathrin; hemoglobin; endocytosis
Abstract: Leishmania is auxotroph for heme. Previously, we have shown that Leishmania acquire heme from the degradation of endocytosed hemoglobin (Hb) via a specific receptor located in the
flagellar pocket (J. Biol. Chem. 1999; J. Biol. Chem. 2005). We have also shown that early step of Hb endocytosis in Leishmania is regulated by Rab5 and transport of internalized Hb to the lysosomes is regulated by a Rab7 homologue (EMBO J. 2003;. Proc. Natl. Acad. Sci.USA 2008). Here, we report the cloning and expression of clathrin heavy chain from Leishmania (LdCHC) and provide evidences that Ld-CHC is localized in flagellar pocket and regulates Hbendocytosis in Leishmania. Kinetic analysis of Hb trafficking in GFP-Ld-CHC overexpressed Leishmania reveal that Hb is internalized through Ld-CHC coated region and remains associated with Ld-CHC containing vesicles at early time points of internalization and subsequently starts dissociating from Hb-containing vesicles at later time points indicating that clathrin-coating and uncoating regulates Hb trafficking in Leishmania. Interestingly, overexpression of dominant negative mutant of clathrin heavy chain of Leishmania (GFP-Ld-CHC-Hub) blocks the Hb internalization and causes severe growth defect in parasite. Moreover, we have shown that chlorpromazine, a pharmacological agent, blocks Hb internalization in Leishmania by depolymerizing Ld-CHC and thereby inhibits the growth of the parasites. Taken together, our results have shown that Hb endocytosis in Leishmania is a clathrin dependent process and is essential for the survival of the parasites. - CLINICAL LEISHMANIASIS [1364] P 316 - AN UNUSUAL CASE OF VISCERAL LEISHMANIASIS IN THE SMALL INTESTINE OF A DOG PINTO, A.J.W.; FIGUEIREDO, M.M.; DE AMORIM, I.F.G.; FERREIRA, R.A.; CALIARI, M.V.; TAFURI, W.L. UFMG, BELO HORIZONTE, MG, BRAZIL.
Keyword:canine visceral leishmaniasis; gastro-intestinal tract; histopathology
Abstract: Canine visceral leishmaniasis (CVL) is a severe zoonotic disease caused by an obligate intracellular protozoan Leishmania (Leishmania) infantum chagasi transmitted by sandflies (Phlebotomus sp.). Gastro-intestinal tract (GIT) disorders occur in human and dogs infection with Leishmania sp. Some studies described a chronic inflammatory exudate throughout the mucosa (lamina propria) of the all GIT segments in a dog naturally infected with L. infantum chagasi. Some woks with dogs naturally infected with L. infantum chagasi, have identified amastigotes of Leishmania inside macrophages of the lamina propria of all GIT segments. These authors reported that parasites were present in all intestinal segments and layers of the intestinal wall (mucosa, muscularis, and submucosa) irrespective of clinical status. However, the parasite load was significantly higher in the cecum and colon than in other segments of the GIT. A mixed breed dog of unknown age carried out by Control Zoonosis Center of the Municipality of Ribeir達o das Neves, Minas Gerais State, Brazil was positive for Leishmania using an enzymelinked immunosorbent assay, direct examination of parasites through bone marrow aspiration, and immunohistochemistry of ear and liver biopsy. Samples of spleen, liver, lung, kidney, heart, cervical and mesenteric lymph nodes; ear, snout, abdominal skin and GIT segments (esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, and rectum) were fixed in 10% neutral buffered formalin and hematoxylin and eosin staining. Immunohistochemistry using the streptoavidin peroxidase reaction was carried out to identify amastigotes of Leishmania. For morphometric analysis, samples were submitted to immunohistochemistry for identification of Leishmania amastigotes using the program KS400. Macroscopic and microscopic examination of necropsy samples showed no severe alterations of the mucosa in any gastro-intestinal segment. A conspicuous parasite load was observed in the lamina propria of the duodenum, jejunum, ileum, cecum, colon, and rectum. The parasite load in duodenum was higher than the other segments and it was diffusely distributed in the lamina propria. Parasite distribution in the small intestine was diffuse through the lamina propria, whereas in the large intestine it was
concentrated near the muscularis and distant from the intestinal lumen. These unusual cases of parasites localization in the small intestine suggest that atypical pathology alteration in dogs. - CELLULAR AND MOLECULAR BIOLOGY [1373] P 317 - HISTOLOGICAL HEPATIC ALTERATIONS OF DOGS NATURALLY INFECTED WITH LEISHMANIA (LEISHMANIA) INFANTUM AFTER COMBINED TREATMENT WITH LIPOSOME-ENCAPSULATED MEGLUMINE ANTIMONIATE AND ALLOPURINOL TAFURI, W.; AMORIM, I.; CASTRO, R.; SILVA, S. UFMG, BELO HORIZONTE, MG, BRAZIL.
Keyword:canine visceral leishmaniasis ; histology liver ; liposome-encapsulated meglumine antimoniate
Abstract: This work aimed to evaluate the collagen deposition and inflammation in livers of fifty two dogs naturally infected with Leishmania (L.) infantum chagasi treated with liposome-encapsulated meglumine antimoniate and/or allopurinol. Treated dogs were divided in six groups as follow: Group I composed by eight dogs treated with meglumine antimoniate encapsulated in liposomes (LMA) associated with allopurinol (Allop) denominated LMA+Allop; Group II composed by eight dogs treated with LMA; Group III composed by eight dogs treated with allopurinol (Allop); Group IV composed by eight dogs treated with empty liposome (LEMP) associated with Allop denominated group LEMP+Allop; Group V composed by eight dogs treated with LEMP. Group VI composed by twelve dogs treated with saline solution (SG). Liver sections stained with HE and Gomori ammoniacal silver stains were morphometrically analyzed to characterize the hepatic intralobular granulomas and hepatic intralobular collagen deposition, excluding perivascular collagen. In all cases 20 randomly microscope optic fields, using an Axiolab light microscope (Zeiss) with ร 440 resolution, were done. The images were transferred to a computer video screen using software and relayed to a computer-assisted image analysis system (Kontron Elektronic/Carl Zeiss, Oberkochen, Germany). Using a digital pad, the total diameter of granulomas was measured and the results were expressed in square micrometers ( 2). Also, the total area occupied by stained collagen fibers was measured from real images and segmented to generate binary images expressed in square micrometers. (Caliari, 1997; Gonรงalves et al., 2003; Melo et al. 2009). The results showed that the LMA+Allop group decreased deposition of collagen fibers and number of granulomas, compared to SG. Parasite load tissue was significantly lower in all treated groups compared to SG group. However, LMA+Allop group was the most effective protocol to reduce the parasite load in the liver, demonstrated by negative immunohistochemistry test in all dogs treated with this protocol. In addition, our results demonstrate that treatment with allopurinol decreases fibrosis and granuloma formation in the liver of dogs naturally infected with L. infatum chagasi. - CELLULAR AND MOLECULAR BIOLOGY [1376] P 318 - LEISHMANIA DONOVANI MEMBRANE HISTIDINE ACID ECTOPHOSPHATASE: NEW INSIGHT IN THE STRUCTURE AND FUNCTION BOLETI, H.1; PAPADAKI, A.1; SMIRLIS, D.1; KOUROU, K.2; POLITOU, A.2 1.HELLENIC PASTEUR INSTITUTE, ATHENS, GREECE; 2.MEDICAL SCHOOL OF IOANNINA,, IOANNINA, GREECE.
Keyword:membrane histidine acid phosphatase; ectophosphatase; leishmania donovani
Abstract: The need for new and effective leishmanicidal agents is urgent and identification of novel drug targets is a strong challenge for biomedical research. One class of parasitic molecular targets are
phosphatases. Histidine acid phosphatases constitute an interesting family of Leishmania phosphatases the inhibition of which may have therapeutic value. The histidine phosphatase superfamily is a large functionally diverse group of enzymes across species (Rigden 2008. Biochem J 409(2): 333-48) and various parasites contain superfamily members. We study a membrane bound histidine acid ecto-phosphatase (LdMAcP, GenBank: AF149839.1) from L.donovani, identified by molecular cloning (Shakarian et al. 2002. J Biol Chem 277(20): 17994) as a protein homologous to the secreted (LdSAcP1) histidine acid phosphatase from Leishmania donovani. It was initially suggested to confer the tartate resistant acid ecto-phosphatase activity implicated in promastigote virulence. We re-examined the molecular properties and complemented the molecular characterization of the LdMacP, compared it to its homologues from other Leishmania sps by an in silico analysis and established its plasma membrane localization and acid ecto-phosphatase activity. We generated transgenic LdMacP-RFP L.donovani parasites with episomal overexpression of the LdMacPRFP chimera and established the extracellular surface expression of the endogenous LdMacP and the chimeric LdMacP-RFP by surface membrane biotinylation. The surface acid ectophosphatase activity of LdMacP was also confirmed by heterologous expression of LdMacPRFP in Leishmania tarentolae parasites and of LdMacP-8His in a mammalian cell culture system. The significant homology of LdMacP with the human prostate acid phosphatase that has a resolved crystal structure permitted us to perform 3D molecular modeling. For the first time it is demonstrated directly that LdMacP confers acid ecto-phosphatase activity at the surface of L.donovani and L.tarentolae parasites and of a heterologous eucaryotic cell system, without however excluding the possibility that another membrane acid phosphatase contributes as well to this activity at the surface of the L.donovani parasites. Analysis of the characteristics of the LdMacP-mRFP L.donovani strains for growth characteristics, infectivity and survival in macrophages is in progress.
- CLINICAL LEISHMANIASIS [1385] P 319 - DRAMATIC RESPONSE OF DIFFUSE LEISHMANIASIS TO THE ASSOCIATION OF MILTEFOSINE WITH PENTAMIDINE COSTA, C.H.N.; CANTINHO JUNIOR, J.J.; E SILVA, A.A.S.; CLARK, S.C.; VIEIRA FILHO, J.N. FEDERAL UNIVERSITY OF PIAUĂ?, TERESINA, PI, BRAZIL.
Keyword:spleen; kala-azar; immunology
Abstract: A 32 years old lady, born in the city of Urbano Santos, MaranhĂŁo State, Brazil, developed diffuse leishmaniasis by Leishmania amazonensis when she was 5 years old, diagnosed after an infiltrative lesion in her left arm. Four months later the lesions had spread to the ears, face, arms and legs, but remained stable thereafter. She had been submitted to many therapies with Sbv, amphotericin B, paramomycin, and interferon-gamma with temporary improvements. Lately, she developed chronic renal failure with a stable creatinine around 2.0 mg/dL. In 2010, she got her third pregnancy and the lesions worsened with the appearance of nodules 24 cm wide and coalescent ulcers in the lower limbs, with recurrent bacterial infection. The immunomodulator glucan had no effect. In July of 2011 we made an international pledge via the email list Leish-l asking for miltefosine and suggestions to her treatment. After that, we got the donation of miltefosine from Paladin Labs and from the Colombian Government, a radiofrequency thermotherapy equipment from Thermosurgery Technologies Inc, a portable sauna by Rotary Club, and the promises of paramomycin by OneWorldHealth and
GlandPharma. The latter was denied in the end. The lesions neither responded to burn, nor to the use of sauna. After waiting in vain for paramomycin, we started miltefosine with a course of pentamidin in May/2012; the clinical response was dramatic with disappearance of all nodules and scarring of the ulcers. However, in December we noticed the reappearance of skin infiltrates, plenty of parasites at the skin biopsy. In January, we just started a new course of amphotericin B and pentamidine.
- GENETICS, EVOLUTION AND TAXONOMY [1405] P 320 - MICROSATELLITES FOR LEISHMANIA MEXICANA COMPLEX: DEVELOPMENT OF NEW MARKERS FERREIRA, G.E.M.1; MAZZONI, C.2; HARTMANN, S.3; DA GRAÇA, G.C.4; CUPOLILLO, E.1 1.FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 2.BEGENDIV - IZW, BERLIN, GERMANY; 3.BEGENDIV - UNIPOTSDAM, BERLIN, GERMANY; 4.FIOCRUZ - OPAS, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:leishmania mexicana complex; microsatellites; leishmania amazonensis
Abstract: In the last decade advances on molecular techniques have been responsible for a strong increase in the knowledge of genetic diversity and population structure of Leishmania species. Among PCR based methodologies, microsatellites or short tandem repeats analysis accounts for at least 43 original publications since 2002. The approach became widespread as multilocus microsatellite typing (MLMT) and most of the markers sets proposed presented a good discriminative power at the strain level, making this approach one of the most efficient for molecular epidemiology. Despite the popular use of MLMT, no markers were developed for the Leishmania mexicana species complex. Here we present the strategy we used for a rapidly development of microsatellite markers and a new set of markers for L. mexicana and L. amazonensis that have been tested in our lab. We have sequenced the whole genome of L. mexicana and L. amazonensis sheared DNA using the Illumina MiSeq. Primers were constructed using very stringent criteria in a Perl script, Primer3 software. Primers that were common to both species but presented different number of repeats between them were chosen, as this would potentially increase their discriminatory power. Only loci with simple dinucleotide repeats were elected, in order to fit standard assumptions for future population genetics analyses. We found 73 variable loci from the in silico analysis between the two genomes. In order to reduce the costs of experiments the 5’ end of each forward primer pair was synthetized with a M13 tail for posterior fragment analyzes. Nine of the 73 primer pairs were tested in real PCRs and all of them amplified the expected band. Reactions were performed with DNA templates from the same L. mexicana and L. amazonensis strains that were sequenced and many tests are being conducted to a proper validation of the markers set for MLMT studies on L. mexicana species complexes. We believe that this approach may contribute to the knowledge of molecular epidemiology of cutaneous leishmaniases and the population genetics of this complex in a near future. - CLINICAL LEISHMANIASIS [1408] P 321 - DISSEMINATED CUTANEOUS LEISHMANIASIS IN ELDERLY: A CASE REPORT MOURA, V.A.1; TELES-JUNIOR, E.P.1; VIEIRA BRANQUINHO, H.C.1; DE CASTRO FREITAS, T.M.1; JANOTTI, A.2; COUTINHO COSTA VELOSO, O.M.3; DE PÁDUA MILAGRES, F.A.3 1.UNIVERSIDADE FEDERAL DO TOCANTINS, PALMAS, TO, BRAZIL; 2.HOSPITAL GERAL PÚBLICO DE PALMAS, PALMAS, TO, BRAZIL; 3.UNIVERSIDADE FEDERAL DO TOCANTINS AND HOSPITAL GERAL PÚBLICO DE PALMAS, PALMAS, TO, BRAZIL.
Keyword:leishmaniasis; cutaneous; disseminated
Abstract: Introduction: In Tegumentary Leishmanisis American (TLA) a spectrum of clinical forms can develop in dependence on the characteristics of the cell mediated immune response. In anergic pole of this spectrum is a rare form Disseminated Cutaneous Leishmaniasis rare form (DCL). This is characterized by multiple lesions of varying aspect, and sometimes secondary infection can turn the lesions more painful, inflamed and purulent. Case Report: VLS, female, 66, from Guaraí-TO. Two months ago showed initial ulcerated lesion on the calcaneal region of right foot associated with itching and local pain, progressing with other similar lesions in the calcaneal region and big toe of the left foot and on the fourth finger of the right hand, characterizing the disseminated form of TLA. The ulcerated area of lesions of the feet increased reaching five inches in diameter, with purulent and erythematosus center and edges with necrotic areas and inflammation signs. The lesions on the fingers of the hand and feet became bullous and hyperchromic with ulceration later. The patient was Diagnosed with TLA, after positive direct testing for Leishmania of shaved of the initial lesion. Started the treatment with amphotericin B deoxycholate (50 mg / day), progressing to nausea, dizziness, chills and palpitations after infusion, despite the administration of dipyrone 1g and hydrocortisone 100 mg IV earlier. Because of injuries with signs of inflammation and signs of secondary infection debridement was done on foot ulcers and was prescribed ceftriaxone 1g IV 12/12h and clindamycin 600 mg IV 8/8 h for 7 days. Responded favorably to treatment for TLA beyond resolution of secondary infection and gradual decrease of pain intensity. She was discharged from the hospital after completing treatment with a total dose of amphotericin B deoxycholate (1g). The patient developed clinical improvement of pain and significant improvement of ulcerated lesions after treatment with amphotericin B deoxycholate, being referred to outpatient following. - CLINICAL LEISHMANIASIS [1409] P 322 - AN ATYPICAL PRESENTATION OF CUTANEOUS LEISHMANIASIS WITH RESISTENCE TO GLUCANTIME: A CASE REPORT MOURA, V.A.1; ARAÚJO NETO, A.R.1; BRITO, J.F.1; SIQUEIRA FORMIGA, C.C.1; PARREIRA, B.L.1; COUTINHO COSTA VELOSO, O.M.2; JANOTTI, A.3; DE PÁDUA MILAGRES, F.A.2 1.UNIVERSIDADE FEDERAL DO TOCANTINS, PALMAS, TO, BRAZIL; 2.UNIVERSIDADE FEDERAL DO TOCANTINS AND HOSPITAL GERAL PÚBLICO DE PALMAS, PALMAS, TO, BRAZIL; 3.HOSPITAL GERAL PÚBLICO DE PALMAS, PALMAS, TO, BRAZIL.
Keyword:atypical ; leishmaniasis; glucantime
Abstract: Introduction: Cutaneous leishmaniasis (CL) is endemic in Brazil and presents regional particularities, such as the occurrence of severe forms and resistance to treatment. There are few drugs available for CL treatment, but Glucantime still the main drug in Brazil. Some cases of therapeutic failure, absence of lesion epithelialization after treatment, or clinical reactivation have been reported in some endemic areas. Case report: E.S.S., Male, 29 years old, from Palmas-TO. Started three years ago, in the right malar region, with papular lesions that progressed to brownish and infiltrated plaques (approximately 10 centimeters), with welldefined and raised margin, crusted lesion with central ulceration and detachment of small whitish plaques. There were also similar lesions in the right submandibular and left retroauricular region. Scraping of the lesions was negative to Leishmania, but biopsy suggested the diagnosis of CL. It began the treatment with Glucantime, but there was no improvement in the lesions. There was no clinical or laboratory signs of immunosuppression, including seronegative for HIV. Due to the resistance to Glucantime, was combined treatment with allopurinol (400 mg) and sulfadiazine (1000 mg/dia) for 60 days, with partial improvement in the appearance of lesions in the face, the center has become more clear in relation to the edge Later he was referred to the reference Public General Hospital of Palmas and starting
amphotericin B deoxycholate 50 mg/day due to refractory at prior therapy and the positive scraping at the lesion for Leishmania, preceded by dipyrone and hydrocortisone, however the patient showed signs of intolerance, such as fever, chills, vomiting, palpitations, chest pain, dyspnea and tachyarrhythmia. The treatment staying for 18 days (total dose 900 mg), so was initiated Liposomal Amphotericin B (04 mg/kg/day), with good response to treatment and improves of appearance of the lesions (partial healing). The patient was discharged with a total dose of Liposomal Amphotericin B (1750 mg) and was followed as outpatients. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1415] P 323 - SERO-IMMUNOPROTEOMIC APPROACHES REVEAL DIFFERENCES OF FUNCTIONAL PROPERTIES BETWEEN ANTIBODIES FROM SYMPTOMATIC AND ASYMPTOMATIC INDIVIDUALS INFECTED WITH LEISHMANIA INFANTUM GARDINASSI, L.G.A.1; GARCIA, G.R.1; MARUYAMA, S.R.1; SILVA, F.O.2; COSTA, C.H.N.2; ALMEIDA, R.P.3; JESUS, A.M.R.3; SANTOS, I.K.F.M.1 1.1FACULDADE DE MEDICINA DE RIBEIRÃO PRETO - USP, DEPARTAMENTO DE BIOQUÍMICA E IMUNOLOGIA, RIBEIRÃO PRETO, SP, BRAZIL; 2.INSTITUTO DE DOENÇAS TROPICAIS NATAN PORTELLA - UFPI, TERESINA, PI, BRAZIL; 3.3CENTRO DE CIÊNCIAS BIOLÓGICAS E DA SAÚDE – UFS, DEPTO. DE MEDICINA INTERNA E PATOLOGIA, ARACAJU, SE, BRAZIL.
Keyword:leishamaniasis; infection; l. infantum
Abstract: The majority of infections with the etiological agent of visceral leishmaniasis in Brazil, Leishmania infantum, do not result in disease. The scientific consensus is that antibodies do not have a role in this outcome and may contribute to susceptibility to visceral leishmaniasis; nevertheless recent studies challenge this view. We hypothesized that individuals presenting asymptomatic infections with L. infantum produce antibodies with functional properties that differ from those produced by individuals who develop visceral leishmaniasis. Sera were collected from 208 kala-azar patients, from 197 individuals with asymptomatic infection and from 138 healthy donors living in endemic areas, in order to verify differences of several characteristics of specific antibodies, such as IgG titers, fine specificities and intensity of antigen recognition. Late-log phase promastigotes of L. infantum were used to extract soluble antigens (SLA), membrane antigens (MA) and secreted/excreted antigens (SEA). ELISA showed that kala-azar patients present higher absolute OD of IgG (~10 fold) against L. infantum SA, MA and SEA than asymptomatic individuals. On the other hand, asymptomatic individuals presented with significantly (p<0,001) higher titers of IgG antibodies than healthy uninfected controls. Furthermore, when we normalized the titers of IgG antibodies reactive with SLA and MA by calculating the ratio of OD for membrane antigens / OD for soluble antigens for each serum sample, we observed that asymptomatic individuals presented higher specific reactivity with MA, which suggests that MA may be more suitable for the identification of asymptomatic infection by immunoassays. MA and SEA were separated by 2D polyacrylamide gel, and western blots were performed with sera from kala-azar patients or infected asymptomatic individuals. The reactivity of sera from asymptomatic individuals recognized a set of antigens that differed both by specificity and intensity compared to kala-azar patients. Thus, further refinement of the pattern of reactivities with these antigens and of IgG properties (such as their subclasses, glycosylation profiles and allotypes) may confirm a protective role of antibodies against the development of visceral leishmaniasis and identify new antigens that could be useful for discrimination of clinical outcome as well as vaccine candidates. Financial Support: FAPESP (2011/23819-0), CAPES.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [19] P 324 - NEW MOLECULAR TARGETS AND ANTILEISHMANIAL DRUG LEADS IDENTIFIED THROUGH SCREENING A LIBRARY OF NATURAL PRODUCTS PREPARED BY HIGH THROUGHPUT FRACTIONATION PARADIGM TEKWANI, B.L. UNIVERSITY OF MISSISSIPPI, UNIVERSITY, UNITED STATES.
Keyword:natural products; na,k-atpase; topoisomerase
Abstract: Visceral leishmaniasis, caused due to infection with a protozoan parasite Leishmania donovani, is a prominent disease in certain tropical regions of the world. Toxicity and suboptimal efficacy of current antileishmanial drugs and severely depleted antileishmania drug discovery pipeline necessitate the discovery of new antileishmanial drugs with better efficacy and safety profiles. Natural products remain an unmatched source of drugs with novel chemotypes. However conventional natural products drug discovery approach, which includes screening of crude natural products extracts followed by bio-assay guided fractionation, suffers from certain limitations. Polyphenols, often present in considerable quantities in ethanol extracts of plants, can cause false-positive results in in vitro screening procedures due to non-selective inhibition. The chemical diversity found in a single extract may represent several different classes of molecules that exhibit different (and sometimes opposing) biological activities. Biologically active compounds may be present in crude extracts at extremely low concentrations that are below the detection threshold for bioactivity screening. Pre-fractionation technologies have been useful in overcoming these limitations. A library of >30,000 natural product fractions have been generated through a high throughput fractionation paradigm. This natural product collection represents significant biodiversity. Each fractions, based on weight, were pooled, transferred to 96 well plates, and analyzed via QC-UPLC MS/MS. 7387 fractions from 671 plant extracts were screened through a newly developed L. donovani amastigotes-THP1 cells parasite rescue & transformation assay and for cytotoxicity against differentiated THP1 cells. A total of 194 fractions were identified with 50% or higher inhibition vs. leishmania amastigote growth in THP1 cells; only 10 of these were toxic against the THP1 cells alone. The active 194 fractions were further screened for dose-response antileishmanial analysis. The selective antileishmanial activity of 65 fractions was confirmed. Active fractions from Thuja occidentalis (IC50-0.25 ug/mL), Asclepias asperula (IC50-0.33 ug/mL), Rhodea japonica (IC50-0.41ug/mL) and Nerium oleander (IC50-0.03 ug/mL) were selected for further investigations. Deoxypodophyllotoxin from Thuja occidentalis, oleandrin from Nerium oleander, rhodexoside from Rhodea japonica and a cardinolide from Asclepias asperula were identified as actives, and potential new antileishmanial drug leads. These diverse compounds, which are potential inhibitors DNA Topiosmerases and Na+,K+-ATPases, were selective active against intracellular amastigotes with so significant effects on leishmania promastigotes. The results indicate Na,KATPase as potential new molecular drug targets and confirm topoisomerases as promising targets for new antileishmanial drug discovery. Supported by US DoD CDMRP grant W81XWH-09-2-0093. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [21] P 325 - BIODEGRADABLE MICROPARTICLES FUNCTIONALIZED WITH ANIMAL TOXINS: CHARACTERIZATION AND POTENTIAL APPLICATION IN THE TREATMENT OF CUTANEOUS LEISHMANIASIS MACEDO, S.R.A.; FERREIRA, A.S.; DE BARROS, N.B.; NICOLETE, R. FIOCRUZ RONDﾃ年IA, PORTO VELHO, RO, BRAZIL.
Keyword:leishmania amazonensis; biodegradable microparticles; chemotherapy
Abstract: Leishmaniases are among the highest incidence of infectious diseases in the world. They are caused by protozoa of the genus Leishmania spp. This disease affects over 12 million people in 88 countries, with 350 million at risk of becoming ill. The drugs available for the treatment show high toxicity, none of them is sufficiently effective leading to disease control and treatment failure. In this context, the production of prototype drugs addressing micro/nanotechnology is a promising alternative, since compounds endowed with antiparasitic activity may be directed, through this technology, to target cell containing the protozoa. In this work, microparticles constituted of Poly Lactic Co-Glycolic Acid (PLGA) biodegradable polymer and functionalized with snake toxins (Crotalus durissus terrificus) were produced and in vitro characterized, by testing their diameters and Zeta Potential. The biological activity against Leishmania amazonensis elicited by the encapsulated toxins, as well as the assessment of the in vitro toxicity using peritoneal macrophages from BALB/c mice were evaluated by assaying for viability employing the MTT reagent. Also, it was determined the phagocytic index of peritoneal macrophages incubated with microparticles, by panoptic staining and visualization in bright field microscopy. The size (1.3, 3.0 and 3.5 µm) and Zeta Potential (-12.3, -20.9 and 13.9 mV) values were assessed for control, crotamine and crotoxin-encapsulated particles, respectively. The control particle (without any encapsulated toxin) at a concentration of 100 µg/ml did not show toxicity against L.amazonensis in 24 hs. The same concentration of crotamine-encapsulated microparticle showed an inhibition of 20% at 24 and 48 hs. The particles containing crotoxin showed no toxicity within 24 hs and further an inhibition of approximately 20% (48 hs). Also, when particles’ toxicity was assessed against peritoneal macrophages, none of them showed any significant effect. Regarding the phagocytic index assay, the control particles, crotamine and crotoxin-encapsulated particles presented the values of 61.7%, 84.7% and 141.7%, respectively. Ours results suggest that micro-structured functionalized particles achieved in this study can contribute for the development of promising new therapies against cutaneous leishmaniasis. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [28] P 326 - PROMISING ANTI-LEISHMANIAL AGENT REVEALED THROUGH PROTEOMIC ANALYSIS OF LEISHMANIA-INFECTED MACROPHAGES GUEDES, C.E.S.1; SIMÕES DIAS, B.R.1; PETERSEN, A.L.O.A.1; CRUZ, K.P.1; ALMEIDA, N.J.1; VERAS, P.S.T.2 1.CENTRO DE PESQUISA GONÇALO MONIZ, FIOCRUZ-BA, SALVADOR, BA, BRAZIL; 2.CENTRO DE PESQUISA GONÇALO MONIZ, FIOCRUZ-BA; INCT-DT, SALVADOR, BA, BRAZIL.
Keyword:tspo; anti-leishmanials; leishmania amazonensis
Abstract: Leishmaniasis is a major neglected disease with an increasing number of cases related to host susceptibility due to co-infection with HIV. Currently available leishmanials, including pentavalent antimonials and amphotericin B, are highly toxic. The efficacy of these drugs has come under scrutiny due to severe side effects, reducing treatment adherence and thus favoring the selection of resistant strains. Substantial research has focused on the role of macrophages (M ) in the immune response to Leishmania infection. Previously, we demonstrated that CBA mouse M control L. major, yet are permissive to L. amazonensis infection, constituting an interesting infection model to study host resistance to infection. Using a proteomic approach, we identified proteins whose expression is modulated in CBA M infected with L. amazonensis compared to M infected with L. major. The translocator protein (TSPO), mainly found on the outer mitochondrial membrane, was selected as a potential target for treatment intervention. We hypothesized that control of L. amazonensis infection would be obtained by modulating TSPO. Thus, the present study aimed to assess the effect of TSPO modulation by treating L.
amazonensis-infected M with MTB2, a ligand for TSPO. First, in order to evaluate the direct effect of MTB2 on L. amazonensis promastigotes, parasite cultures were exposed to increasing concentrations for 24-72h, followed by direct parasite counts. MTB2 inhibited promastigote proliferation in axenic cultures at concentrations between 25-100 M, which have demonstrated non-toxicity in M . To evaluate the effect of MTB2 on intracellular parasite viability, L. amazonensis-infected macrophages were subjected to drug concentrations of 25-100 M for 648h. These cultures were then fixed and the percentage of infected macrophages was determined. Treatment with 50-100ﾂｵM of MTB2 resulted in a reduction of 75-99%. This inhibitory effect was shown to be irreversible after 6h of exposure. Electron microscopy analysis revealed alterations suggestive of autophagy in intracellular parasites, indicating that Leishmania death might be related to an autophagic process. In addition, MTB2 induced an increase in intracellular superoxide production by infected M , implying that this free-radical may play a role in intracellular parasite death. In sum, these findings demonstrate the promising potential of MTB2 as an anti-leishmanial candidate, possibly by way of a mechanism related to TSPO modulation. Supported by FIOCRUZ, CAPES and CNPq - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [33] P 327 - UTILITY OF HYPERICIN IN THE TREATMENT OF CUTANEOUS LEISHMANIASIS: IN VITRO AND IN VIVO STUDIES MONTOYA, E.A.1; RESTREPO, A.M.1; UPEGUI, Y.A.1; CEDEﾃ前, D.L.2; MUﾃ前Z, D.L.1; VELEZ, I.D.1; ECHEVERRI, L.F.1; ROBLEDO, S.M.1 1.PECET, UNIVERSITY OF ANTIOQUIA, MEDELLIN, COLOMBIA; 2.ILLINOIS STATE UNIVERSITY, NORMAL, UNITED STATES.
Keyword:hypericin; phototherapy; therapeutical response
Abstract: Very few drugs are available for treatment of leishmaniasis and all of them are highly toxic, costly, and their efficacy is becoming lower. Therefore, new drugs are needed. Hypericin is the main component of Hypericum perforatum and it has traditionally been used throughout the history of folk medicine. Studies report several biological activities including antidepressive, antineoplastic, antitumor and antiviral activities. Hypericin is also considered one of the most powerful type II photosensitizer found in nature, which is useful to photodynamic therapy as an alternative treatment for cutaneous leishmaniasis (CL). Here, Hypericin was evaluated for its in vitro and in vivo leishmanial activities. In vitro studies showed that hypericin is highly active against Leishmania amastigotes with EC values of 0.9 + 0.14 M. On the other hand, topical treatment of hamsters with CL after experimental infection with L. amazonensis in the dorsal skin (6 applications of hypericin in presence of light, twice a week) produced complete cure in 4 of 5 animals. The last hamster showed a decrease in the lesion size of 79,8%. In absence of light cure was observed only in 2 of 5 animals. No toxicity associated to dose was observed with Hypericin. These results suggest that hypericin has a great potential as drug candidate against CL.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [34] P 328 - POTENTIAL UTILITY OF QUATERNARY N -(HALOMETHYL) AMMONIUM SALTS IN THE TREATMENT OF CUTANEOUS LEISHMANIASIS: IN VITRO AND IN VIVO STUDIES
ROBLEDO, S.M.1; RIOS, L.A.2; RESTREPO, A.M.1; MUﾃ前Z, D.L.1; CEDEﾃ前, D.L.3; DUQUE, S.M.2; OCAMPO, R.2; JONES, M.A.3; PULIDO, S.A.1; VELEZ, I.D.4
1.PECET, UNIVERSITY OF ANTIOQUIA, MEDELLIN, COLOMBIA; 2.UNIVERSITY OF CALDAS, MANIZALES, COLOMBIA; 3.ILLINOIS STATE UNIVERSITY, NORMAL, UNITED STATES; 4.PECET, AGRARIAN SCIENCESUNIVERSITY OF ANTIOQUIA, MEDELLIN, COLOMBIA.
Keyword:quaternary n -(halomethyl) ammonium salts; leishmanicidal activity; cutaneous leishmaniasis, therapeutical response
Abstract: There are very few available drugs to treat cutaneous leishmaniasis (CL) and all of them are highly toxic, costly, and their efficacy is becoming lower. Therefore, there is a need for new drugs and better drugs. To search new drugs to treat CL, a series of new quaternary n (halomethyl) ammonium salts (compounds C6, K, D and H) were synthesized and evaluated in vitro for leishmanicidal activity and and in vivo for the therapeutic response by comparing the size of the lesion before and after the treatment. All compounds were active in vitro against intracellular amastigotesof L. (V) panamensis with EC50 < 40 mg/mL. In the hamster model for CL, topical treatment (once a daday during 15 days) with compounds C6 and K produced cure in 6 of 6 animals and 4 of 6 animals at three months after the end of treatment, respectively. With compound D, cure was observed in 3 o 6 animals and compound H produced cure in only 2 of 6 animals. No toxicity associated to dose was observed with any compound. These results suggest that this serie of compounds exhibits a great potential as drug candidates against CL. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [42] P 329 - OVER EXPRESSION OF DIPEPTIDYL CARBOXYPEPTIDASE OF LEISHMANIA DONOVANI MODULATES PARASITE INFECTIVITY GANGWAR, S.; GOYAL, N. CENTRAL DRUG RESEARCH INSTITUTE, LUCKNOW, INDIA.
Keyword:overexpression; dipeptidylcarboxypeptidase; infectivity
Abstract: Leishmaniasis is a tropical disease caused by parasites of the genus Leishmania, with clinical manifestations ranging from localized cutaneous ulcers to systemic visceral organ damage. Treatment of leishmaniasis relies mainly on chemotherapy and is far from ideal because of high costs, high toxicity and long term treatment requirements. Increasing incidences of therapeutic failures and emergence of drug resistant parasites now warrants the urgent need for new improved drugs. The peptidases of protozoan parasites are important for their role in parasite survival and pathogenecity. Leishmania donovani dipeptidylcarboxypeptidase (LdDCP), an angiotensin converting enzyme (ACE) related metallopeptidase, was identified and characterized as novel drug target from our lab. The present study aims to determine the role of LdDCP in parasite survival and pathogenesis. LdDCP gene was transfected in wild type promastigotes. The transfectants exhibited slow multiplication rate and higher infectivity as compared to wild type strain. Over-expression of the gene did not affect parasite susceptibility towards the available standard antileishmanial drugs, namely Sb(III), amphotericin B, pentamidine, miltefosine and paromomycin. However, the transfectants were more susceptible to inhibitors of ACE (captopril) and LdDCP than the wild type.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [43] P 330 - QUANTITATIVE REAL TIME PCR ASSAYS FOR THE DETECTION OF LEISHMANIA (VIANNIA) BRAZILIENSIS IN ANIMALS AND HUMANS GONÇALVES, S.C.1; DE MORAIS, R.C.S.1; E SILVA, R.P.1; DANTAS-TORRES, F.1; DE BRITO, M.E.F.1; OTRANTO, D.2; BRANDÃO-FILHO, S.P.1; DE PAIVA-CAVALCANTI, M.1 1.AGGEU MAGALHAES FIOCRUZ-PE, RECIFE, PE, BRAZIL; 2.UNIVERSITÁ DEGLI STUDI DI BARI, BARI, ITALY.
Keyword:molecular targets ; diagnosis; leishmania spp
Abstract: American cutaneous leishmaniosis (ACL) caused by Leishmania (Viannia) braziliensis is a neglected disease of humans in the New World that may also cause irreversible skin and eventually mucocutaneous lesions. Methods currently available for the diagnosis of ACL have low sensitivity and may be time-consuming, representing a limit for treatment expedition of ACL. Quantitative real time PCR assays (qPCR) for the detection of L. (V.) braziliensis in canine blood samples were herein developed and the detection limit and specificity of different molecular targets (kDNA and rDNA) evaluated. Among protocols screened, two qPCR assays, one targeting the kDNA and other the SSU rDNA of L. (V.) braziliensis, showed better performances, with detection limits of 100 fg and 10 pg, respectively. Protocols were also evaluated on skin samples from humans with suspected ACL. The results indicate that the qPCR protocols here developed represent an advance for the diagnosis of ACL in dogs and humans from this region and provide a rapid, non-invasive and accurate diagnosis of the infection by L. (V.) braziliensis. Considering the quantitative nature of the assays, they will also be useful for monitoring treatment and preventing relapses in human patients. Finally, molecular assays here described may represent new tools for future basic and applied research on species identification, host-parasite associations, and infection dynamics in host and vector populations. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [44] P 331 - GLUCOSE TRANSPORT AS A MARKER OF CHEMO-RESISTANCE IN ISOLATES FROM PATIENTS WITH DCL PONTE-SUCRE, A.; PADRON-NIEVES, M. UNIVERSIDAD CENTRAL DE VENEZUELA, CARACAS, VENEZUELA.
Keyword: drug resistance cellular markers; diffuse cutaneous leishmaniasis; glucose transport
Abstract: Therapeutic failure in leishmaniasis is a common problem in endemic areas. This could be attributed to altered drug pharmacokinetics, re-infection, or immunologic compromise of the host. However, strong indicators suggest that it may be partly due to drug resistance. Until now, whether drug resistance in Leishmania compromises the parasite proficiency, e.g. in terms of infectivity or metabolism, has not been systematically evaluated. If confirmed and validated, these changes can become cellular and molecular indicators feasible to be used to identify the drug-resistant phenotype of the infecting parasites. Herein we have evaluated the time course of glucose utilization as an indicator of cellular viability closely associated with metabolic activity in isolates from DCL patients that failed to respond to Glucantime. The results obtained with the isolates were compared with those found in WHO-reference Leishmania strains. The data analysis demonstrates that the rate of glucose utilization is very low in the isolates VE2000MM and VE98MR compared to that determined in its reference strain L. amazonensis (LTB0016). The third one, VE96ZC has a slope of glucose utilization similar to its reference L. mexicana strain Bel21. They also suggest that assessment of the rate of glucose utilization can be useful for differentiating patient’s isolates according to their ability to incorporate glucose into the cell,
regardless of its subsequent metabolism. Of note, the patient infected with VE96ZC was the only one that cured after several combined therapeutic interventions (glucantime plus immunotherapy). The validation of the data presented herein would confirm if this methodology could be included in a set of markers to identify whether a patient treatment failure is due to infection with a chemo- resistant parasite.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [45] P 332 - A SIMPLE COLORIMETRIC ASSAY FOR HIGH-THROUGHPUT SCREENING OF DRUGS AGAINST LEISHMANIA INTRACELLULAR AMASTIGOTES. SCHALLIG, H.; BOGAART, E.V.D.; FABER, D.; SCHOONE, G.; ADAMS, E. ROYAL TROPICAL INSTITUTE, AMSTERDAM, NETHERLANDS.
Keyword:drug; development; in-vitro
Abstract: Development of safe and efficacious therapies able to counteract the significant problems posed by current anti-leishmanial drugs has been strongly advocated. Crucial to the search for new anti-leishmanial drugs is the availability of high-throughput methods to screen chemical compounds against the relevant stage for disease pathogenesis, the intracellular amastigotes. Recent progress in automated microscopy and genetic recombination have produced powerful tools for drug discovery. However, a simple and efficient test for measuring cytotoxicity against Leishmania clinical isolates is still lacking. A quantitative colorimetric assay has been developed, whereby activity of a Leishmania native enzyme is used to assess its viability. Enzymatic reduction of disulphide-trypanothione, monitored by a microtiter plate reader, was used to quantify the growth of Leishmania donovani intracellular amastigotes. An excellent correlation was found between the optical density, measured at 412nm, and number of parasites inoculated. Validation of this assay was performed with a selected drug-panel against standard microscopy. The activity of several anti-leishmanial reference drugs, as measured by this assay, corroborated with microscopy results and previously published data. This simple and inexpensive assay provides a reliable, accurate method for screening anti-leishmanial agents, at high-throughput. The basic equipment and manipulation required to perform the assay makes it easy to implement, simplifying the methodology for scoring inhibitor assays.
- GENETICS, EVOLUTION AND TAXONOMY [48] P 333 - ENZYMES THAT OVEREXPRES IN DRUG RESISTANT LEISHMANIA LACKS 3D STABILITY IN COMPARISON TO THE HOUSE KEEPING ENZYMES THAT DO NOT : IN-SILICO EVIDENCE DEBNATH, S.1; ADDYA, S.2 1.DEPT. OF MEDICAL LABORATORY TECHNOLOGY , WOMEN'S POLYTECHNIC, AGARTALA, INDIA; 2.GENERAL MEDICINE PRACTITIONER, AGARTALA, INDIA.
Keyword:drug resistance; protein disorder; over expression
Abstract: Induced drug resistance in Leishmania sp. has conclusively shown amplification & overexpression of several genes as DHFR -Thymidylate Synthase(DHFR-TS) /Pteridine Reductase 1(PR1) in Methotraxate resistance,UDP-N acetylglucosamine phosphotransferase(NAPAPT) in Tunicamycin resistance,IMPDH in Mycophenolic acid resistance & Onithine Decarboxylase(OD) in A-difluoromethylornithine resistance.We evaluated Protein disorder in terms of 3D structural stability of these enzymes in comparison to housekeeping enzymes.Protein disorder characterized by lack of stable 3D structure & is considered to be involved in regulatory & signalling events.An elevated evolutionary rate is seen in disordered proteins.We have used POODLE server (http://mbs.cbrc.jp/poodle/poodles.html) to predict disordered region from Amino Acid (AA) sequences of Enzymes, that overexpress & that do not (Enzymes of Glycolysis as Hexokinase HK,Enolase E,Cytosolic & Glycosomal Glyceraldehyde 3-Phosphate Dehydrogenase G3PD & of TCA Cycle as Pyruvate Dehydrogenase PD, Aconitase A,Citrate Synthase CS & Succinyl-CoA Synthetase alpha SCSA).Enzyme sequences for Leishmania donovani ASM22713v2 (http://www.ncbi.nlm.nih.gov/genome/3516) retrieved in FASTA & deposited to POODLE that predicts disordered region from AA sequences using physicochemical features & reduced AA set of a position-specific scoring matrix.Server returned probability of disordered region graphically from N-terminus as well as on a 0 to 1.0 scale with 0.5 as threshold.Numbers of disordered domains were counted from the matrix & numbers of AAs in these domains calculated in percentage of total number of constituent AAs in the enzyme.Disorder scored of >0.5 to <0.7 termed as minimum disorder for simplicity of representation though domains even with minimum disorder were also counted. AA entity conferring highest probability of disorder is mentioned with its position in the peptide.All enzymes causeing Drug resistance by amplification & overexpression in Leishmania showed higher magnitude of protein disorder compared to housekeeping counter parts.OD (293aa) has highest 3D disorder (25.59%) in 03 domains (highest at AA position G11,F123,A180 & A181).IMPDH (514aa) has 3D disorder of 22.76% in 08 domains (highest at AA - M1,A2 & E429).DHFR-TS(520aa) has 3D disorder of 14.80% in 04 domains (highest at AA - M1 & R213). PR1 (288 aa) has 3D disorder of 9.375% in 05 domains (highest at AA - G76,A77,P82,V130 & A288).In contrary house keeping enzymes are far more stable with highly ordered 3D.For example Cytosolic G3PD (331aa) have no disorder.Enzyme E (429aa) has 01 domain with 4 AAs(0.93%) in C-ter end.HK (471aa) has 02 domains (2.12%) in extreme C & N-ter.N-ter end has minimum disorder.It is so evident that overexpressed enzymes conferring drug resistance are more disordered & unstable which may be necessary for more evolutionary adaptation. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [49] P 334 - PHOSPHOLIPASE A1 AND LIPIDS FROM LEISHMANIA BRAZILIENSIS. ITS POSSIBLE PARTICIPATION IN THE PATHOGENESIS OF LEISHMANIASIS BELAUNZARAN, M.L.; GIMENEZ, G.; BOTT, E.; LAMMEL, E.M.; ISOLA, E.L.
INSTITUTO DE INVESTIGACIONES EN MICROBIOLOGÍA Y PARASITOLOGÍA MÉDICA (IMPAM, UBA-CONICET). FACULTAD, BUENOS AIRES, ARGENTINA.
Keyword:phospholipase a1; lipids; autolysis
Abstract: Leishmania spp, an intracellular parasite that belongs to the Trypanosomatidae family is transmitted by phlebotomine sandflies as metacyclic promastigotes that infect macrophages in the vertebrate host, where they multiply as amastigotes. In Argentina, the coexistence of L. braziliensis, L. guyanensis and L. amazonensis has been detected, being the major causative agents of human leishmaniasis.
Phospholipase A1 (PLA1) specifically hydrolyses acyl groups from phospholiplids (PL) at the sn-1 position and is considered a virulence factor for many pathogens, trypanosomatids among them. On the other hand, the limitation of initial infection depends on the activation of the innate immunity and there is a growing body of evidence pointing to an important modulatory role of microorganisms' lipids in this process. In this regard, we have previously reported in T. cruzi a rapid accumulation of free fatty acids (FFA) and lysophospholipids upon parasite autolysis, as result of PLA1 activity. These compounds function as second messengers and have been implicated in inflammatory processes. As we previously demonstrated that L. braziliensis also possesses this enzymatic activity, in the present work we performed its biochemical characterization and analyzed the lipid profiles of autolysing parasites as well as the effect of these lipids in nitric oxide release from macrophages. Results indicated that in amastigotes and promastigotes PLA1 activity was mainly associated to the soluble fractions. Interestingly, the activity levels were significantly high in the infective amastigotes, being in the soluble and in the organelle enriched fractions 2- and 8-fold higher respectively, compared to non-infective promastigotes. In both stages, PLA1 activity was independent of the bivalent cations Ca2+ and Mg2+, with an optimum pH of 6.3 and only for amastigotes, the enzyme was activated by Triton X-100. On the other hand, the lipid analysis of autolysing amastigotes showed a significant PL degradation with an important increase in FFA, indicating the action of parasite PLA1. Moreover, total lipids from these parasites were able to induce in macrophages nitric oxide release, a soluble factor implicated in parasite growth inhibition. In conclusion, PL degradation in autolysing amastigotes due to parasite PLA1 activity generated FFA, second lipid messenger that could participate in several signaling pathways related to inflammatory processes in Leishmaniasis. Supported by FONCYT/CONICET
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [51] P 335 - USE OF A PLASMID (PUC 18) AS A DNA EXTRACTION CONTROL IN A MULTIPLEX PCR ASSAY FOR THE DIAGNOSIS OF AMERICAN CUTANEOUS LEISHMANIASIS SILVA, R.P.1; GONÇALVES, S.C.2; MORAIS, R.C.3; SILVA, L.T.1; BRANDÃO-FILHO, S.P.2; DANTAS-TORRES, F.2; CAVALCANTI, M.P.2 1.UFPE, RECIFE, PE, BRAZIL; 2.CPQAM/FIOCRUZ, RECIFE, PE, BRAZIL; 3.UFRPE, RECIFE, PE, BRAZIL.
Keyword:"diagnosis ; leishmaniasis; extraction control"
Abstract: American cutaneous leishmaniasis (ACL) is a parasitic disease, clinically heterogeneous, caused by protozoa of genus Leishmania. Losses of parasite’s genetic material during DNA extraction processes, even if minimal, may significantly impair its detection by PCR and lead to falsenegative results. This work was aimed at developing and standardizing a multiplex PCR able to detect small amounts of L. braziliensis DNA with an internal control (G3PD gene of mammals), including a new external control (i.e., the plasmid pUC18), in the same reaction. Two primer pairs were designed to detect plasmid pUC18. PCR products from pUC18 DNA resulted in bands of 368 (system P1) and 316 (system P2) base pairs (bp). The P1 set of primers showed the best performance, and was chosen for further standardization. A concentration of 10 pmol/ L of these primers were able to detect 10 pg of pUC18, being thus chosen to be added on the multiplex PCR assay. The triplex PCR was optimized after some modifications on cycling temperatures and concentrations of reagents. Due to lack of reproducibility of the plasmid
pUC18 in positive samples, tests using an alternative target as external control (DNA of Malassezia pachydermatis) were performed. The tests showed an excellent performance of the new target, including those performed on biological samples (in these cases, the DNA of the yeasts was added during the extraction process). Using a concentration of 10 pmol/ L of the primers, a better detection limit was obtained (100 fg per reaction), comparing the reaction optimized for the plasmid pUC18. With the standardization of the triplex PCR, a very important step for the implementation of a reliable diagnostic tool was concluded. After validation, this new tool may assure the quality of the results, allowing a more accurate diagnosis and faster treatment initiation in infected patients. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [56] P 336 - BIOCHEMICAL ROLES OF LEISHMANIA INFANTUM RAD51 PARALOGS IN HOMOLOGOUS RECOMBINATION GENOIS, M.1; ÉTHIER, C.2; ROY, G.3; UBEDA, J.4; MUKHERJEE, A.5; POIRIER, G.2; OUELLETTE, M.3; MASSON, J.1 1.GENOME STABILITY LABORATORY, LAVAL UNIVERSITY CANCER RESEARCH CENTER, HÔTEL-DIEU DE QUÉBEC, QUÉBEC, CANADA; 2.CANCER RESEARCH UNIT, LAVAL UNIVERSITY MEDICAL RESEARCH CENTER, CHUQ-CRCHUL, QUÉBEC, CANADA; 3.CENTRE DE RECHERCHE EN INFECTIOLOGIE, CHUL, UNIVERSITÉ LAVAL, QUÉBEC, CANADA; 4.CENTRE DE GÉNÉTIQUE ET DE PHYSIOLOGIE MOLÉCULAIRE ET CELLULAIRE, LYON, FRANCE; 5.DEPARTMENT OF IMMUNOLOGY AND INFECTIOUS DISEASES, HARVARD SCHOOL OF PUBLIC HEALTH, BOSTON, UNITED STATES.
Keyword:lirad51 paralogs; drug resistance; dna repair
Abstract: The parasite Leishmania often amplify and delete specific portion of its genome by homologous recombination (HR) at the level of DNA repeated sequences that are widespread in the genome. In fact, Leishmania take advantage of this pathway for drug resistance. However, the interplay between HR and this adaptation system is poorly understood. Inactivation of the Leishmania Rad51 gene reduces the rate of HR at the level of direct repeated sequences leading to extrachromosomal circles. RAD51 protein, the homolog of RecA in E. coli, is a key enzyme promoting DNA strand exchange during HR. In contrast to RecA, RAD51 has significantly lower activity, and the presence of "mediators" are required to stimulate HR by RAD51. Among them, include RAD51 paralogs forming two complexes termed BCDX2 and CX3. Based on bioinformatic analyses, three RAD51 paralogs are conserved in Leishmania, named LiRad51-3 (RAD51-C), LiRad51-4 ( XRCC2) and LiRad51-6 (RAD51-D). To better understand their functions in HR, biochemical studies of the paralogs were undertaken using similar strategies developed for study on LiRad51(Genois et al.,NAR, 2012). First, the gene encoding each protein were cloned into vectors allowing the expression of recombinant proteins in insect cells. We succeded to purify each LiRad51 paralog individually. We show by DNA binding assays and surface plasmon resonance (SPR) that the paralogs interact together and bind DNA. We analyzed on which type of DNA substrates the paralogs preferentially bind, and we report that stable complexes are formed with ssDNA, tailed DNA and D-loop substrates. Consistently on our previous work, the paralogs might help LiRad51 through their ability to promote DNA strand exchange. Our work provides important clues on how HR occurs in the parasite to eventually counteract drug resistance.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [64] P 337 - TREATMENT OF DISSEMINATED LEISHMANIASIS WITH LIPOSOMAL AMPHOTERICIN MACHADO, P.R.L.; ROSA, M.E.; GUIMARÃES, L.H.; SCHRIEFER, A.; CARVALHO, E.M. UFBA, SALVADOR, BA, BRAZIL.
Keyword:disseminated leishmaniasis; liposomal amphotericin b; treatment
Abstract: Disseminated leishmaniasis (DL) is a severe and emerging form of American tegumentary leishmaniasis. DL has been characterized by more than 10 multiple and pleomorphic cutaneous lesions, distributed in more than two noncontiguous parts of the body. DL is a difficult to cure disease and failure to pentavalent antimony therapy is observed in at least 60% of cases. Desoxycholate Amphotericin B is an alternative drug for cases where pentavalent antimony is not effective or cannot be used. However, the administration of Desoxycholate Amphotericin B is limited by its toxicity and high dose needed to cure. We have evaluated Liposomal Amphotericin B in 20 patients with DL. The dose ranged from 21 to 35 mg/kg. The cure rate was higher than 70% and the drug were well tolerated in all cases. We conclude that Liposomal Amphotericin B is a safe and effective therapy for DL. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [66] P 338 - CHEMOTACTIC ACTIVITY OF BETULINIC ACID DERIVATIVES IN LEISHMANIA BRAZILIENSIS SILVA-LOPEZ, A.1; ALCAZAR, W.1; ALAKURTTI, S.2; YLI-KAUHALUOMA, J.2; DIAZ, E.1; PADRON-NIEVES, M.1; PONTE-SUCRE, A.1
1.LABORATORIO DE FISIOLOGÍA MOLECULARY, IME, ESCUELA LUIS RAZETTI, FACULTAD DE MEDICINA, UNIVERSIDAD, CARACAS, VENEZUELA; 2.DIVISION OF PHARMACEUTICAL CHEMISTRY, FACULTY OF PHARMACY, UNIVERSITY OF HELSINKI, FINLAND, HELSINKI, FINLAND.
Keyword:betulinic acid; chemotaxis; l. braziliensis
Abstract: Leishmaniasis is a public health problem in tropical and subtropical areas of the world, including Venezuela. The incidence of treatment failure, and the number of cases with Leishmania-HIV co -infection, stresses the importance of developming alternative, effective and economical therapies. The betulin (lup-20(29)-ene-3(beta),28-diol) is an abundant birch bark triterpene. Its terpenoids derivatives are active against various parasites including Leishmania [1]. The infection begins with Leishmania migration towards the host cell, skin macrophage. In this interaction chemotaxis is a fundamental event. Herein we evaluated the leishmanicidal effect of 30 betulin acid derivatives, as well as the chemotaxis induced by four candidates with demonstrated leishmanicidal activity (IC50 = submicromolar and micromolar range). Chemotaxis was assessed by a modified two chamber capillary technique [2,3]. Our results demonstrated that the derivatives are negative chemotactic on Leishmania. The compounds were classified as follows: B10 and B17, with moderate negative (at 10-10 M), and B11 and B18, with marked negative (at 10-8 M and lower concentrations) chemotactic activity. Additionally, the derivatives herein tested had a clear cut-off concentration dependent chemotactic and leishmanicidal effect. On the contrary, parasite survival was dramatically affected by B17 after 30 min incubation. Finally, our results indicate that B11 inhibits parasites migration ( 40%, 108 - 10-12 M) with an IC50 of 0.25 µM, suggesting that B11 is shaping up as a potential candidate. It prevents host-parasite interaction (nanomolar concentrations) and is leishmanicidal (submicromolar concentrations). To confirm these data we are presently evaluating the leishmanicidal activity of B11 on the macrophage-amastigote model. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [72] P 339 - MOLECULAR STUDY OF THE MRPA TRANSPORTER AND ANALYSIS OF THE INTRACELLULAR ANTIMONY LEVELS IN ANTIMONY–SUSCEPTIBLE AND –
RESISTANT LINES OF FOUR LEISHMANIA SPECIES MOREIRA, D.S.1; MONTE NETO, R.L.2; ANDRADE, J.M.1; FREZARD, F.J.G.2; MURTA, S.M.F.1
1.CENTRO DE PESQUISAS RENÉ RACHOU-FIOCRUZ, BELO HORIZONTE, MG, BRAZIL; 2.DEPARTAMENTO DE FISIOLOGIA E BIOFÍSICA, INSTITUTO DE CIÊNCIAS BIOLÓGICAS, UFMG, BELO HORIZONTE, MG, BRAZIL.
Keyword:drug resistance; potassium antimonyl tartrate; mrpa transporter
Abstract: ATP-binding cassette (ABC) transporters are involved in the ATP-dependent transport of a variety of molecules across biological membranes, including chemotherapeutic drugs. Some members of this superfamily have been associated with drug resistance in various diseases. The MRPA transporter, a member of ABCC subfamily, is involved in the resistance by sequestering metal-thiol conjugate in vesicles close to the flagellar pocket of Leishmania parasite. In this study, promastigote forms of antimony-susceptible and -resistant lines of four Leishmania species, L. (V.) guyanensis, L. (L.) amazonensis, L. (V.) braziliensis and L. (L.) infantum chagasi, were analyzed for: chromosomal location, analysis of amplification and mRNA levels of the MRPA gene, Pgp protein expression and determination of intracellular antimony level. These lines were selected in vitro to potassium antimony tartrate (SbIII) and the resistance index varied from 4 to 20-fold higher than of their wild-type counterparts (Liarte & Murta, 2010). PFGE analysis revealed an association of chromosomal amplification of MRPA gene with the drug resistance phenotype in SbIII-resistant L. (V.) braziliensis and L. (L.) infantum chagasi lines. Southern blot assays with the BamHI endonuclease showed that MRPA gene probe recognized a fragment with higher intensity in the antimony-resistant line of L. (V.) braziliensis, when compared to the susceptible pair. Levels of the mRNA MRPA gene determined by realtime RT-PCR showed an increased expression of two-fold in the antimony-resistant in L. (L.) amazonensis and L. (V.) braziliensis lines compared with their respective susceptible counterparts. Western blot analysis showed that the monoclonal antibody C219 recognized a protein of the expected size 170 kDa. This antibody detected the Pgp protein only in the antimony-resistant L. (V.) guyanensis and L. (L.) amazonensis lines, but not in the susceptible lines of these species. The intracellular level of antimony quantified by graphite furnace atomic absorption spectrometry showed a reduction in the accumulation of this element of approximately seven-, five- and two-fold in the antimony-resistant L. (V.) guyanensis, L. (L.) amazonensis and L. (V.) braziliensis lines, when compared to their susceptible pairs, respectively. Our results indicate that the mechanisms of antimony-resistance of the MRPA gene are different among species of Leishmania analyzed. Financial Support: CNPq, FAPEMIG, CPqRR and UNICEF/UNDP/World Bank/WHO/TDR.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [74] P 340 - DERMOSCOPIC FEATURES OF CUTANEOUS LEISHMANIASIS TAHERI, 1.1; PISHGOOEI, 2.2; MALEKI, 3.2; MASHAYEKHI, 4.2; KIAFAR, 5.2; BANIHASHEMI, M.2 1.RESEARCH CENTER OF CUTANEOUS LEISHMANIASIS, MASHHAD, IRAN, ISLAMIC REPUBLIC OF; 2., RESEARCH CENTER OF CUTANEOUS LEISHMANIASIS,, MASHHAD, IRAN, ISLAMIC REPUBLIC OF.
Keyword:cutaneous leishmaniasis; dermoscopy; dermoscopic features
Abstract: Background: Previous studies have demonstrated that dermoscopy improves accuracy in diagnosing pigmented and non-pigmented tumors. Recently, there has been increasing evidence that dermoscopy can also be useful in the diagnosis of some skin infections.
Objective: We sought to describe the dermoscopic features of cutaneous leishmaniasis (CL). Method: Dermoscopic examination (using the DermLite FOTO, 3Gen) of 144 CL lesions in 82 patients was performed. This study was conducted in the Imam Reza and Ghaem hospitals from October 2010 to September 2011. Results: We observed the following dermoscopic features: generalized erythema (81.9%), white star bursts (60.4%), yellow hue (43.8%), yellow tears (41.7%), central ulcers (59%), hyperkeratosis(33.3%), and milia-like cysts (4.9%).We also observed vascular structures, including dotted vessels(61.1%), hairpin vessels(37.5%), linear irregular vessels(30.6 %), comma-shaped vessels(29.9%), glomerular vessels(22.9 %), arborizing telangiectasia (10.4 %), and corkscrew vessels(4.2%). Limitations: Biopsy and pathologic evaluation were not performed due to ethical considerations. Conclusion: Important vascular patterns seen in melanocytic and non-melanocytic tumors were frequently observed in this infection. Dermoscopy may be a promising tool to predict the clinical course in cases of cutaneous leishmaniasis.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [76] P 341 - SERODIAGNOSIS OF VISCERAL AND TEGUMENTARY LEISHMANIASIS USING L. INFANTUM RECOMBINANT HEAT SHOCK PROTEIN 83 CELESTE, B.J.1; SANCHEZ, M.C.A.1; ASSIS LIMA COSTA, F.2; ANGEL, S.O.3; CASTRO, L.G.M.4; GOTO, H.1 1.INSTITUTO DE MEDICINA TROPICAL, SÃO PAULO, SP, BRAZIL; 2.UNIVERSIDADE FEDERAL DO PIAUÍ, TERESINA, PI, BRAZIL; 3.DEPARTAMENTO DE PARASILTOLOGIA, ANLIS, DR. CARLOS G. MALBRAN, BUENOS AIRES, ARGENTINA; 4.DEPARTAMENTO DE DERMATOLOGIA FMUSP, SÃO PAULO, SP, BRAZIL.
Keyword:leishmania infantum; rhsp83; serodiagnosis
Abstract: For routine serodiagnosis of leishmaniasis, mainly of tegumentary diseases, a defined Leishmania antigen is lacking. In this work the use of Leishmania infantum-recombinant heat shock protein (Hsp) 83 as antigen for ELISA-based diagnosis was studied and compared with ELISA using L. major-like promastigote total antigen. We analyzed sensitivity in the sera from cutaneous (CL; N=12), mucocutaneous (MCL; N=14) and visceral (VL; N=32) leishmaniasis, having 30 healthy blood bank donors (CN) as control and the test specificity, including sera from Chagas’ disease (CH=23) and other infectious disease (OP=56) patients. The cut-off point chosen based on the ROC curve were 0.070 for rHsp83-ELISA and 0.190 for L.major-likeELISA that resulted in a maximum specificity (95% CI) 100.0% (88.43-100.0%). The rHsp83ELISA sensitivity (95% CI) was 96.55 (88.09-99.58) while L.major-like –ELISA was 82.76 (70.57-91.41) (McNemar's test, p=0.013). Considering leishmaniasis and control samples, an excellent agreement was observed using these two antigens, kappa=0.8136 (0.6904-0.9368). Studying samples from other infectious diseases, the overall specificity (95% CI) was 97.47% (91.15-99.69%) using rHsp83 and 70.89% (59.58-80.57%) using L.major-like antigens (McNemar's test, p<0.001), but specifically with samples of patients with Chagas’ disease, the specificity was significantly higher using rHsp83 (McNemar's test, p<0.001). Non-specific reactions with rHsp83 antigen occurred with samples from Aspergilosis and Toxoplasmosis; of the 23 sera reacting with L.major-like antigen, 21 (91.30%) were from Chagas’ disease patients. Then excluding Chagas’ disease in other pathologies, both tests had the same specificity = 96.43% (95% IC=87.69-99.56%) (McNemar's test, p=0.617). In tegumentary leishmaniasis
antibody titer variation was also studied in different periods, before therapy (T0), during the treatment (T1, 6 months), and after the therapy (T2, 12 months). In this assessment using rHsp83-ELISA no significant difference (Friedman Repeated Measures ANOVA on Ranks) was observed in MCL (p=0.607) and CL (p=0.205). In turn, with L.majorlike-ELISA the median values were significantly higher (p<0.05) in T0 as compared to T2 (MCL and CL) and as compared to T1 (MCL). Therefore, we consider the L.infantum-rHsp83 a good antigen for use for serodiagnosis of leishmaniasis.
Supported by FAPESP, CNPq, INCT-INAMI, LIM-38 (HC-FMUSP) - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [81] P 342 - EFFECT OF CERAGENIN CSA-13 ON INFECTION WITH LEISHMANIA (L.) MEXICANA IN THE SEARCH FOR NEW THERAPEUTIC AGENTS AGAINST CUTANEOUS LEISHMANIASIS SANCHEZ, M.A.1; MONTILLA-MATOS, M.L.A.1; MORENO, M.1; AGUILAR, E.1; MILANI, N.1; SAVAGE, P.B.2 1.BIOLOGĂŒA CELULAR LAB, INSTITUTO DE BIOMEDICINA-UCV, MPPS, CARACAS, VENEZUELA; 2.DEPT OF CHEM AND BIOCHEM, BRIGHAM YOUNG UNIVERSITY,, UTHA, UNITED STATES.
Keyword:ceragenins; experimental treatment; leishmaniaisis
Abstract: The high toxicity of drugs currently used against leishmaniasis, and the development of resistant parasites demonstrate the importance of evaluating new therapeutic agents with leishmanicidal activity safe and effective. Previous studies demonstrated in vitro antileishmanicidal activity of the Ceragenin 13 (CSA-13) a Cationic Steroid Antibiotic against Leishmania (L.) mexicana. Therefore, here we evaluate the effect of CSA-13 on Leishmania (L.) mexicana infection in a murine model. 103 amastigotes of L. (L.) mexicana (MHOM/BZ/BEL21) were inoculated in the footpad of female BALB / c mice,. 3 weeks post-infection CSA-13 was applied to groups of mice at different doses(4, 8, 16, 32, 54, 72, and 10x103 5x103 ng/g body weight/day) subcutaneously, intralesional, for 3 consecutive days. We evaluated the progress of the disease for 10 weeks by measuring the parasite load in the cutaneous lesions using the limiting dilution technique and the footpad swelling (lesion size) was determined weekly by digital morphometry using tpsDig2 program. Additionally we assessed the influence of the CSA-13 on the weight of the experimental animals and blood chemistry parameters. RESULTS: A reduction of the parasitic load in the groups of mice treated with 4, 8, 16, 32, 54 and 72 ng/g weight/day CSA-13 was evidenced, after 3 weeks post-treatment, with respect the control group (PBS), we also determined that the dose 4 and 8 ng/g weight/day of CSA-13 were more effective in terms of decreasing the size of the cutaneous lesions in BALB/c mice infected with L. (L.) mexicana. Moreover, the CSA-13 did not affect the weight of BALB/c mice but causes a slight alteration of blood levels of transaminases, and blood glucose amylase, indicating a possible hepatic and pancreatic compromise of these experimental animals. This antibiotic might represent a useful tool in combination with new drugs against cutaneous leishmaniasis. Funded by: CDCH UCV PSU-09-7878-2009/1; FONACIT G-2005000375 MS - CLINICAL LEISHMANIASIS [88] P 343 - ARGINASE I, PGE2 AND TGF- 1 IN DIFFUSE CUTANEOUS LEISHMANIASIS PATIENTS ARE ASSOCIATED WITH IMMUNOSUPPRESSIVE RESPONSE TO LEISHMANIA
FRANÇA-COSTA, J.; BOAVENTURA, V.; MALTA, H.; LUZ, N.; CAMPOS, D.; OLIVEIRA, M.; BARRAL, A.; COSTA, J.; BORGES, V.M. BFEDERAL UNIVERSITY OF BAHIA/INCT/III, SALVADOR, BA, BRAZIL.
Keyword:arginase; diffuse cutaneous leishmaniasis; leishmania amazonensis
Abstract: Diffuse Cutaneous Leishmaniasis (DCL) is a rare clinical manifestation of tegumentary leishmaniasis caused by Leishmania amazonensis. It is characterized by an inefficient parasitespecific cellular response and heavily parasited macrophages. It has been demonstrated “in vitro” that murine macrophage infection by L. amazonensis increases arginase I, TGF- 1 and PGE2 contributing to parasite proliferation enhancement. However, the relevance of these mediators for DCL pathogenesis remains unknown. Here, we evaluate systemic and local expression of inflammatory mediators in DCL patients and the role of arginase inhibition in vitro. Plasma from 12 active DCL and 29 Localized Cutaneous Leishmaniasis (LCL) patients were evaluated for arginase I, TGF- 1, PGE2, TNF, IL-12 and MCP-1 levels by ELISA. Whereas Arginase I, TGF- 1 and PGE2 were increased, TNF, IL-12 and MCP-1 were decreased in DCL plasma when compared with LCL, supporting an anti-inflammatory immune profile in DCL patients. Moreover, arginase I and cicloxigenase 2 expression were higher in DCL lesions than LCL lesions. In order to know if arginase could alter Leishmania infection, human monocytederived macrophages from health volunteers were infected with L. amazonensis and treated with NOHA (arginase inhibitor). The infectivity index 72 h post infection in the group treated with NOHA was lower than unstimulated (UNST) infected group. Interestingly, arginase inhibition with NOHA decreased TGF- 1 and PGE2 production when compared with the UNST group. Our data suggest that arginase, PGE2 and TGF- 1 might be implicated in the inability of DCL patients to mount an efficient immune response against L. amazonensis.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [93] P 344 - IN VITRO BIOLOGICAL ASSAYS OF CROTON LECHLERI MUELL.ARG AND MORINDA CITRIFOLIA L. AGAINST PROMASTIGOTE LEISHMANIA (L.)AMAZONENSIS. DOS SANTOS, A.P.A.; PACHECO, S.G.; TELES, C.B.G. FACULDADE SÃO LUCAS, PORTO VELHO, RO, BRAZIL.
Keyword:chemotherapy ; croton lechleri
Abstract: Leishmaniasis is an infectious disease caused by protozoa of the genus Leishmania, it is transmitted to humans by the bite of the phlebotomine sandflies. The chemotherapy used is pentavalent antimonials glucantime and pentamidine, the drugs used are not fully effective and may cause side effects because their levels of toxicity. The species Croton lechleri Muell Arg (Dragon's blood) and Morinda citrifolia L. (Noni) are plants known by presenting antiinflammatory potential, popularly used in combating various diseases. This study was in vitro assay of extracts of Croton lechleri Muell. Arg and Morinda citrifolia L. against promastigote Leishmania (L.) amazonensis (IFLA/BR/67/PH8). The plant extracts were obtained using native plant material (tree bark and leaf) and introduced plant material (tree bark) of C. lechleri Muell Arg and introduced Morinda citrifolia L. (fruit), both donated by the community Santa Marcelina, Porto Velho-RO. To evaluate leishmanicidal extracts were resuspended in PBS at
concentrations of 0.0025 g/ml and 0.005 g/ml. The tests were carried out using PBS as a negative control for 5 consecutive days, aliquots of the cultures were diluted in RPMI containing the dye Erythrosine B. The counting of the strain is made in a Neubauer hemocytometer chamber with the aid of optical microscope with 400X magnification. The extracts showed antileishmanial action at a concentration of 0.005 g/ml. The results were released in statistical analysis and found 90% of inhibitory activity for the tree bark extract introduced and 85% inhibitory to native leaf extract of C. lechleri Muell Arg, the rate of growth inhibition for the stratum of Morinda citrifolia L. was 84%. The tests showed promising, because the tested extracts showed inhibitory action of up to 80% in the growth of Leishmania amazonensis. Thus indicating that further studies should be performed, seeking to improve the results, which may contribute to the emergence of a new alternative therapy with leishmanicidal activity of minor side effects and greater economic viability in the treatment of leishmaniasis. - GENETICS, EVOLUTION AND TAXONOMY [94] P 345 - CHARACTERIZATION OF PHLEBOTOMINE INFECTION WITH THE LEISHMANIA BRAZILIENSIS COMPLEX USING MULTIPLEX PCR IN A BRAZILIAN HIGH ENDEMIC AREA.ASSIS BRASIL, STATE OF ACRE, WESTERN AMAZON, BRAZIL. TELES, C.B.G.1; DOS SANTOS, A.P.A.2; CUNHA, P.N.A.2; DE ARAÚJO, E.A.C.1; FREITAS, R.A.3; OGAWA, G.M.4; MEDEIROS, J.F.5; PESSOA, F.A.C.6; CÁCERES, O.C.1; CAMARGO, L.M.A.4 1.DEPARTAMENTO DE MEDICINA, FACULDADE SÃO LUCAS, PORTO VELHO, RO, BRAZIL; 2.FACULDADE SÃO LUCAS, PORTO VELHO, RO, BRAZIL; 3.LABORATÓRIO DE LEISHMANIOSE E DOENÇA DE CHAGAS, INPA, MANAUS, AM, BRAZIL; 4.DEPARTAMENTO DE PARASITOLOGIA, INSTITUTO DE CIÊNCIAS BIOMÉDICAS, UNIVERSIDADE DE SÃO PAULO., MONTE NEGRO, RO, BRAZIL; 5.COORDENAÇÃO SOCIEDADE, AMBIENTE E SAÚDE, INPA, MANAUS, AM, BRAZIL; 6.GRUPO DE PESQUISA EM ECOLOGIA DE DOENÇAS TRANSMISSÍVEIS NA AMAZÔNIA, INSTITUTO LEÔNIDAS E MARIA DEAN, MANAUS, AM, BRAZIL.
Keyword:leishmania; sand flies; pcr-multiplex
Abstract: Cutaneous Leishmaniasis(CL) is an infectious disease caused by parasitic protozoa of the genus Leishmania. It is transmitted to humans by the bite of hematophagous insects of the subfamily Phlebotominae fed on hosts previously infected with the parasite. It´s among one of the parasitic diseases of highest incidence in the world, with annual records reaching up to 2 million cases. In Brazil, CL occurs in all states, with an increasing incidence rate in the north region, mainly in thecity of Assis Brasil (state of Acre) with an average incidence rate of 1, 098 cases of CL per 100,000 inhabitants between the years 2001 to 2010 (10-times Brazilian incidence rate). This study aimed to detect the local etiological agent and the vector (s) involved in the epidemiological cycle of CL in this municipality. Sand flies were captured with the use of CDClight traps, every 2 months, for a 10-day capture period, between 2009 and 2010. The females were identified and were grouped in 282 pools (2 to 20 specimens/vial). Afterwards, DNA extraction was performed and the PCR technique was applied in the investigation of the presence of Leishmania (Kinetoplastida: Trypanosomatidae) parasites in pools of phlebotomine sandflies. Of the 282 pools, 44 pools were positive: 25 pools with Lutzomyia davisi and 19 with L. auraensis. To determine the possible complex involved, the samples of DNA extracted from pools of the 44 naturally infected sand flies were analyzed by Multiplex-PCR assay was targeted to the spliced leader RNA (mini-exon) gene repeats of these organisms and can detect all three complexes simultaneously generating differently sized products for each complex of New World Leishmania (Leishmania braziliensis, Leishmania mexicana, and Leishmania donovani). All the 44 tested pools were positive for the LB-3C, complex which corresponds to the Leishmania braziliensis complex. Further studies are being carried out in order to try to identify other etiological agents, vectors and natural reservoirs.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [103] P 346 - IN VITRO AND IN VIVO EFFECTS OF APIGENIN AGAINST LEISHMANIA (LEISHMANIA) AMAZONENSIS SILVA, F.F.; INACIO FILHO, J.D.; CANTO-CAVALHEIRO, M.M.; ALMEIDA-AMARAL, E.E. FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:effects; apigenin; leishmania amazonensis
Abstract: Leishmaniasis is included in the list of neglected tropical diseases, has been reported in 88 countries and affects more than 12 million people worldwide. The development of new, safer, cheaper and orally available drug treatments for leishmaniasis is urgently needed. Apigenin is a flavonoid present in common fruits and vegetables and has been shown to possess diverse therapeutic potentials. However, its mechanism of protozoan action has not been studied yet. In this present study, we report the mechanism for the antileishmanial activity of apigenin against both forms of Leishmania amazonensis. Apigenin inhibited promastigote and amastigote growth in a dose-dependent manner - with 24 hours of treatment reaching 74% of inhibition at the concentration of 96µM (IC50 = 23.68 M) and with 72 hours of treatment reaching 71% of inhibition at the concentration of 12µM (IC50 = 4.33 M) - respectively. We observed the activity of apigenin on amastigotes at nontoxic concentrations for macrophages. Reactive oxygen species (ROS) production was increased in a dose-dependent manner in L. amazonensis promastigotes treated with increase concentration of apigenin (3 – 96 µM) and in infected macrophages at the concentration of 12µM. Pre-incubation of both forms of L. amazonensis with reduced glutathione (GSH) or N-Acetyl-cysteine (NAC) significantly reduced apigenininduced cell death. In addition, apigenin caused mitochondrial dysfunction due to collapse of mitochondrial membrane potential in L. amazonensis promastigotes. Apigenin has been described as a pro-oxidant, generating ROS which are responsible for cell death in some cancer cells. Mitochondrial membrane potential loss can be brought about by ROS added directly in vitro or induced by chemical agents. For in vivo studies, apigenin was administered orally (1 and 2 mg/Kg/day, every day) to L. amazonensis-infected BALB/c mice lasting 45 days. Apigenin was able to control lesion and parasite growth in these mice, without altering serological markers of toxicity (alanine aminotransferase, aspartate aminotransferase and creatinine). Apigenin (2mg/Kg/day) was more effective than pentavalent antimonial in preventing lesion development. Taken together, our results demonstrate the in vitro and in vivo antileishmanial activity of apigenin on L. amazonensis due to the generation of ROS and disrupted parasite mitochondrial function. These characteristics encouraging and suggests the apigenin as a possible prototype for the clinical treatment of cutaneous leishmaniasis. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [107] P 347 - IMMUNODIAGNOSIS OF CANINE VISCERAL LEISHMANIASIS USING PHAGE DISPLAY TECHNIQUE MACHADO, C.M.T.; FUJIWARA, R.T.; OLORTEGUI, C.D.C. UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL.
Keyword:immunodiagnosis; synthetic peptides; phage display
Abstract: Visceral leishmaniasis (VL) is a widespread zoonosis in tropical and subtropical regions of the planet, which causes high numbers of deaths. In Brazil, the country with the highest register of cases of Canine Visceral Leishmaniasis (CVL) in the Americas (WHO, 2008), the disease is caused by the protozoan parasite Leishmania chagasi (syn. Leishmania infantum) and is transmitted by phlebotomine sandflies of the Lutzomyia genus. In canine visceral leishmaniasis,
the abnormalities most commonly observed in clinical examination on the animals are lymphadenomegaly and skin lesions. Dogs are the main domestic reservoir of the disease. Visceral leishmaniasis diagnosis has been largely based on microscopic examination of thin smears of bone marrow, although serological and molecular methods constitute less invasive alternatives currently in use. In this study, three antigens were selected through Phage Display technique for the immunodiagnosis of canine visceral leishmaniasis. The DNAs were extracted, sequenced and synthetized through chemical synthesis in its soluble forms. These peptides were linked to a Glutharaldeyde (GLU) molecule and then evaluated through indirect ELISA using dog’s positive and negative serum to visceral leishmaniasis. The cross-reactivity was also evaluated using dog’s serum infected with Trypanossoma cruzi parasite. All three peptides were highly sensitive and specific for the diagnosis of visceral leishmaniasis when compared to soluble antigen. Thus, Phage Display technique represents a valuable tool for diagnosis of CVL and can be used on routine to make a diagnosis of leishmaniasis. - CONTROL PROGRAMS [112] P 348 - NEW TOOLS FOR MONITORING DRUG RESISTANCE AND TREATMENT RESPONSE IN VISCERAL LEISHMANIASIS IN THE INDIAN SUBCONTINENT DUJARDIN, J. INSTITUTE OF TROPICAL MEDICINE, ANTWERPEN, BELGIUM.
Keyword:kala azar elimination programme; drug resistance; monitoring
Abstract: Early treatment is a major pillar of the program for Visceral Leishmaniasis (VL) elimination in the Indian sub-continent (ISC). While waiting for new drugs or therapeutic schemes, the effectiveness of current drugs needs to be safeguarded. In the frame of the Kaladrug-R initiative (EC project: EC-FP7-222895) we contributed to this effort and aimed to understand drug resistance and develop tools to monitor it. 1. We developed simple clinical and epidemiological tools for monitoring the effectiveness of VL drug treatments in routine conditions. 2. We report a substantial decline in the efficacy of MIL (90.48% cure rate at 6 months in India and 73.3% at 12 months in Nepal). Although MIL-tolerant strains occur, the observed decline of MIL effectiveness is not related to MIL resistance of the parasite. MIL blood levels at the end of treatment were similar for cured and relapsed patients, but lower in children <12yrs. Incidence risk for relapse in the Nepal study was 3 times higher in children compared to adolescents and adults, possibly due to underdosing.. We are currently exploring other factors like the virulence of the parasites. We are also checking if any MIL-resistance phenotype might not be lost in culture. 3. The decreasing efficacy of MIL could be an heritage of the past pressure of antimonials (SSG). Mathematical modelling predicted that SSG-resistant (SSG-R) parasites were likely more fit than sensitive ones. Different experimental lines confirmed this prediction and showed that SSG-R parasites had better survival skills and might be more virulent than their sensitive counterparts. 4. Therefore, we further focused research on SSG-resistant strains. Using next generation sequencing, we deciphered the complete genome of 200 clinical isolates and described different signatures of resistance (SNPs and structural variation), opening the way to simplified molecular assays for resistance screening. Metabolomic profiling evidenced molecular adaptations which could jeopardise the efficacy of the other drugs (a.o. membrane fluidity). We also showed that SSG-R parasites better manipulated the macrophage for their own survival. 5. We experimentally induced resistance to MIL and Paromomycin on strains from the SSG era; results from genome and metabolome studies will be presented. 6. We described the evolution of L. donovani through the SSG and MIL eras and situated it in a worldwide context. Our research demonstrates the complexity of the problem of clinical drug resistance in VL and highlights the need of multidisciplinary approaches to tackle it (see specific talks or posters for details).
- GENOMICS, TRANSCRIPTOMICS, PROTEOMICS, METABOLOMICS [115] P 349 - COMPARATIVE PROTEOMIC ANALYSIS OF MILTEFOSINE-SENSITIVE AND MILTEFOSINE-RESISTANT LEISHMANIA CHAGASI CLINICAL ISOLATES CARNIELLI, J.B.T.1; PIRES, S.F.2; ANDRADE, H.M.2; MONTI-ROCHA, R.1; PANSINI, L.N.N.1; DIETZE, R.1; FIGUEIREDO, S.G.1; LEMOS, E.M.1 1.UNIVERSIDADE FEDERAL DO ESPÍRITO SANTO, VITÓRIA, ES, BRAZIL; 2.UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL.
Keyword:miltefosine resistance; leishmania chagasi; proteomics analysis
Abstract: Visceral leishmaniasis is a serious disease caused by protozoan parasites called Leishmania. The treatment for this disease is difficult, as it requires prolonged and painful applications of toxic drugs that are poorly tolerated. Therefore, one of the most significant advances in the leishmaniasis therapy was the identification of an effective and safe oral drug, the miltefosine. However, different sensitivities to miltefosine by Leishmania spp have been observed in clinically relevant species and the biological mechanism whereby Leishmania clinical isolates acquire drug resistance is poorly understood. In this context, comparative two-dimensional gel electrophoresis and mass spectrometry methodologies were applied to highlight and identify proteins that are differentially expressed between miltefosine-sensitive and miltefosine-resistant L. chagasi clinical isolates. It is described here a high-resolution proteome for L. chagasi promastigotes comprising an average of 459 spots, which corresponds to 5,7% of gene products predicted for Leishmania spp. Following comparison of the whole proteome profiles between sensitive and resistant strains, 67 differentially expressed spots were detected. Eighteen spots were found to be specific of a sensitive group and only one of a resistant group, while 48 spots changed in intensity between these groups. MALDI/TOF-TOF mass spectrometry allowed the identification of 48 spots (71,6%), corresponding to 39 distinct proteins of which seven are hypothetical proteins. Among the proteins identified, the comparative proteomics screen highlighted the peroxidoxin, a serine/threonine protein phosphatase, a calpain-like cysteine peptidase and a 4-3-3 protein-like protein that have been reported to play a role in the druginduced programmed cell death in parasites, suggesting that resistance mechanisms to miltefosine are possibly associated with a lesser susceptibility to apoptosis. Our findings demonstrate that proteomic screen has great potential to identify protein differences between closely related parasite strains and can be used to distinguish parasite phenotypes of clinical relevance. Supported by: CNPq, CAPES and FAPES.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [116] P 350 - USE OF A RECOMBINANT CYSTEINE PROTEINASE FROM LEISHMANIA (LEISHMANIA) CHAGASI FOR IMMUNOCHEMOTHERAPY OF CANINE VISCERAL LEISHMANIASIS FERREIRA, J.H.L.1; KATZ, S.2; BARBIERI, C.L.2
1.UNIVERSIDADE FEDERAL DO PIAUÍ, TERESINA, PI, BRAZIL; 2.ESCOLA PAULISTA DE MEDICINA - UNIVERSIDADADE FEDERAL DE SÃO PAULO, SÃO PAULO, SP, BRAZIL.
Keyword:leishmania (leishmania) chagasi; cysteine proteinase; canine visceral leishmaniasis
Abstract: A recombinant cysteine proteinase from Leishmania (Leishmania) chagasi, rLdccys1, was previously shown to be a useful immunological marker for stages of visceral leishmaniasis (VL) in humans and dogs, as well as a potential tool for the diagnosis of human and canine VL (Pinheiro et al., 2005; Dias et al., 2005; Pinheiro et al., 2009). Furthermore, immunization with rLdccys1, as well as with the gene encoding the cysteine proteinase Ldccys1, induced significant protective immune responses in a mouse model of VL (Ferreira et al., 2008). Preliminary results also revealed that immunization with rLdccys1 resulted in a significant protection in dogs after challenge with L. (L.) chagasi (Pinheiro, 2009). These results encouraged us to ask if rLdccys1 could be used in immunotherapy of naturally infected dogs from an endemic region of VL, Teresina, Piauí state of Brazil. Thirty symptomatic naturally infected mongrel dogs were divided in three groups. One group received three doses, with one month interval, of rLdccys1 plus Propionibacterium acnes as adjuvant, a second group received three doses of P. acnes only and a third group received saline. Improvement of clinical parameters (alopecia, onychogryphosis, cachexia, anorexia, apathy, skin lesions, hyperkeratosis, ocular secretion, increasing of lymph nodes and weight loss), besides a significant reduction of spleen parasite load were observed within three months in dogs that received rLdccys1 + P. acnes, in comparison to controls which received either P. acnes only or saline. Evaluation of humoral and cellular immune responses was performed one month after the end of treatment. A more intense delayed type hypersensitivity reactions against L. (L.) chagasi lysate were observed in dogs treated with rLdccys1 when compared to controls. Furthermore, a higher IgG2 and a decreased IgG1 response followed by a high IFN-gamma and a low IL-10 concentration were detected in serum from rLdccys1-treated dogs, pointing to a Th1 cell mediated response. In contrast, dogs which received P. acnes only or saline exhibited low serum levels of IgG2 and IFN-gamma and high concentrations of IgG1 and IL-10. All of the dogs treated with rLdccys1 until 12 months after treatment survived, whereas control dogs which received either saline or P. acnes only died between 3 and 6 months. These findings support the potential use of recombinant L. (L.) chagasi cysteine proteinase for immunotherapy of canine visceral leishmaniasis. - OTHER SPECIAL TOPICS [122] P 351 - ISOLATION OF ANTILEISHMANIAL SECONDARY METABOLITES FROM THE OCTOCORAL LEPTOGORGIA PUNICEA: AN IN VITRO STUDY MARTINS, L.F.; ALEXANDRE, T.R.; MIGOTTO, Á.; TEMPONE, A.G. INSTITUTO ADOLFO LUTZ, SÃO PAULO, SP, BRAZIL.
Keyword:natural products ; drug prototypes; leishmania
Abstract: Metabolites from marine invertebrates has been considered a promising source of drug prototypes and has been originating clinically used drugs as AZT (AIDS), Cytosar-U (leukemia), Vira-A (herpes) and Acyclovir (herpes). Considering the small number of drugs in clinical use against Leishmaniasis and their high toxicity, the need for new therapeuticals is urgent. This work aimed the discovery of new marine secondary metabolites with antileishmanial activity, isolated from the octocoral Leptogorgia punicea. This octocoral was collected by scuba diving in North cost of São Paulo state, Brazil, dried and triturated in organic solvent. The methanolic extract of L. punicea showed antileishmanial activity against promastigotes, with a 50% Inhibitory Concentration value of 93.30 g/mL. By using liquidliquid partition, the methanolic extract was fractionated and the ethyl acetate fraction, containing most of medium polarity compounds, showed 100% killing of promastigotes of Leishmania (L.) infantum at 300 g/mL (named LPEAc). The chromatographic bio-guided fractionation of LPEAc using molecular exclusion chromatography (Sephadex LH-20) resulted in 10 new
subfractions (LPEAcLH 1-10), including two active fractions (LPEAcLH 4 and 5), which killed 100% of parasites at 100 g/mL. RP-HPLC-PDA chromatography of LPEAcLH-4 resulted in 14 new subfractions (LPEAcLH/HPLC 1-14), which were tested against the promastigotes. By using the tetrazolium colorimetric assay with MTT, LPEAcLH/HPLC-5 and LPEAcLH/HPLC11 showed a leishmanicidal effect at concentrations below 100 g/mL. The structural elucidation of isolated compounds is under investigation by Liquid Chromatography-Mass Spectrometry (LC-MS-TOF) and NMR analysis. These results indicate the potential of the marine natural products as novel drug prototypes for neglected diseases. Support: FAPESPBIOTA/CAPES - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [125] P 352 - EVALUATION OF THE SYNERGIC EFFECTS OF ERGOSTEROL BIOSYNTHESIS INHIBITORS IN LEISHMANIA AMAZONENSIS MACEDO SILVA, S.T.1; URBINA, J.A.2; DE SOUZA, W.1; RODRIGUES, J.C.F.1 1.INSTITUTO DE BIOFÍSICA CARLOS CHAGAS FILHO, RIO DE JANEIRO, RJ, BRAZIL; 2.INSTITUTO VENEZOLANO DE INVESTIGACIONES CIENTÍFICAS, CARACAS, VENEZUELA.
Keyword:ergosterol; leishmaniasis; synergism
Abstract: Leishmaniases are among the most prevalent neglected tropical diseases and are endemic in 98 countries. The treatments include the pentavalent antimonials, miltefosine, amphotericin B, and pentamidine. However, such drugs are unsatisfactory due to toxicity, limited efficacy, cost and administration. Therefore, there is an urgent need to develop new drugs that are efficacious, safe, and more accessible. Trypanosomatids have an essential requirement for ergosterol and other 24-alkyl sterols, which are absent in mammalian cells. The inhibition of ergosterol biosynthesis (EB) has been recognized as a promising target for the development of new chemotherapeutic agents. Thus, the aim of this work was to investigate the effects of E5700, a squalene synthase inhibitor, in combination with itraconazole (ITZ) or posaconazole (POSA), two known inhibitors of the sterol C14 -demethylase (CYP51), against Leishmania amazonensis in vitro. E5700, ITZ and POSA alone produced a marked reduction in the viability of L. amazonensis promastigotes, with MIC (minimum inhibitory concentration) values of 30 nM, 1 µM, and 1 µM, respectively. Several combinations were tested and the most efficient was the combination of 1.25 nM E5700 with 40 nM ITZ or 0.625 nM E5700 with 5 nM POSA, which resulted in FIC (fractional inhibitory concentration) values of 0.082 for the combination of E5700 with ITZ and 0.026 for E5700 with POSA, indicating a potent synergistic effects. Against intracellular amastigotes, the MICs for E5700, ITZ and POSA alone were 30 nM, 1 µM, and 1 µM, respectively. The results indicated again strong synergism, with MICs of 2.5 nM E5700 plus 20 nM ITZ (FIC=0.103) and 2.5 nM E5700 plus 2.5 nM POSA (FIC=0.085). Differential interference contrast microcopy (DIC) revealed a significant alteration on the shape of promastigotes after treatment with E5700 in combination with POSA, more than that observed with ITZ. Transmission electron microscopy of treated-parasites showed several alterations such as: 1) Presence of lipid bodies; 2) Intense mitochondrial swelling followed by the loss of matrix content; and 3) Presence of autophagosome-like structures. In summary, our results indicate that combinations of EB inhibitors acting at different steps of the pathway have synergistic activity against L. amazonensis and open up the possibility of a novel combination therapies for the treatment of leishmaniasis. Supported by CNPq, CAPES, and FAPERJ. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [127] P 353 - SEROLOGIC SURVEY OF LEISHMANIASIS IN IBN SINA PATIENTS, SIRTE, LIBYA BAGAR, S.R.A.
INFECTIOUS DISEASE, SIRTE, LIBIA.
Keyword:leishmaniasis, sirte; leishmaniasis, libia; leishmaniasis, hospital
Abstract: Background : Since the leishmaniasis was a huge problem in North Africa for this reason we carrying out this study , Serologic survey of leishmaniasis in Ibn Sina patients, Sirte, Libya Material and Methods : The clinical and parasitological features of leishmaniasis were investigated, retrospectively, in 162 patients who attended the out-patient clinic of Ibn Sinha hospital during January 2009 and June 2010. The aim : was to evaluate the epidemiological characteristics of leishmaniasis. Sera from all the suspected patients were examined for antibodies against Leishmania spp. by IFA and enzyme linked immunosorbent assay (ELISA). Results : Seventy one of 162 (43.82%) had antibodies against Leishmania spp. of which, 49 (69%) were males and 22 (31%) females. Discussion & Conclusion The most affected age group was found to be between 21 and 30; the least affected was age group below 10 years. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [134] P 354 - PHOTODYNAMIC TREATMENT USING SILICON BIS (DIMETILAMINOETANOXI)-PHTHALOCYANIN AND LED IRRADIATION REDUCES CELL VIABILITY OF J774.1 CELLS INFECTED WITH L. AMAZONENSIS. MITTMANN, J.; CORTEZ MARCOLINO, L.M.; DE OLIVEIRA SILVA, E.P.; JUNIOR, M.B.; DE OLIVEIRA, M.A. UNIVERSIDADE DO VALE DO PARAÍBA, SÃO JOSÉ DOS CAMPOS, SP, BRAZIL.
Keyword:pdt; leishmania; phtalocyanin
Abstract: The available antileishmanial drugs are toxic, expensive, have limited effectiveness and the treatment frequently fails due to parasite drug resistance. Because cutaneous leishmaniasis (CL) is characterized by skin lesions, application of photodynamic therapy (PDT) may be a treatment option. This therapeutic modality has been proposed as a promising alternative therapy for the treatment of CL. The results showed leishmania eradication, low scar formation and superior esthetic effects when compared to conventional treatments. Despite the promising results obtained in clinical trials, the lack of knowledge about the mechanism of action of this therapeutic modality for CL control indicates that additional studies are necessary. Previous studies in our laboratory suggest that silicon phtalocyanins are able to kill leishmania promastigotes. Here we investigated the reduction of cell viability of J774.1 cells infected with L. amazonensis using a Silicon bis (dimetilaminoetanoxi)-phthalocyanine (SiPc) as
photosensitizer and LED as light source. Infections with 10:1 promastigotes in stationary-phase per J774.1 (1x104 cells/mL) in 96-well plates cells were performed. After internalization (24 h) cells were were incubated with SiPc at 0.1 or 1.5 M for 1 h, then supernatant was discarded and the wells washed twice with PBS to remove unabsorbed SiPc. Cells were irradiated with Biotable (Biopdi) at 660 nm and fluence of 10 J/cm2 (maximum of 25 mW/cm2). After irradiation, the PBS was removed and RPMI medium plus 10% FBS was added and the culture was maintained for 20 h in standard conditions. The controls were cells without treatments, cells incubated with SiPc and cells only irradiated. Cell viability was determined using the MTT method. Two independent assays were performed in quintuplicates. The SiPc was not toxic in the dark to J774.1 with or without intracellular amastigotes. The percentage of viable cell was reduced after irradiation when compared with controls (not irradiated cells). Although, infected J774.1 cells were more resistant to PDT (p < 0,01).Tthe percentage of viable cells in infected J774.1 was 22.8% and uninfected cells was 5.08%. Leishmanias are able to evade the immune system, and this survival process are correlated with the increased macrophage survival. The increase in macrophage viability indicated that more than one PDT session would be required to remove all the infected cells killing the intracellular amastigotes. - CLINICAL LEISHMANIASIS [137] P 355 - FOLLOW-UP OF VISCERAL LEISHMANIASIS CASES TREATED WITH MEGLUMINE ANTIMONIATE AND BAD PROGNOSTIC FACTORS IN KAIROUAN, CENTRAL TUNISIA AOUN, K.1; AISSI, W.1; HABBOUL, Z.2; BEN ABDA, I.1; BENHELEL, K.2; BEN SGHAIER, I.1; AMRI, F.2; BOURATBINE, A.1 1.RESEARCH LAB LR 11-IPT-06 “MEDICAL PARASITOLOGY, BIOTECHNOLOGY & BIOMOLECULES�, INSTITUT PASTEUR, TUNIS, TUNISIA; 2.PEDIATRICS DEPARTMENT, KAIROUAN, TUNISIA.
Keyword:visceral leishmaniasis; prognosis factors; tunisia
Abstract: Visceral leishmaniasis (VL) is emergent in Tunisia. Children less than 5 years are the most affected. The case fatality rate is still high (5-8%). The aim of this study was to evaluate VL cases response to Meglumine antimoniate (MA) and to ascertain bad prognostic factors. Data of 205 VL cases were collected in the pediatric department of Kairouan hospital. All patients received a 28 days MA course as recommended by WHO. Statistic analysis was performed by Stata 11. The median age was 18 months. The most frequent symptoms on admission were splenomegaly (97.6%), fever (81.3%) and hepatomegaly (46%). Biological disturbances were anemia (91.2%), thrombocytopenia (83.1%) and leucopenia (55.9%). Parasite loads were over 1000 parasites/ml of blood among 70 patients. All isolated strains (n=67) corresponded to Leishmania infantum species. Patients were classified, according to the disease course, into cured (n=185), unresponsive (n=9) and dead (n=11). The lethality rate was 5.4%. In cured patients, fever has disappeared within 3 days to a week. Splenomegaly decline was slow; 164 of the 185 cured patients (88.6%) still had this sign at the end of the treatment. A drastic increase in hemoglobin and platelets levels was observed.
Bad prognostic factors were hemorrhage (p<0,001), 4 times higher serum hepatic transaminases levels (p=0.001), severe anemia (p=0.003), low age (p=0.018), severe thrombocytopenia (p=0.022) and severe leucopenia (p=0.025). Compared to previous reports, VL is better managed in Tunisia (shorter diagnosis delays and lower fatality rate). However, more efforts should be supplied. Low age, severe anemia and thrombocytopenia should be considered as alarming signs. It is also mandatory to introduce alternative treatments such as liposomal Amphotericine B for a shorter hospitalization and better efficiency. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [143] P 356 - THE EFFECT OF BUTHIONINE SULPHOXIMINE ON THE SURVIVAL OF DIFFERENT LEISHMANIA SPECIES DORO, B.1; WESTROP, G.1; WIESE, M.1; WILLIAMS, R.2; SHAW, C.1; BURCHMORE, R.3; MULLEN, A.1; CARTER, K.1 1.STRATHCLYDE INSTITUTE OF PHARMACY AND BIOMEDICAL SCIENCES, GLASGOW, UNITED KINGDOM; 2.SCHOOL OF SCIENCE, UNIVERSITY OF THE WEST OF SCOTLAND, PAISLEY, UNITED KINGDOM; 3.FACULTY OF BIOMEDICAL AND LIFE SCIENCES, UNIVERSITY OF GLASGOW, GLASGOW, UNITED KINGDOM.
Keyword:enzyme; drug; biochemistry
Abstract: Glutathione (L- -glutamyl-L-cysteinyl-glycine; GSH) is the precursor of dithiol trypanothione [(bis(glutathionyl) spermidine], the major intracellular thiol antioxidant in Leishmania and other trypanosomatids. Trypanothione helps to maintain a cell’s redox potential and functions in many other biological processes such as protein and DNA synthesis; detoxification of free radicals, peroxides, and endogenous and exogenous toxins. Buthionine sulphoximine (BSO) is an irreversible inhibitor of gamma glutamyl cysteine synthetase ( GCS), an enzyme which catalyses the rate-limiting step in GSH biosynthesis. Previous studies have shown that BSO can inhibit the survival of Leishmania donovani both in vitro and in vivo. In this study experiments were carried out to determine whether BSO had a similar effect on L. mexicana and L. major, which are known to interact differently with their host cells. Therefore the effect of BSO treatment on the survival of promastigotes and intracellular amastigotes of L. donovani, L. mexicana, and L. major, and the inhibition of recombinant GCS from each species were aligned using the ClustalW2 Multiple Sequence Alignment Tool . The proteins of all three species showed more than 87.6%) % amino acid identity suggesting a similar degree of inhibition by BSO. Treatment with BSO significantly affected the survival of all three Leishmania species (n=4), with L. donovani having the highest susceptibility to BSO at both the promastigote (mean IC50±SE, for L. donovani, 1.3±0.29mM; L. mexicana, 2.4±0.68 mM , L. major, 1.8±0.017mM;) and intracellular amastigote stage. These results were also reflected by the effect of BSO on the recombinant proteins of all three species with L. mexicana GCS being most resistant to inhibition (mean % inhibition of specific activity with 1 mM BSO (n=4); L. donovani, 86%±0.02; L. mexicana, 45%±0.19; L. major, 63%±0.17). The results of this study indicate that inhibition of GSH production to treat leishmaniasis requires development of species-specific inhibitors. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [145] P 357 - HUMAN TEGUMENTARY LEISHMANIASIS DUE TO LEISHMANIA (VIANNIA) BRAZILIENSIS IN CORRIENTES, ARGENTINA REA, M.J.; CAMPAGNARO, G.; BORDA, C.E.; STEINDEL, M. CENTRO NACIONAL DE PARASITOLOGÍA Y ENFERMEDADES TROPICALES (CENPETROP), FACULTAD MEDICINA, UNNE, CORRIENTES, ARGENTINA.
Keyword:leishmania braziliensis ; corrientes; argentina
Abstract: American tegumentary leishmaniasis ATL is endemic in the Province of Corrientes, located in the Northeast of Argentina. In the Cenpetrop 147 ATL cases, including the mucosal and mucocutaneus forms in 25 patients have been diagnosed during 1988-2011 of which 15% were children. In previous reports L. (V.) braziliensis and L. (V.) guyanensis were isolate in two endemic ATL localities of Corrientes. Since an increased number of autochthonous cases have been documented in the last few years, it has become important to identify the Leishmania species involved. We confirmed the preliminary diagnosis of ATL in clinical samples of patients with positive Montenegro skin test. Skin biopsies were obtained under aseptic conditions and with local anesthesia and the parasites were detected by Giemsa-stained smears, culture and hamster inoculation. Isolation of Leishmania in hamsters from 2 patients (a 54 years old male and a female of 22 years old with 3-months clinical evolution) from Bella Vista Department was performed. After 6 months of inoculation, kidney, liver, spleen and skin tissue samples from infected hamsters were subjected to DNA extraction by standard phenol-chloroform method. After isopropanol precipitation, DNA was suspended in 50 µl of ultra-pure water containing RNAse A (10mg/mL), incubated at 37oC for 1h and stored at -20oC until use. A 120 bp fragment of Leishmania k-DNA mini-circle was PCR amplified using primers 150 and 152 in a final volume of 20 µl containing amplification buffer (10mM Tris-HCl, pH 8.4, 50mM KCl, 1.5mM MgCl2), 200µM dNTP, 10 pmol of primers, 1 U Taq DNA polymerase (Invitrogen) and 2 µl DNA sample diluted at 1:10 and 1:100. DNA amplification was done as described by Volpini et al 2004. Five µl of the amplified DNA fragment was digested with restriction endonucleases AvaI and HaeIII and subjected to electrophoresis in a 10% polyacrylamide gel and stained with ethidium bromide, visualized under UV light and digitally recorded. L. (L.) amazonensisLTB0016 and L. (V.) braziliensis- M2904 DNA were used as control. The amplified DNA fragment was transferred to charged nylon membrane and hybridized overnight at 42oC with L. braziliensis and L. amazonensis k-DNA horseradish peroxidase labelled probes and revealed using an ECL kit. The expected 120bp was observed in all samples and confirmed as L. braziliensis by hybridization. Our data confirms that L. (V.) braziliensis constitutes the predominant etiologic agent of ATL in patients from this area. Financial support by CNPq
- CLINICAL LEISHMANIASIS [148] P 358 - COMPARATIVE EVALUATION OF DIRECT AGGLUTINATION TEST, RK39 DIPSTICK, IFAT AND QPCR FOR THE DIAGNOSIS OF VISCERAL LEISHMANIASIS AMONG HIV-INFECTED PATIENTS COTA, G.F.1; DE SOUSA, M.R.2; GOMES, L.I.1; OLIVEIRA, E.1; ASSIS, T.S.M.1; NOGUEIRA, B.M.F.3; MENDONÇA, A.L.P.3; PINTO, B.F.1; SALIBA, J.W.1; RABELLO, A.1
1.LABORATÓRIO DE PESQUISAS CLÍNICAS, CENTRO DE PESQUISAS RENÉ RACHOU, FUNDAÇÃO OSWALDO CRUZ-CPQRRFIOC, BELO HORIZONTE, MG, BRAZIL; 2.POST-GRADUATE PROGRAM IN ADULT HEALTH SCIENCES - UNIVERSIDADE
FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL; 3.EDUARDO DE MENEZES HOSPITAL, FUNDAÇÃO HOSPITALAR DO ESTADO DE MINAS GERAIS-FHEMIG, BELO HORIZONTE, MG, BRAZIL.
Keyword:hiv infection; visceral leishmaniasis; diagnosis
Abstract: Visceral leishmaniasis (VL) has emerged as an opportunistic condition in HIV infected patients. In immunocompromised patients, serological investigation is considered not an accurate diagnostic method and molecular techniques seem especially promising. So we compared the sensitivity of the indirect fluorescence antibody test (IFAT) using 1:40 and 1:80 cut-off titles, the direct agglutination test produced at the Clinical Research Laboratory of CPqRR (DATLPC), a commercial rK39 dipstick test (Kalazar Detect®) and quantitative real-time PCR (qPCR) performed in peripheral blood for VL diagnosis among HIV-infected patients in an urban area in Brazil. Clinical suspicion for VL was defined as a history of fever of 14 days or more and/or splenomegaly and/or cytopenia. A questionnaire with clinical and epidemiologic data was filled out at enrollment and diagnostic procedures were performed: bone marrow aspiration and a venous blood sample were taken. The diagnosis of VL was further confirmed when amastigotes were seen in Giemsa-stained smears or when promastigote forms were isolated from culturing bone marrow aspirates (BMA). Between March 2011 and September 2012, 37 (42%) patients had a diagnosis of VL: in 33, confirmed by direct exam (89%) and in 4 patients (11%), by culture of BMA. The mean age of the patients was 41.7 years (21-65 years), and the female-to-male ratio was 5:1. Only fifteen (40.5%) VL patients had specific antibodies detectable by all 3 serological techniques. Results for sensitivity and 95% confidence interval of tests (parasitological test as gold standard) were the following: IFAT 1:40 81.1% (68.2-93.9%); IFAT 1:80 62.2% (46.2-78.0%); DAT-LPC 94.6% (87.2-100%); rK39 45.9% (29.6-62.3%) and qPCR 86.5% (75.2-97.8%). There weren't statistical significant differences between IFAT 1:40 and DAT-LPC and between DAT-LPC and qPCR (two-by-two analysis). Differences were significant and favorable to DAT-LPC against IFAT 1:80 (p=0.0019) and against rK39 (p=0.00001). In conclusion, our results show that the DAT-LPC has high sensitivity, similar to qPCR, in diagnosis of VL among HIV-infected patients. IFAT 1:40 has high sensitivity, but results of its specificity, along with other tests, are being evaluated in a delayed-type cross-sectional study. Negative rK39 and IFAT 1:80 do not rule out VL in HIV infected patients. Funding: CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), FAPEMIG (Fundação de Amparo à Pesquisa no Estado de Minas Gerais)
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [157] P 359 - METACASPASE MEDIATED CELL DEATH INDUCED BY NOVEL BETULIN DERIVATIVES TARGETING TYPE IB DNA TOPOISOMERASE OF LEISHMANIA DONOVANI. MAJUMDER, H.K.; CHOWDHURY, S.; JAISANKAR, P. CSIR-INDIAN INSTITUTE OF CHEMICAL BIOLOGY, KOLKATA, INDIA.
Keyword:triterpenoids; programmed cell death; topoisomerase ib
Abstract: Toward developing antileishmanial agents with mode of action targeted to DNA topoisomerases of Leishmania donovani, we have synthesized a large number of derivatives of betulin. The compound, a natural triterpene isolated from the cork layer of Betula spp. plants exhibits several pharmacological properties. Three compounds (disuccinyl betulin, diglutaryl dihydrobetulin, and disuccinyl dihydrobetulin) inhibit growth of the parasite as well as relaxation activity of the enzyme type IB topoisomerase (LdTOP1LS) Leishmania donovani. Mechanistic studies suggest that these compounds interact with the enzyme in a reversible manner. The stoichiometry of these compounds binding to LdTOP1LS is 1:1 (mole/mole) with a dissociation constant of the order of 10-6 M. In processive mode of relaxation assay, these compounds slow down the strand rotation event, which ultimately affects the relaxation of supercoiled DNA. It is noteworthy that these compounds reduce the intracellular parasite burden in macrophages infected with wild-type L. donovani as well as with sodium antimony gluconate resistant parasite (GE1). Amongst the three derivatives, disuccinyl betulin (DiSB) is most potent growth inhibitor of Leishmania promastigotes. It induces parasitic cell death by generating oxidative stress. The physiological and biochemical characteristic feature of death process is like mammalian programmed cell death (PCD). Generation of reactive oxygen species (ROS), followed by mitochondrial membrane depolarization (D m) causes loss of ATP production. This further gives positive feedback to produce high amount of ROS, which in turn causes oxidative DNA lesions and genomic DNA fragmentation. Loss of D m leads to release of cytochrome c into the cytosol. The PCD is insensitive to zVAD-fmk, but metacaspase activity has been identified. Activation of metacaspase activates LdEndoG which is translocated from mitochondria to nucleus. There is a concomitant nuclear depletion of deoxynucleotidyltransferase-mediated dUTP end labelling (TUNEL) which indicates abrogation of DNA synthesis process. As a result, promastigote replication is hampered and the cell eventually dies. Conditional knock-down of metacaspase and endonulease G subvert DiSB mediated cell death. Taken together, our data suggest that these betulin derivatives can be exploited as potential drug candidates against threatening drug resistant leishmaniasis. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [160] P 360 - TREATMENT OF CUTANEOUS LEISHMANIASIS WITH LUCILIA SERICATA LARVAE POLAT, E.1; KUTLUBAY, Z.2; SIREKBASAN, S.3 1.DEPARTMENT OF MEDICAL MICROBIOLOGY, CERRAHPASA MEDICAL FACULTY, ISTANBUL UNIVERSITY, ISTANBUL, TURKEY; 2.DEPARTMENT OF DERMATOLOGY, CERRAHPASA MEDICAL FACULTY, ISTANBUL UNIVERSITY, ISTANBUL, TURKEY; 3.DEPARTMENT OF TUBITAK RESEARCH AND DEVELOPMENT LABORATORY OF BIOTHERAPY, ISTANBUL UNIVERSITY, ISTANBUL, TURKEY.
Keyword:leishmaniasis; lucilia sericata larvae; treatment
Abstract: Sodium stibogluconate and meglumine antimonate which are pentavalent antimony compounds, were developed in 1940. Although they are still the first drug used in the treatment of cutaneous and visceral leishmaniasis in our country, resistance to these drugs has been reported by various researchers. In our studies, we demonstrated that the secretions larvae of Lucilia sericata had effects on Leishmania tropica in invitro and invivo conditions. Larvae of Lucilia sericata, with the enzymes they secrete, dissolve the necrotic tissue, disinfect and stimulate the granulation tissue formation. With this effect, larvae of Lucilia sericata have been successfully used in the treatment of cutaneous leishmaniasis. Materials were taken from the patients with suspicion of leishmaniasis. Culture was made and stanied with Giemsa. Leishmania deemed patients’ treatment was started. Leishmania parasites reproduced in culture from these patients’ materials. Patients with cutaneous leishmaniasis
lesions located on the face were treated with Glucantime, the patients with the lesions on hands and arms were treated with Lucilia sericata larvae. Glucantime was injected intradermally into the lesion for twice a week, totally 5 doses until the lesion has been completely whitened. After one month from the completion of treatment, patients’ controls were done but cure was not detected in the lesions. Whereupon the second cure treatment was applied. But there was not any recovery in the patients’ lesions. Lesions in a patient which were initially on both cheeks were spread and they caused to formation of new lesions on patients’ foot and arms. The specimens prepared from materials taken from lesions in these patients were stained with Giemsa and culture of leishmaniasis was done. In Giemsa staining, leishmaniasis amastigote forms were seen but leishmania parasite did not be reproduced in culture. Patients having cutaneous leishmaniasis lesions located on hands and arms were treated with Lucilia sericata larvae six-seven of larvae were directly put on 1cm2 wound. In this way, the moving larva can hold on to all the necrotic area on the wound and enter depths of necrotic tissue. According to the state of necrotic tissue on the wound, larval therapy was applied daily, weekly, or once-twice in a week. After larvae were held into 48-72 hours on the wound, they were removed from the wound. This process was continued until necrotic tissue was completely cleansed on the wound. Cutaneous leishmaniasis lesions applied the larval treatment 2-3 times, were healed in as few as 10 days. The lesions of cutaneous leishmaniasis located on faces not responding to the treatment of Glucantime were treated with Lucilia sericata larvae. Lesions in these patients were treated in a short time like 6 days as a result of two larval treatments. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [161] P 361 - TRAINING AIDS TO REDUCE READER ERROR FOR THE DIRECT AGGLUTINATION TEST. ADAMS, E.1; JAQUET, D.2; SCHOONE, G.1; GIDWANI, K.3; BOELAERT, M.2; CUNNINGHAM, J.4 1.KIT, AMSTERDAM, NETHERLANDS; 2.ITM, ANTWERP, BELGIUM; 3.3BANARAS HINDU UNIVERSITY, VARANASI, INDIA; 4.WHO/TDR, GENEVA, SWITZERLAND.
Keyword:dat; training materials; diagnosis
Abstract: The Direct Agglutination Test (DAT) has a high diagnostic accuracy and remains, in some geographical areas, part of the diagnostic algorithm for Visceral Leishmaniasis (VL). However, subjective interpretation of results introduces potential for inter-reader variation. We report an assessment of inter-laboratory agreement and propose a pictorial-based approach to standardize reading of the DAT. In preparation for a WHO/TDR coordinated evaluation of diagnostics for VL, a proficiency panel of 15 well-characterized sera, DAT-antigen from a single batch and common protocol was sent to nine laboratories in Latin-America, East-Africa and Asia, all members of the Visceral Leishmaniasis Laboratory Network (VL-LN). Agreement (i.e. equal titre or within 1 titer) with reading by the reference laboratory was computed. Subsequently, photos of training plates were made, and end-titres were agreed upon by the reference laboratories and experienced users of DAT within the VL-LN. Overall concordance in DAT results with the reference laboratories was only 50%, although agreement on negative sera was high (94%). After refresher training the concordance increased
to 84% with the reference laboratories; agreement on negative controls increased to 98%. Variance in readings significantly decreased after training as shown by a two-sample Wilcoxon rank-sum (Mann-Whitney) test (z = -3,624 and p = 0.0003). Using pictorials as training materials may be a useful tool to reduce disparity in results and to promote more standardized reading of DAT, without compromising its diagnostic sensitivity. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [163] P 362 - IN VITRO ANTILEISHMANIAL ACTIVITY OF QUERCETIN 3-METHYL ETHER AND STRYCHNOBIFLAVONE PURIFIED FROM STRYCHNOS PSEUDOQUINA AGAINST LEISHMANIA AMAZONENSIS CARVALHO, P.S.L.1; ANDRADE, P.H.R.2; DUARTE, M.C.1; COSTA, T.R.3; VALADARES, D.G.; CHAVEZ-FUMAGALLI, M.A.1; COSTA, L.E.1; MARTINS, V.T.4; LAGE, D.P.2; REGIS, W.B.5; FILHO, J.D.S.6; PADUA, R.M.3; TAVARES, C.A.P.4; LEITE, J.P.V.7; COELHO, E.A.F.2 1.FACULDADE DE MEDICINA UFMG, BELO HORIZONTE, MG, BRAZIL; 2.COLEGIO TECNICO UFMG, BELO HORIZONTE, MG, BRAZIL; 3.FACULDADE DE FARMACIA UFMG, BELO HORIZONTE, MG, BRAZIL; 4.INSTITUTO DE CIENCIA BIOLOGICAS UFMG, BELO HORIZONTE, MG, BRAZIL; 5.PUC MINAS AND MINAS FUNGI DO BRASIL, BELO HORIZONTE, MG, BRAZIL; 6.DEPARTAMENTO DE QUIMICA UFMG, BELO HORIZONTE, MG, BRAZIL; 7.UNIVERSIDADE FEDERAL DE VISCOSA, VICOSA, MG, BRAZIL.
Keyword:strychnos pseudoquina; tegumentary leishmaniasis; antileishmanial activity
Abstract: Leishmaniasis is a major public health problem, and the alarming spread of parasite resistance underlines the importance of discovering new therapeutic products. In this study, a bioassayguided fractionation using the water extract of Strychnos pseudoquina led to the isolation of two purified bioactive flavonoids. These products, quercetin 3-methyl ether and strychnobiflavone were investigated in their capacity of present in vitro antileishmanial activity against Leishmania amazonensis. Studies were performed in order to establish their minimum inhibitory concentrations (IC50), leishmanicidal effects on intra-macrophages Leishmania amastigotes, as well as their cytotoxic effects on murine macrophages (CC50), and on human red blood cells (RBC50). The results showed that the quercetin 3-methyl ether and strychnobiflavone proved to be effective against promastigote and amastigote-like stages of L. amazonensis, presenting IC50 values of 1,52 M and 0,15 M, respectively, for promastigote stage, and of 25,64 M and 33,11 M, for amastigotes intra-macrophages, with the selectivity index (CC50/IC50) of 16,9 and 220,7, respectively. The applied compounds presented a low cytotoxicity in murine macrophages and a null hemolytic activity in type O+ human red blood cells. The data presented showed the potential of two compounds, quercetin 3-methy ether and strychnobiflavone, which were purified from Strychnos pseudoquina, as alternative for the treatment of tegumentary leishmaniasis. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [181] P 363 - EXPERIMENTAL PAROMOMYCIN RESISTANCE IN LEISHMANIA DONOVANI IS ASSOCIATED WITH INCREASED MEMBRANE FLUIDITY, DRUG EFFLUX AND PARASITE FITNESS BHANDARI, V.1; SUNDAR, S.2; DUJARDIN, J.C.3; SALOTRA, P.1 1.NATIONAL INSTITUTE OF PATHOLOGY, NEW DELHI, INDIA; 2.INSTITUTE OF MEDICAL SCIENCES, VARANASI, INDIA; 3.INSTITUTE OF TROPICAL MEDICINE, ANTWERP, BELGIUM.
Keyword:drug resistance; paromomycin; visceral leishmaniasis
Abstract: Paromomycin (PMM) is a promising drug for VL treatment since the phase IV trials confirmed its safety and efficacy. In order to safeguard the drug it’s vital to understand the mechanism of resistance. We induced experimental resistance up to 97µM PMM in L. donovani promastigotes (PMM-R parasite), resulting in more than 9 fold decrease in the susceptibility to PMM compared to the wild type parasite (PMM-S). Resistance induced at promastigote stage was also evident at the amastigote stage as depicted by >6 fold decrease in PMM susceptibility at amastigote stage of PMM-R (meanIC50±SD=61±7µM) compared to that of PMM-S (meanIC50±SD=11±1µM), while susceptibility to sodium antimony gluconate or miltefosine remained unaltered. The PMM-R parasite showed increased (2.5 fold) membrane fluidity, and decreased (3 fold) intracellular PMM accumulation as compared to PMM-S. Analysis of gene expression by real time PCR revealed an increased expression of ABC transporters; MDR1 (8.95±6.08 fold) as well as MRPA (11.47±0.22 fold) and protein phosphatase 2A gene (4.44±0.71 fold) in PMM-R parasite, evincing increased efflux of PMM. Full length gene sequencing of L. donovani rRNA gene failed to detect any mutations in the PMM-R strain of L donovani, unlike the reported observation in PMM resistant E. coli. Additionally, we evaluated the tolerance of PMM-R parasite towards nitrosative, oxidative and complement mediated stresses. PMM-R strain was more tolerant to nitrosative stress induced by SIN-1(above 2 fold) and SNAP (9 fold) as compared to PMM-S isolate, however, the tolerance to oxidative stress remained unaltered. The PMM-R parasites showed significantly higher resistance to complement mediated lysis. Macrophages infected with PMM-R parasites elicited higher (1.8 fold) IL-10 levels in comparison with PMM-S, indicating a better survival capacity of PMM-R. The study shed light on the mechanism of PMM resistance and fitness levels of PMM-R Leishmania donovani. This work was carried out under European Commission funded Kaladrug-R project (EC-FP7222895).
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [182] P 364 - DECLINE IN IN VITRO DRUG SUSCEPTIBILITY OF CLINICAL ISOLATES OF LEISHMANIA DONOVANI FROM MILTEFOSINE TREATED CASES OF VISCERAL LEISHMANIASIS AND POST KALA-AZAR DERMAL LEISHMANIASIS (PKDL) BHANDARI, V.1; KULSHRESTHA, A.1; DEEP, D.K.1; STARK, O.2; PRAJAPATI, V.K.3; RAMESH, V.4; SINGH, R.1; SUNDAR, S.3; SCHONIAN, G.2; DUJARDIN, J.C.5; SALOTRA, P.1 1.NATIONAL INSTITUTE OF PATHOLOGY, NEW DELHI, INDIA; 2.INSTITUTE OF MICROBIOLOGY AND HYGIENE, BERLIN, GERMANY; 3.INSTITUTE OF MEDICAL SCIENCES, VARANASI, INDIA; 4.DEPARTMENT OF DERMATOLOGY, NEW DELHI, INDIA; 5.INSTITUTE OF TROPICAL MEDICINE, ANTWERP, BELGIUM.
Keyword:visceral leishmaniasis; post kala azar dermal leishmaniasis; miltefosine
Abstract: With widespread resistance to antimonials in the Indian subcontinent, Miltefosine (MIL) has been introduced as the first line therapy for visceral leishmaniasis (VL), however decline in treatment efficacy is reported. MIL has also been successfully used for treatment of post kalaazar dermal leishmaniasis (PKDL), a sequel of VL that constitutes an important parasite reservoir. In a set of VL/PKDL cases recruited for MIL treatment, we determined the in vitro susceptibility of L. donovani parasites obtained pre- and post-treatment at amastigote stage. MIL susceptibility of post-treatment isolates from cured VL cases (mean IC50±SD=2.43±1.44µM), was comparable (p>0.05) to that of the pre-treatment group (mean IC50 =1.86±0.75µM), while
that from relapses (mean IC50=4.72±1.99µM) was significantly lower (p=0.04). Similarly, PKDL isolates from relapse cases exhibited significantly higher (p=0.03) MIL tolerance compared to pre-treatment isolates (IC50=16.13±2.64µM and 8.63±0.94µM, respectively). Interestingly, PKDL isolates were more tolerant towards MIL than VL isolates. The inter-stage MIL susceptibility correlated significantly for parasite isolated from VL (r=0.70, p=0.0018); PKDL (r=0.78, p=0.046) and MIL induced parasites with high MIL tolerance (r=0.92, p=0.0001) when macrophage based amastigote assay and high-throughput resazurin-based promastigote assay were compared. The data suggests that the resazurin assay with promastigotes is applicable as a simplified biological tool for high throughput MILsusceptibility monitoring in clinical isolates of VL. Point mutations in the miltefosine transporter (LdMT) and its beta subunit (LdRos3) genes as well as changes in the mRNA expression of these genes that previously correlated with experimental resistance to MIL, were not evident in the clinical isolates. This work was carried out under European Commission funded Kaladrug-R project (EC-FP7222895).
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [183] P 365 - MECHANISM OF RESISTANCE IN FLAVONE RESISTANT LEISHMANIA DONOVANI TO CIRCUMVENT THE FLAVONE-MEDIATED CELL DEATH: ROAD TOWARDS PHENOTYPIC TO GENOTYPIC GAIN OF FUNCTION CHOWDHURY, S.; MAJUMDER, H.K. CSIR-INDIAN INSTITUTE OF CHEMICAL BIOLOGY, KOLKATA, INDIA.
Keyword:induced resistance; transporter; topoisomerase
Abstract: In parasites, ATP-binding cassette (ABC) transporters represent an important family of proteins related to drug resistance and other biological activities. Resistance of leishmanial parasites to therapeutic drugs continues to escalate in developing countries and in many instances it is due to overexpressed ABC efflux pumps. Progressively adapted baicalein (BLN)-resistant parasites (pB25R) show overexpression of a novel ABC transporter, which was classified as ABCC2 or LdMrp2. The localization is primarily in the flagellar pocket region and in internal vesicles. Overexpressed LdABCC2 confers substantial BLN resistance to the parasites by rapid drug efflux. The BLN-resistant promastigotes when transformed into amastigotes in macrophage cells cannot be removed by treatment of macrophages with BLN. Amastigotes resistance is concommitant to the overexpression of macrophage multidrug resistance protein 2 (MRP2) transporter. Reporter analysis and site-directed mutagenesis assays demonstrated that antioxidant response element (ARE)-1 is activated followed by infection. The expression of this phase II detoxifying gene is regulated by Nrf2 (NFE2 – related factor 2) mediated Antioxidant Response Element (ARE) activation. In view of the fact that the signalling pathway of Phospho Inositol 3-kinase controls microfilament rearrangement and translocation of actin-associated proteins, the current study correlates with intricate pathway of PI3-kinase–mediated nuclear translocation of Nrf2 which activates Mrp2 expression in macrophages. In contrast, phalloidin, an agent that prevents actin filaments from depolymerization, inhibited Nrf2 translocation and Mrp2 activation by pB25R infection. Parasites exposed to higher drug concentration (75 µM) show no difference in drug accumulation or efflux. Earlier it was found that flavones interact with LdTOP1LS to abrogate DNA transaction mediated life processes. Scanning of LdTOP1LS gene revealed that there are some mutations near to the active site of the enzyme. To our surprise this mutatnt enzyme is cross resistant to certain levels of other relative flavones like luteolin and quercetin. Taken together, these results provide an insight into the mechanisms by
which resistant clinical isolates of Leishmania donovani induces intracellular events relevant to drug resistance. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [184] P 366 - DETECTION OF LEISHMANIAL ANTIGENS IN PATIENT URINE BY QUANTITATIVE ELISA AND ITS POTENTIAL AS A TEST-OF-CURE ALBERTINI, A.1; HOMMEL, M.; ELLIS, S.2; HAQUE, R.3; HAQUE BHUIYAN, A.T.R.3; RAHMAN, R.4; FAIZ, M.A.5; MONDAL, D.3; CHILDERSTONE, M.6; PURCELL, N.6; NDUNG'U, J.1; PERKINS, M.D.1 1.FIND, GENEVA, SWITZERLAND; 2.DNDI, GENEVA, SWITZERLAND; 3.ICCDDR,B, DHAKA, BANGLADESH; 4.SHAHEED SUHRAWARDY MEDICAL COLLEGE AND HOSPITAL, DHAKA, BANGLADESH; 5.MALARIA RESEARCH GROUP, CHITTAGONG MEDICAL COLLEGE, DHAKA, BANGLADESH; 6.KALON BIOLOGICAL, GUILFORD, UNITED KINGDOM.
Keyword:diagnostics; antigen detection; vl
Abstract: Early identification of treatment failure in visceral leishmaniasis (VL) patients is critical to the successful control and elimination of the disease. An assay that could do this would also have potential as a pharmacodynamic marker. Both test indications would require a sensitive and quantitative assay. We report on the development of an antigen affinity purified (AAP) polyclonal double-antibody sandwich ELISA assay for leishmania antigens in urine, and its testing on patient and endemic control samples from South-East Asia. The AAP ELISA uses the same antigen as the KAtex latex agglutination test but is quantitative and does not require boiling of urine samples before testing. Urine samples were collected by DNDi in Bangladesh during a phase III/IV drug study, conducted at the Community Based Medical College (CBMC) of Mymenshingh, to determine the feasibility of implementing new treatment regimens for VL recommended by WHO (miltefosine (1.5-2.5 mg/kg in 1 or 2 doses/day) + paromomycin (11mg/kg/day) for 10 days, AmBisome® (5mg/kg) + miltefosine for 7 days, AmBisome® (5mg/kg) + paromomycin for 10 days, AmBisome® 15mg/kg) in a primary healthcare setting. The samples were obtained from parasitologically confirmed VL patients (collected at pretreatment, and on days 7, 15, 45 and 180 after initiation of treatment) and from healthy endemic controls. When a cut-off of 1 UAU/ml (arbituary units of urinary antigen per ml) was used, the AAP ELISA prototype was positive in 95.3% (102/107) of leishmania patient samples before treatment (baseline), while all the endemic controls (48/48) were negative. During the first 45 days after initiation of treatment, the level of leishmania antigen in urine decreased by 95% in majority of the patients. However, the cut-off of 1 UAU/ml still classified more than 50% of the samples as positive 45 days after initiation of treatment. While this assay has good potential as a test of cure, the appropriate time for testing after treatment needs to be defined, in line with patient follow-up visits. Further testing of this non-invasive prototype assay on samples from Eastern Africa is planned, before large-scale evaluation in the field. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [188] P 367 - EXPERIMENTAL STUDIES OF LIPOSOMAL BUPARVAQUONE USING DIFFERENT ROUTES OF ADMINISTRATION AND REGIMENS AGAINST LEISHMANIA (L.) INFANTUM DA COSTA SILVA, T.A.1; GARCIA-PEREZ, A.2; DEL VALLE, S.G.2; KESSLER, A.2; TEMPONE, A.G.1 1.DEPARTMENT OF PARASITOLOGY INSTITUTO ALDOLFO LUTZ, SÃO PAULO, SP, BRAZIL; 2.GSK DISEASES OF THE DEVELOPING WORLD, TRES CANTOS, SPAIN.
Keyword:buparvaquone; liposomes; leishmania (l.) infantum
Abstract: Buparvaquone is a veterinary drug used to treat Theileriosis and has shown promising activities against protozoan parasites, including Leishmania spp. Despite its in vitro anti-leishmanial activity, in vivo experiments with L. (L.) donovani showed only a weak suppression of the parasite burden. Our previous studies demonstrated that intraperitoneal buparvaquone entrapped in phosphatidylserine-liposomes (BPQ-PS-LP) resulted in a significant suppression of Leishmania (L.) infantum in a hamster model. In this study the effectiveness of BPQ-PS-LP was evaluated at 0.4 mg/kg/day in a L. (L.) infantum-hamster model (n= 5/group) using different routes of administration (subcutaneous, intramuscular and intravenous) and regimens (5 and 10 consecutive days). The treatment was quantified by real-time PCR, using the detection of living amastigotes (RNA) in the spleen and the liver. BPQ-PS-LP showed the most significant reduction of the parasite burden when administered by subcutaneous route, reducing the amastigotes in spleen and liver by 98% (p<0.05) and 96% (p<0.05), respectively. BPQ-PS-LP by the intravenous route was effective but to a lesser extent, reducing the amastigotes in spleen and liver by 88% and 35%. BPQ-PS-LP failed to reduce the parasite burden of liver and spleen when administered by intramuscular route. Finally, BPQ-PS-LP was administered by subcutaneous route using a short course treatment of 5 consecutive days; a limited efficacy was observed at 0.4 mg/kg/day, showing a reduction of the parasite burden in spleen and liver by 42% and 38%, respectively. Considering the future possibility of a clinical use for liposomal buparvaquone against Visceral Leishmaniasis, the subcutaneous route at low doses may represent a higher advantage to increase patient compliance. Supported by GlaxoSmithKline (Trust in Science initiative). - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [192] P 368 - OPTIMIZATION OF SINGLE-TUBE NESTED PCR FOR DIAGNOSING VISCERAL LEISHMANIASIS SILVA, M.A.L.; SOARES, C.R.; MEDEIROS, Z.; MELO, F.L. CENTRO DE PESQUISAS AGGEU MAGALHĂƒES, RECIFE, PE, BRAZIL.
Keyword:pcr; visceral leishmaniasis; diagnosis
Abstract: Conventional nested PCR is a very sensitive and specific method for diagnosing visceral leishmaniasis. However, this type of PCR is notorious for contamination problems relating to passage of the product from the first to the second round. In order to have a PCR method that is just as efficient, but without the risk of contamination, we proposed to optimize a single-tube nested PCR (STNPCR) method. We used the targets of the small subunit ribosomal RNA in the first step and the ribosomal internal transcribed spacer in the second stage. The performance of STNPCR and nested PCR for detecting DNA from Leishmania chagasi was compared. For STNPCR, the inner primers were immobilized on the internal interface of the cap by means of adsorption microtubes, and these were made soluble after the first reaction. This procedure eliminated opening the microtube, which could have led to false-positive results through crosscontamination. The detection limit for purified L. (L.) chagasi DNA was 1 fg using nested PCR and 10 fg using STNPCR. We also tested the specificity of the system against other parasites, and observed that Trypanosoma cruzi DNA was amplified, reaching detection of up to 1 pg. This study not only presents a promising tool for diagnosing visceral leishmaniasis, but also provides a new target to be exploited in diagnosing Chagas disease, either in mono-infection with T. cruzi, or co-infection with Leishmania spp.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [193] P 369 - A COMPARISON OF FOUR DNA EXTRACTION PROTOCOLS FOR ANALYSIS OF URINE FROM PATIENTS WITH VISCERAL LEISHMANIASIS SILVA, M.A.L.; SOARES, C.R.; DIONÍSIO, E.; MEDEIROS, Z.; MELO, F.L. CENTRO DE PESQUISAS AGGEU MAGALHÃES, RECIFE, PE, BRAZIL.
Keyword:urine; visceral leishmaniasis; diagnosis
Abstract: Obtaining a good quantity of high quality genomic DNA is a crucial step for successful amplification using the polymerase chain reaction (PCR) and, therefore, for research and diagnostic testing. A comparative performance evaluation was carried out of methods for extracting DNA from urine of the individuals with leishmaniasis visceral using various protocols, to determine which produces the best quality DNA suitable for PCR amplification. Two commercially available extraction kits and two in-house DNA extraction methods for the release of DNA from Leishmania infantum chagasi in urine were assessed for sensitivity, purity, duration and cost. All commercially available kits helped to shorten the duration of DNA extraction. The QIAamp DNA Mini Kit was the commercially available assay that yielded the same sensitivity and purity of Leishmania DNA release as the in-house protocol but was the most expensive method. Our results suggest that differences exist between methods for extracting DNA from urine, and this should be considered when assessing the parasite rate blood and determining the clinical profile of the individual. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [199] P 370 - IN VITRO COMBINATION OF CALCIUM CHANNEL BLOCKERS AND THEIR INTERFERENCE IN THE METABOLISM OF LEISHMANIA (L.) INFANTUM MESQUITA-LIMA, R.T.; MESQUITA, J.T.; FERREIRA, D.D.; REIMÃO, J.Q.; TEMPONE, A.G. DEPARTMENT OF PARASITOLOGY - INSTITUTO ADOLFO LUTZ, SÃO PAULO, SP, BRAZIL.
Keyword:amlodipine; fendiline; leishmania (l.) infantum
Abstract: Visceral Leishmaniasis is a neglected disease endemic in more than 68 countries with 200 million people at risk. Efforts to find new therapies for Leishmaniasis remain a priority. Calcium Channel Blockers (CCB) are drugs used for controlling blood pressure and other heart diseases. This class of drugs acts by selective inhibition of calcium influx through cell membranes or on the release and binding of calcium in intracellular pools. Considering that drug repurposing is a promising approach to discover novel drug candidates to treat neglected diseases, the objective of this work was to study the action of fendiline and amlodipine in the metabolism and plasma membrane of Leishmania (L.) infantum promastigotes. Besides, in vitro combinations were performed to identify interactions between fendiline and standard drugs against promastigotes. By using the fluorimetric probe SYTOX Green, neither fendiline nor amlodipine induced plasma membrane disturbance after 60 minutes incubation. The Mitotracker Red assay showed that fendiline and to a lesser extent, amlodipine, significantly (p<0.05) affected the mitochondrial membrane potential of Leishmania when compared to untreated parasites. Both drugs induced a rapid up-regulation of Reactive Oxygen Species (ROS) in promastigotes, but amlodipine resulted in at least 7-fold increase (p<0.05) in ROS production when compared to fendiline. Graphical isobologram analysis of the combination of fendiline and amphotericin B, miltefosine and pentamidine showed overall mean of sum of Fractional Inhibitory Concentrations ( FIC) of 1.13, 1.30 and 1.22 respectively, indicating indifferent interaction. CCB induced metabolic alterations in Leishmania and the association of fendiline with standard drugs showed no interference in their in vitro activity, suggesting future possibilities for
experimental studies. If adequately studied, CCB could also be used as tools for discovering new drug targets in Leishmania. Supported by FAPESP, CNPq and CAPES. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [201] P 371 - EFFECT OF SYNTHETIC HETEROCYCLIC NITRO DERIVATIVES ON PROMASTIGOTES OF L. (L.) INFANTUM PETRI, S.C.S.1; BERL, F.P.2; SOARES, S.R.C.1; TAVARES, L.C.2; LINDOSO, J.A.L.1 1.FACULDADE DE MEDICINA DA UNIVERSIDADE DE SAO PAULO, SAO PAULO, SP, BRAZIL; 2.FACULDADE DE CIENCIAS FARMACEUTICAS DA UNIVERSIDADE DE SAO PAULO, SAO PAULO, SP, BRAZIL.
Keyword:l. (l.) infantum; synthetic heterocyclic nitro derivatives ; promastogotes
Abstract: Visceral leishmaniasis (VL) belongs to the group of neglected tropical disease prioritized to drug development by World Heath Organization. Despite of spread of the disease, treatment for visceral leishmaniasis did not obtain progress and few drugs are available; pentavalent antimony and amphotericin B. The drugs available present several side effects, increase in the resistance and relapse in immunosuppressed patients. In this way the search for new molecules to treat neglected tropical disease is necessary. Derived from nitro-heterocyclic compounds has shown promise against promastigotes of L. (L) amazonensis and Trypanosoma cruzi epimastigotes. In this work we evaluated in vitro bioactivity of synthetic heterocyclic nitro derivatives upon L (L) infantum promastigotes. Culture of promastigotes was obtained by purification of amastigotes taken out from spleen of hamsters infected with L (L.) infantum. After seven days in culture, promastigotes were used in the stationary phase to perform the experiment. 106 L (L.) infantum promastigotes in M199 medium supplemented with Fetal Bovine Serum (10%) and urine (5%) were plated in 96 well plates and added four different synthetic heterocyclic nitro derivatives and control drugs (liposomal amphotericin B and pentavalent antimonial) in a spread of concentration from 0.04 to 50 uM for 48 hours and incubated in CO2 incubator at 37 ° C. Enzimatic method assay of bromate salt conversion (MTT) was used to detected death of parasites. The nytro-heterocycles synthetics derivate were able to dead Leishmania (L.) infantum promastigotes. The compound A showed IC50 uM 8.730; compound B showed IC50 2.125 uM; compound C showed IC50 4.097 uM and compound D showed IC50 9.952 uM. These data show that the compounds have an effective action at low dosages as compared with the control drugs that require high doses to obtain an effective action. The compound B presented the best action against promastigotes of L. (L.) infantum. The results are promising, and the compounds are considered candidates for more extensive studies to become candidates for possible antileishmanial drugs. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [203] P 372 - EVALUATION OF A LEISHMANIAL DYRK KINASE AS A MOLECULAR TARGET FOR THE DEVELOPMENT OF ANTILEISHMANIAL DRUGS ROCHA, V.1; EFSTATHIOU, A.2; SOARES, M.B.P.1; SMIRLIS, D.2 1.LABORATORY OF TISSUE ENGINEERING AND IMMUNOPHARMACOLOGY, GONÇALO MONIZ RESEARCH CENTER, OSWALDO CRUZ, SALVADOR, BA, BRAZIL; 2.LABORATORY OF MOLECULAR PARASITOLOGY, MICROBIOLOGY DEPARTMENT, HELLENIC PASTEUR INSTITUTE, ATHENS, GREECE.
Keyword:leishmania; drug; dyrk
Abstract: The leishmaniases comprise a broad spectrum of infectious diseases caused by parasites belonging to the Leishmania genus. The illness is endemic in 88 countries worldwide. The firstline treatment performed with the pentavalent antimonials is potentially toxic and related to
therapeutic failure caused by the low sensitivity of some species of Leishmania parasites. Based on this scenario, the rational drug design and the search for new dug targets are of prime importance. Parasitic protein kinases are attractive molecular drug targets because they are involved in phosphorylation pathways critical for parasitic survival and virulence. Proteins belonging to the CMGC group of kinases are relatively abundant in trypanosomatids, possibly reflecting the essentiality of them in the parasite viability or virulence. DYRK (dual-specificity yak-related kinases) are related to cell survival, cell differentiation and gene transcription in mammalians cells. To our best knowledge there is no information about DYRK and neglected tropical diseases, including leishmaniases. The objective of this study is to evaluate the parasitic protein kinase DYRK as a molecular target for the development of antileishmanial drugs. Harmine, a specific inhibitor of mammalian DYRK1A, displayed antiparasitic activity (IC50=10 ) against L. infantum and L. amazonensis promastigotes, suggesting that the parasitic target(s) of harmine are essential for viability. Database search of the leishmanial genome predicts the presence of 7 putative DYRK homologues. Amongst these, one putative DYRK gene (LinJ.15.0180 or LiDYRK) encodes a protein that has the highest homology to the DYRK signature (DYRK homology box) and the DYRK kinase domain (DYRK1A template) species. Alignment of the parasitic LiDYRK and human DYRK proteins identified differences in the active site, suggesting LiDYRK as a promising candidate for drug target validation. To this end we cloned the L. amazonensis and L. infantum genes, in frame with a C terminal histidine tag in a bacterial expression vector. We generated and purified the two recombinant proteins. Future work involves the knockout of these genes in Leishmania to assess the essentiality in the viability or virulence of Leishmania and its molecular characterization. Our ultimate goal is to validate DYRK as a drug target for the treatment of Leishmaniases. - GENOMICS, TRANSCRIPTOMICS, PROTEOMICS, METABOLOMICS [213] P 373 - METABOLOMIC APPROACH FOR LEISHMANIA DONOVANI MILTEFOSINE TREATMENT AND RESISTANCE EVALUATION. CANUTO, G.A.B.1; CASTILHO-MARTINS, E.A.2; RIVAS, L.3; TAVARES, M.F.M.1; LÓPEZGONZÁLVEZ, Á.2; BARBAS, C.2 1.UNIVERSITY OF SÃO PAULO, SÃO PAULO, SP, BRAZIL; 2.UNIVERSIDAD CEU SAN PABLO, MADRID, SPAIN; 3.CENTRO DE INVESTIGACIONES BIOLÓGICAS, MADRID, SPAIN.
Keyword:miltefosine; leishmania donovani; metabolomics
Abstract: Leishmaniasis is a public health problem in at least 88 countries, including Brazil. The therapeutic arsenal against the disease is limited and consists of highly toxic drugs. One of the most commonly used drugs is miltefosine, an alkylphosphocholine, which has been applied as an effective oral treatment for the visceral and cutaneous leishmaniasis. The biochemical mechanism of action of miltefosine is not completely clear. Therefore, methods to assess compounds of biological importance at metabolic levels are highly demanded. Metabolomics is becoming popular because it measures various metabolites directly in biological matrices, with excellent speed and accuracy. In this work, the mechanism of action and resistance of Leishmania donovani to miltefosine was studied using gas chromatography, liquid chromatography and capillary electrophoresis all coupled to mass-spectrometry. After data processing and statistical analysis, 82 different compounds have been found and related to the Leishmania’s metabolic pathways. An increase on 18 of 20 amino acids was found in resistant parasites. Treated promastigotes showed an increased level for a pentose with a molecular mass corresponding to ribitol, xylitol or arabitol, this m/z is decreased on resistant parasites. Metabolites from L-arginine metabolism pathway are increased on resistant parasites and decreased on treated ones. This route is responsible for providing spermidine to glutathione pathway, where it is used with glutathione to produce glutathionyl-spermidine and trypanothione, the main anti-oxidant generated from the parasites against host cell oxidative
burst. Glutathionyl-spermidine and trypanothione disulfide are increased in resistant parasites. Another interesting result about the action of miltefosine was a decrease in the content of ergosterol. Miltefosine interacts with the ergosterol, allowing the permeability of ions and other molecules in the cellular membrane, killing the cell. These results suggest an involvement of the thiol pathway on drug´s action related to it’s the resistance mechanism of Leishmania donovani, reinforcing previously reported apoptosis-like death on treated parasites. The use of separation methods coupled to mass spectrometry allowed the reliable detection of low molecular analytes in a complex matrix, without extensive sample preparation, aiding in the understanding of the mechanism of drug action and discovery of the metabolic pathways responsible for parasite resistance. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [216] P 374 - DIRECT AGGLUTINATION TEST: INTERNAL VALIDATION OF A PROTOTYPE KIT FOR THE DIAGNOSIS OF VISCERAL LEISHMANIASIS OLIVEIRA, E.; SALIBA, S.W.; SALIBA, J.W.; RABELLO, A. CENTRO DE PESQUISAS RENÉ RACHOU-FIOCRUZ, BELO HORIZONTE, MG, BRAZIL.
Keyword:visceral leishmaniasis; serological diagnosis; direct agglutination test-dat
Abstract: Despite the drawbacks of requiring fluorescence microscopes and relatively well-equipped laboratories and the lack of specificity, IFAT has been widely used for the diagnosis of visceral leishmaniasis (VL) in the Brazilian public health laboratories. Investments have been made to develop a direct agglutination test using L. infantum promastigotes for the diagnosis of LV in Brazil. Herein, we present a prototype direct agglutination kit produced with freeze-dried antigen and all necessary reagents for the laboratory diagnosis of VL, named DAT-LPC. To evaluate its diagnostic validity, 103 samples from VL patients and 110 samples from patients with other parasitic infections, and healthy subjects were simultaneously assayed with DATLPC and DAT-KIT (Royal Tropical Institute, Amsterdam, NL). Additionally, the results of 103 samples of patients with LV by the two agglutination tests were transformed in Log10 and correlated. The DAT-LPC presented sensitivity of 99% (CI: 93.9 - 99.9%), specificity of 98.2% (CI: 92.9- 99.7%) and diagnosis validity of 98.6% (CI: 95.6-99.6%), which were similar to those found by the DAT-KIT (p> 0.05). Moreover, positive correlation was observed between the positive titers obtained by DAT-LPC and DAT-KIT (Spearman correlation coefficient of 0.75 p = 0.0001). DAT-LPC has presented thermal stability and diagnostic performance similar to the DAT-KIT. Our results suggest that DAT-LPC is a robust, simple, equipment-independent and efficient tool for the diagnosis of VL and should thus replace the IFAT as routine diagnostic test in the Brazilian public health system. Funding: CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnológico), Brasília, DF, Brazil (grants 350594/1995-3 and311641/2009-1) and FIOCRUZ, Rio de Janeiro, RJ, Brazil (PDTIS RID06). - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [223] P 375 - SPECIFIC SERODIAGNOSIS OF CANINE VISCERAL LEISHMANIASIS USING PEPTIDES SELECTED FROM HYPOTHETICAL PROTEINS IDENTIFIED BY AN IMMUNOPROTEOMIC APPROACH FUMAGALLI, M.1; MARTINS, V.1; LAGE, D.1; COSTA, L.1; LAGE, P.1; DOS REIS, A.2; SOTO, M.3; TAVARES, C.A.1; FERNANDES, A.P.1; COELHO, E.A.F.1 1.UFMG, BELO HORIZONTE, MG, BRAZIL; 2.UFOP, OURO PRETO, MG, BRAZIL; 3.UAM, MADRID, SPAIN.
Keyword:immunoproteomics; serodiagnosis; elisa
Abstract: The present work analyzed the potential of synthetic peptides mapped from hypothetical proteins identified by an immunoproteomic approach for the specific serodiagnosis of canine visceral leishmaniasis (CVL). From previously identified leishmanial hypothetical proteins, eight were selected, considering that no homologies could be identified between these proteins and others from the sequence databases of the closely related Trypanosoma cruzi and T. brucei species. The sequences of these hypothetical proteins were mapped to identify linear B-cell epitopes, and the corresponding peptides were synthesized. In this context, seventeen peptides were constructed and tested in ELISA for CVL serodiagnosis. From these, three peptides (PepLi1, PepLi2 and PepLi7) presented the sensitivity and specificity values higher than 75% and 90%, in order to differentiate PCR(+)/sero(-) and symptomatic animals from T. cruziinfected and healthy animals, respectively. When peptides were combined in different mixed formats, high sensitivity values could be observed in the diagnosis of canine clinically symptomatic visceral leishmaniasis. In addition, high specificity was reached, even when the sera from T. cruzi-infected dogs were included in the analysis. The study’s findings suggest that these three top peptides, when used in isolation or in combination, can constitute a potential tool for a more sensible and specific CVL serodiagnosis. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [224] P 376 - IN VITRO EFFICACY OF ZINGIBER OFFICINALIS ROSCOE AGAINST LEISHMANIA AMAZONENSIS DUARTE, M.C.1; LAGE, D.P.2; FUMAGALLI, M.C.1; RIBEIRO, T.G.; OLIVEIRA, J.S.3; COSTA, L.E.1; MARTINS, V.T.3; LAGE, P.S.1; ANDRADE, P.H.R.2; SANTORO, M.M.3; RÉGIS, W.C.4; CASTILHO, R.O.5; TAVARES, C.A.P.3; COELHO, E.A.F.2 1.PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE: INFECTOLOGIA E MEDICINA TROPICAL, FACULDADE DE MEDI, BELO HORIZONTE, MG, BRAZIL; 2.DEPARTAMENTO DE PATOLOGIA CLÍNICA, COLTEC, UFMG, BELO HORIZONTE, MG, BRAZIL; 3.DEPARTAMENTO DE BIOQUÍMICA E IMUNOLOGIA, ICB, UFMG, BELO HORIZONTE, MG, BRAZIL; 4.PUC MINAS AND MINASFUNGI DO BRASIL LTDA, BELO HORIZONTE, MG, BRAZIL; 5.PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS FARMACÊUTICAS, FACULDADE DE FARMÁCIA, UFMG, BELO HORIZONTE, MG, BRAZIL.
Keyword:leishmania amazonensis; treatment; zingiber officinalis roscoe
Abstract: Leishmaniasis is a major public health problem, and the alarming spread of parasite resistance underlines the importance of discovering new therapeutic products. The present study aims to investigate the in vitro antileishmanial activity of Zingiber officinalis Roscoe water extract and its purified fraction (namely F10) against Leishmania amazonensis. The water extract and F10 proved to be effective against stationary-phase promastigotes of L. amazonensis, with IC50 values of 80 and 48 µg/ml, respectively. The percentages of infection with L. amazonensis in murine macrophages after treatment with the water extract or F10 was 50% and 12,7%, respectively, while the control cells had 75% of infection. The characterization of F10 showed the presence of tannis, anthraquinones, triterpenoids, coumarins, alkaloids, saponins, and cardiotonic glycoside. The applied extract and fraction presented a low cytotoxicity in murine macrophages and a null hemolytic activity in type O+ human red blood cells. Our studies also showed that fraction F10 induces the production of nitric oxide (NO) after stimulation of murine macrophages, suggesting that this could be the reason for the in vitro elimination of parasites. In conclusion, the results demonstrate that the F10 purified from Zingiber officinalis Roscoe can potentially be used as a new therapeutic alternative on its own, or in association with other drugs, to treat disease evoked by L. amazonensis.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [232] P 377 - ANTIMICROBIAL PEPTIDES: A NEW CLASS OF ANTI-LEISHMANIAL DRUG CANDIDATES CEN, S.; MARR, A.K.; LOUTET, M.; HANCOCK, R.E.W.; MC.MASTER, R.W. UNIVERSITY OF BRITISH COLUMBIA, VANCOUVER, CANADA.
Keyword:antimicrobial peptide; drug therapy; library screen
Abstract: Current drug approaches to treating cutaneous and visceral Leishmaniasis have been hampered due to toxicity of the drugs to the host, and the development of drug resistance. A promising class for new therapeutics are antimicrobial peptides (AMPs). AMPs are cationic peptides that result in killing of microorganisms through different mechanisms such as disruption of membranes and/or modulation of immune signaling events, thereby playing an important role in the innate immune response of the host. Previously we characterized the mode of action of the AMP - BMAP28 – in the killing of promastigotes and intracellular amastigotes. In this study we have screened a library of additional AMPs for their anti-leishmanial activity, both in the promastigote and intramacrophage amastigote form of the parasite. We identified 4 AMPs that demonstrate antileishmanial activity against promastigotes with an LD50 20 µM and incubation times of 5 hours. The 4 AMPs were also effective in reducing intramacrophage amastigotes numbers with a 65% reduction in amastigotes in infected macrophages after 24 hours incubation in 20 µM AMPs, in comparison to untreated infected macrophages. In summary, we have identified new drug candidates against Leishmania. Such AMPs may provide a much-needed alternative to existing drug therapies.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [233] P 378 - ANTI-LEISHMANIA AMAZONENSIS ACTIVITY OF PERILLIC ACID PASCHOAL, M.; SOARES, D.C.; FERREIRA, C.; SARAIVA, E.M. UFRJ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:leshmanicidal activity; perillic acid; natural products
Abstract: Pentavalent antimonials, amphotericin B and more recently, miltefosine are the drugs used for leishmaniasis therapy. These drugs have high toxicity and several side effects leading to patients’ withdrawal and to the increased incidence of resistant cases. The search for new substances, which are efficient against the parasite and with lower cytotoxicity to host, is important to improve the leishmaniasis therapy. Perillic acid (AP) is a monocyclic monoterpene derived from limonene, which shows immunomodulatory and antitumoral activities. In this study we first evaluated the cytotoxicity of AP for host cells by XTT and Trypan blue exclusion methods, which verify mitochondrial activity and membrane integrity, respectively. Our results demonstrated low cytotoxicity for macrophages treated with AP at concentrations until 500 µM. Macrophage phagocytic activity was not affected by treatment with 200µM of AP. The data above indicated the safety of AP for macrophages. Thus, antipromastigote activity of AP was evaluated and our results showed AP at concentration 500 µM inhibits 65% the growth of Leishmania amazonensis. AP was also efficient against Leishmania amazonensis amastigotes, displaying a dose-dependent activity with IC50 63.8 µM after 24h treatment. To evaluate if the
leishmanicidal activity of AP was due to increase of Nitric Oxide (NO) production, an important mediator of parasite death in phagosomes, we verify the level of NO by the Griess method. Our data shows that AP at 200 µM do not modulate NO production in infected and uninfected macrophages, stimulated or not with IFN- . Then, we evaluated the arginase activity. Arginase transforms L-arginine in polyamines that were important for parasite growth. We observed a 34% decrease of the arginase activity in macrophages stimulated with IL-4 after treatment with 200 µM of AP. In conclusion AP shows leishmanicidal activity independent NO production, but this effect could be attributed, at least partly to inhibition of arginase activity, although we cannot rule out a direct effect of AP in amastigotes. Support: CAPES, FAPERJ e CNPq.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [238] P 379 - EVIDENCES OF THE EXISTENCE OF SPHINGOSINE KINASE ACTIVITY IN THE PARASITE LEISHMANIA MEXICANA FERMIN, Z.; GAMERO, M.D.; GIL-ZAMBRANO, G.C.; PEREZ, J.L. LABORATORIO DE INMUNOLOGIA CELULAR, INSTITUTO DE BIOMEDICINA, UNIVERSIDAD CENTRAL DE VENEZUELA., CARACAS, VENEZUELA.
Keyword:sphingosine kinase; lipid metabolism ; leishmania mexicana
Abstract: Membrane sphingolipids are a source of molecules involved in cell signaling. The enzyme sphingosine kinase (SK) catalyzes the phosphorylation of sphingosine, generating the bioactive metabolite sphingosine-1-phosphate (S1P). From yeast to man, SK is involved in the regulation of cellular processes as: proliferation, apoptosis, cytoskeletal architecture, chemotaxis, cell adhesion, neuronal excitation, and Ca+2 homeostasis. In kinetoplastidae, sphingolipid metabolism is closely linked to virulence, pathogenicity, and evasion of the host immune response. In Leishmania parasites, although it has been demonstrated the production of S1P, and the existence of sequences comprising SK-like motifs in the genome, it has not been identified and characterized so far, an enzyme activity which may be responsible for phosphorylation of sphingosine. For this reason, we decided to determine the existence of a SK activity in these parasites and to study its physiological role. SK activity determinations were performed on lysates from Leishmania mexicana promastigotes, using the fluorescent substrate 15-NBD-SphBSA, in the absence or presence of: N,N dimethylsphingosine (DMS), dihydrosphingosine (DHS), and 2-(p-Hydroxyanilino)-4-(p-chlorophenyl) thiazole (SK1 II), known inhibitors of human SKs. Variations in the activity between sub-cellular fractions, during growth in culture, and after differentiation to the amastigote stage, were evaluated. The effect of two of the inhibitors on the promastigote growth in culture was tested as well. We demonstrated the existence of SK activity in L. mexicana promastigotes, which is inhibited by DHS and SK1 II, and, paradoxically, stimulated by DMS. Under resting conditions, in promastigotes this activity is primarily localized in the cytosol, and varies with the phase of culture, increasing progressively as the stationary phase is reached. Specific enzyme activity is considerably greater in the promastigote than in the amastigote form. When evaluated the effect of SK inhibitors on cultures of promastigotes, there was a slight inhibitory effect on the growth of the population from 2.5 µM DMS and 5 µM DHS, and a total inhibition at 10 µM for both compounds. The study of the importance of this enzyme for the parasite's physiology and the analysis of the differences between the parasite and the human enzyme will establish its importance as a potential therapeutic target.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [239] P 380 - DIAGNOSIS OF LEISHMANIASIS USING SYNTHETIC PEPTIDES GENERATED FROM SEQUENCES OF IMMUNOGENIC PROTEINS OF LEISHMANIA AMAZONENSIS NARVAEZ, M.L.A.1; VELASQUEZ, E.1; GAUNA, A.2; BERMUDEZ, H.2; FERMIN, Z.3; HERMOSO, T.1 1.SECCION DE BIOQUIMICA DE PARASITOS, INSTITUTO DE MEDICINA TROPICAL, UNIVERSIDAD CENTRAL DE VENEZUELA, CARACAS, VENEZUELA; 2.LABORATORIO DE SINTESIS DE PEPTIDOS, INSTITUTO DE BIOMEDICINA, UNIVERSIDAD CENTRAL DE VENEZUELA., CARACAS, VENEZUELA; 3.LABORATORIO DE INMUNOLOGIA CELULAR, INSTITUTO DE BIOMEDICINA, UNIVERSIDAD CENTRAL DE VENEZUELA, CARACAS, VENEZUELA.
Keyword:synthetic peptides; diagnosis; leishmania amazonensis
Abstract: Clinical and immunological diagnosis of leishmaniasis has been difficult to interpret, as they can give cross-reactions with other diseases, and their clinical manifestations may be also confused with other illnesses. As proposed in 1989 by the WHO, we have rethinked the use of synthetic peptides designed from sequences with immunogenic value, to contribute toward the improvement of existing immunodiagnostic systems, ensuring a fast and reliable diagnosis and monitoring therapeutic efficiency. We evaluated the antigenic recognition of a group of peptides, from antigenic proteins found in our laboratory by inmunoproteomic, by sera from patients with leishmaniasis and Chagas disease Protein sequences from Leishmania amazonensis were aligned with homologous sequences from other Leishmania species, to select conserved regions, which were then confronted to the Trypanosoma cruzi genome to discard those similar. Possible epitopes were determined using the program ANTHEPROT, and their antigenicity evaluated by the pepscan technique. After this initial approach, the peptides of interest were synthesized, on solid phase, by Fmoc chemistry. The purification of the peptides was done by HPLC and the determination of purity by mass spectrometry. Multiple Antigen Blot Assay (MABA), allowed the evaluation of the specificity and sensitivity of the selected peptides after confrontation with sera from patients and healthy individuals. Diagnostic serum samples were collected at the Department of Dermatology, Hospital Universitario de Caracas and the outpatients clinic of the, Instituto de Medicina Tropical. Four of the fourteen peptides tested gave good responses when confronted with sera from positive patients; peptide 607, 1936, 1937, and 1938 being recognized with a sensitivity of 61%, 56%, 61%, 64% and a specificity of 78.3%, 96.67%, 96,67, and 100%, respectively. The other 10 peptides where discarded for low recognition values or cross-reaction with sera from Chagas disease patients. Negative controls showed no reactivity. Four new proteins found by inmunoproteomic are now under evaluation as possible immunomarkers for leishmanisis. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [242] P 381 - OVEREXPRESSION OF PROLIFERATING CELL NUCLEAR ANTIGEN AUGMENTS THE ANTIMONY RESISTANCE IN SODIUM ANTIMONY GLUCONATE SENSITIVE CLINICAL ISOLATE OF LEISHMANIA DONOVANI TANDON, R.; BAHARIA, R.K.; DAS, S.; MISRA, P.; DUBE, A. CENTRAL DRUG RESEARCH INSTITUTE, LUCKNOW, INDIA.
Keyword:visceral leishmaniasis; sag resistance ; pcna overexpression
Abstract: Amongst the available antileishmanial drugs, Pentavalent antimonials has been the first-line drug for all forms of leishmaniasis for almost seven decades. However, in recent years, a largescale increase in clinical resistance to pentavalent antimonials has been reported. During the last decade several novel formulations of conventional antileishmanials as well as new drugs became available or were under investigation. However, their wide use in poor countries is
hindered mainly due to high costs and also due to concerns of toxicity and emergence of resistance. Most of the knowledge of antimony resistance mechanisms in Leishmania spp. has primarily emerged from the study of laboratory-generated drug-resistant cell lines, but in vitro unresponsiveness does not necessarily translate to clinical resistance. Therefore information collected form the studies on clinical isolates related to Sodium Antimony Gluconate (SAG) resistance are of paramount importance to reveal the mechanism of drug resistance which is sill not very clear till date. Recently microarray and proteomic approaches have been employed to identify the proteins with putative roles in drug resistance. Previously, through differential proteomics, Proliferating Cell Nuclear Antigen (PCNA) was identified to be overexpressed in SAG resistant strain as compared with the SAG sensitive strain of L. donovani. The present study deals with characterization of PCNA and exploration of its role in SAG resistance. PCNA was cloned, overexpressed and purified from the bacterial host system. Polyclonal antibody was raised against rPCNA in Swiss mice. Its differential expression in SAG sensitive and SAG resistant parasites confirmed by western blotting and real time PCR, confirming that PCNA was overexpressed by 3fold in log, stationary, PNA isolated procyclic and metacyclic stages of promastigote and by ~5 fold in amastigote stage of SAG resistant isolate as compared to SAG sensitive isolate. PCNA was overexpressed as GFP fused protein in SAG sensitive clinical isolate of L. donovani and modulation in the drug sensitivity of the transfectants was assessed in vitro which resulted in ~2 fold increase of IC50 of SAG in PCNA overexpressing transfectants. Based on the abovestated studies therefore it was concluded that PCNA has some possible role in SAG resistance in Leishmania donovani clinical isolate and warrants detailed investigation regarding its mechanism.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [246] P 382 - DOES ANTIMONY-RESISTANT LEISHMANIA DONOVANI HAVE A HIGHER FITNESS RELATIVE TO ITS ANTIMONY-SENSITIVE COUTERPARTS? VANAERSCHOT, M.1; STAUCH, A.2; BERG, M.1; ROY, S.3; ADAUI, V.4; RIJAL, S.5; DUJARDIN, J.1 1.INSTITUTE OF TROPICAL MEDICINE, ANTWERPEN, BELGIUM; 2.UNIVERSITY OF TUBINGEN, TUBINGEN, GERMANY; 3.INSITUTE OF CHEMICAL BIOLOGY, KOLKATA, INDIA; 4.INSTITUTO DE MEDICINA TROPICAL ALEXANDER VON HUMBOLDT, LIMA, PERU; 5.B.P. KOIRALA INSTITUTE OF HEALTH SCIENCES, DHARAN, NEPAL.
Keyword:fitness; drug resistance; antimonials
Abstract: Pentavalent antimonials (SSG) have been the mainstay in the treatment of VL but the emergence of SSG-resistant Leishmania affected its efficacy, contributing to high rates of SSG-treatment failure throughout the world (up to 65% in the Indian subcontinent and 39% in South America). SSG acts, among others, through the generation of oxidative stress in Leishmania and through the effector mechanisms of the host cell, the last link of the host immune system, resulting in increased levels of oxidative and nitrosative stress upon the parasite. Leishmania that acquired resistance to SSG might therefore have also acquired a higher adaptive capacity to overcome the immune system defences of the host. This would benefit SSG-resistant (SSG-R) Leishmania above SSG-sensitive (SSG-S) Leishmania even in absence of the drug and might therefore positively affect the relative fitness of SSG-R Leishmania. The fitness of SSG-R L. donovani from the Indian subcontinent has been studied by mimicking several life stages of the parasite in the lab and comparing the in vitro and in vivo survival
capacity of SSG-S and SSG-R L. donovani in various situations. In vitro, SSG-R strains showed a greater capacity to generate infectious forms which likely contributed to the observed higher in vitro and in vivo infection and survival skills of SSG-R L. donovani compared to SSG-S L. donovani. The transmission of L. donovani was also translated into a mathematical model, showing that the prevalence of SSG-resistance in Bihar could not be explained without assuming a higher fitness of SSG-R parasites. Field studies also revealed a high prevalence of SSG-R parasites in natural populations of India despite the low SSG pressure nowadays. Hence, SSG-R L. donovani does indeed display traits of an increased fitness compared to its SSG-S counterparts, which stands in contrast to the fitness cost that is usually related to drug resistant parasites. We will discuss the findings obtained on SSG-R L. donovani from the Indian subcontinent and try to extrapolate them to the South American case where SSG-R L. braziliensis showed to be wide-spread despite the prevalence of untreated animal reservoirs. Might a similar scenario as observed in SSG-R L. donovani also apply for SSG-R L. braziliensis and possibly other Leishmania species in Latin America? This work was supported by the European Commission’s Seventh Framework Programme (Kaladrug-R, grant 222895) and the Gemini consortia (grant ITMA SOFI-B).
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [247] P 383 - THE DETECTION OF ANTIMONY-RESISTANT L. DONOVANI IN ACTIVE VL CASES: APPLICATION OF MOLECULAR MARKERS ON BLOOD, BONE MARROW AND PARASITE DNA VANAERSCHOT, M.1; DE DONCKER, S.1; BHATTARAI, N.R.2; SUNDAR, S.3; RIJAL, S.2; DUJARDIN, J.1
1.INSTITUTE OF TROPICAL MEDICINE, ANTWERP, BELGIUM; 2.B.P. KOIRALA INSTITUTE OF HEALTH SCIENCES, DHARAN, NEPAL; 3.BANARAS HINDU UNIVERSITY, VARANASI, INDIA.
Keyword:antimonials; drug resistance; molecular markers
Abstract: Antimony-resistant (SSG-R) L. donovani was wide spread in the Indian subcontinent at the time that treatment policies changed, introducing miltefosine as the new official first line treatment of VL in 2006. However, epidemiological data on their current prevalence in the field is scarce. Until recently, the standard test to assess SSG susceptibility of Leishmania was a laborious in vitro assay, hampering epidemiological monitoring of the spread of SSG-R strains in the field. However, candidate genetic markers for clinically relevant SSG-resistant L. donovani were recently identified by full genome sequencing and further validated on a larger set of clinical strains, showing that in this dataset 3 genomic locations show a good performance specifically detecting those SSG-resistant parasites that are only present in patients experiencing SSGtreatment failure. Such genetic markers are easy to apply and allow to monitor the spread of SSG-R L. donovani in the field more efficiently. Here we report the assessment of these markers directly on blood and bone marrow samples from confirmed VL patients. Firstly, we performed a meta-analysis combining all available data (both acquired from blood, bone marrow and cultured parasite DNA) to further document the performance of the 3 genetic locations to detect strains that contribute to SSG-treatment failure. This showed that these markers performed better in detecting SSG-non-responsiveness of the infected patient (sensitivity of 71.4%) than SSG-treatment failure in general (including nonresponsiveness and relapse; sensitivity of 56.5%). Secondly, we assessed the prevalence of the parasite markers in the blood or isolated parasites from active VL cases which were sampled between 2009-2011. These results indicate that SSG-R L. donovani that can contribute to SSG-
treatment failure still circulate in the endemic regions in the Indian subcontinent despite the significant drop in SSG-pressure on the parasite population now that MIL has been the official first line treatment for more than 5 years. This work was financially supported by the European Commission’s Seventh Framework Programme (Kaladrug-R project, grant 222895) and the Gemini consortia (grant ITMA SOFIB).
- GENOMICS, TRANSCRIPTOMICS, PROTEOMICS, METABOLOMICS [248] P 384 - METABOLIC ADAPTATIONS OF LEISHMANIA DONOVANI IN RELATION TO DIFFERENTIATION, DRUG RESISTANCE AND DRUG PRESSURE VANAERSCHOT, M.1; JANKEVICS, A.2; CUYPERS, B.1; KHANAL, B.3; RIJAL, S.3; OPPERDOES, F.4; BREITLING, R.2; DUJARDIN, J.1; BERG, M.1 1.INSTITUTE OF TROPICAL MEDICINE, ANTWERPEN, BELGIUM; 2.INSTITUTE OF MOLECULAR, CELL AND SYSTEMS BIOLOGY, GLASGOW, UNITED KINGDOM; 3.B.P. KOIRALA INSTITUTE OF HEALTH SCIENCES, DHARAN, NEPAL; 4.DE DUVE INSTITUTE, BRUSSELS, BELGIUM.
Keyword:antimonials; metabolomics; drug resistance
Abstract: Due to significant improvements in LC-MS technology, metabolomics is increasingly used as a tool to discriminate the responses of organisms to various stimuli or drugs. In this study, we implemented an untargeted LC-MS metabolomics approach to gain insights in metabolic differences between three clinical L. donovani strains with a similar genetic background but different susceptibilities to antimonials (SSG), and this at different stages during promastigote growth and in the absence or presence of drug pressure. Present work clearly revealed dramatic metabolic differences (i) between the different strains, (ii) between different promastigote growth stages within the same strain, and (iii) when a resistant strain is maintained under drug pressure. A number of metabolic changes in resistant strains showed to be constitutive (present in both growth stages), pointing towards a clear link with SSG-resistance, whereas most metabolic changes between SSG-resistant and SSG-sensitive strains are detected only in the stationary growth stage of L. donovani promastigotes, i.e. the moment when they are preparing their metabolism for a possible encounter with the human host and the drug. These stationary stage-specific changes might also reflect the close intertwinement between antimonial resistance and other epi-phenotypes such as an increased metacyclogenesis in resistant parasites. The observed metabolic changes suggest that SSG-resistant parasites have (i) an increased protection against oxidative stress; (ii) a higher fluidity of the plasma membrane; and (iii) a survival kit to better endure the initial stages of infection. Keeping a resistant strain under SbIII drug pressure resulted in a more pronounced metabolic profile of the resistance features detected previously. In conclusion, this study showed the strength of an untargeted LC-MS metabolomics approach to allow an in-depth analysis of the regulation of the metabolome in differentiating organisms with different drug susceptibility such as antimony-sensitive and -resistant L. donovani. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [249] P 385 - A STATISTICAL METHOD FOR THE QUANTIFICATION OF METACYCLOGENESIS OF LEISHMANIA DONOVANI CUYPERS, B.1; VANAERSCHOT, M.1; GARCIA-HERNANDEZ, R.2; GAMARRO, F.2; LAUKENS, K.3; DUJARDIN, J.1; BERG, M.1 1.INSTITUTE OF TROPICAL MEDICINE, ANTWERPEN, BELGIUM; 2.INSTITUTE OF PARASITOLOGY AND BIOMEDICINE 'LOPEZ NEYRA', GRANADA, SPAIN; 3.UNIVERSITY OF ANTWERP, ANTWERPEN, BELGIUM.
Keyword:metacyclogenesis; infectivity; differentiation
Abstract: Parasite development is a continuous event with relicts of the previous, or adaptations to the next stage. Although most of the time this is in fact not the research question, the identification and quantification of life stages of the parasite are crucial to interpret the study results, which is of utmost importance for ‘omics studies. This is particularly the case for the metacyclic life stage, in which most of the molecular adaptations for the intracellular life develop. There are several methods to quantify the percentage of metacyclic parasites, but the fastest one is a statistical determination which works with morphometric input data on cell dimensions and the relative flagella length. Metacyclic parasites typically have a larger value for flagella length/cell length (F/L), and a smaller cell surface (L*W). Cutoff values can be found in literature but are most of the time arbitrary numbers. Even more, these parameters could be population dependent or influenced by the environment (stress) of the parasite. Clearly, there is a need for a fast and objective quantification method. Here we describe a statistical method based on cluster analysis that makes this distinction for us. First, for each of the two parameters, the data is split up in the three best groups by the k-means method. This method minimizes the within group variation, and thus maximizes the between group variation. The parasites are then divided into three populations: metacyclic parasites (F/L > cutoff and L*W < cutoff), procyclic parasites (F/L > cutoff and L*W < cutoff), and transitional forms. When comparing different treatments, p-values are generated with permutation and confidence interval by bootstrapping. This method demands little or no sophisticated equipment, as it requires only parasite smears being photographed, analyzed with freely available software to obtain the morphometric data and then processed using the open source software R. This statistical method can thus be considered as a significant asset to support studies where the level of metacyclogenesis needs to be documented to determine its effect on the generated results. This work was supported by the European Commission’s Seventh Framework Programme (Kaladrug-R project, grant222895) and the Gemini consortia (grant ITMA SOFI-B).
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [250] P 386 - IN VITRO EVALUATION OF METACYCLOGENESIS IN LEISHMANIA DONOVANI ISOLATES IN RELATION TO MILTEFOSINE TREATMENT OUTCOME RAI, K.1; CUYPERS, B.1; BERG, M.1; BHATTARAI, N.R.2; RIJAL, S.2; DUJARDIN, J.1; VANAERSCHOT, M.1
1.INSTITUTE OF TROPICAL MEDICINE, ANTWERP, BELGIUM; 2.B.P. KOIRALA INSTITUTE OF HEALTH SCIENCES, DHARAN, NEPAL.
Keyword:metacyclogenesis; treatment failure; miltefosine
Abstract: Miltefosine (MIL) treatment of visceral leishmaniasis (VL) patients in the Indian subcontinent is currently jeopardized by high rates of MIL-relapse in India (± 10%), Bangladesh (± 15%) and Nepal (± 30%). Several studies assessed the in vitro susceptibility to MIL of the infecting parasite, but could so far not identify clear cut MIL-resistant L. donovani present in MILrelapsed patients. Also the MIL plasma concentration proved similar between cured and relapsed VL patients (Rijal et al., personal communication). Interestingly, previous work on antimony (SSG)-treated patients has shown that also other factors at the parasite level might be of interest when studying treatment failure. As such, SSG-resistant parasites showed an increased capacity to undergo metacyclogenesis, which is the generation of infectious forms during promastigote growth. In this study we therefore aimed to evaluate the metacyclogenic
capacity of clinical parasite isolates from MIL-cured and MIL-relapsed patients from the endemic region in Nepal to determine whether this factor may have influenced MIL-treatment outcome in the Indian subcontinent. Eight strains isolated from MIL-cured and 7 strains isolated from MIL-relapsed patients were grown in vitro and parasite smears for morphometric analysis were prepared during the logarithmic and stationary growth phase. The flagella length, cell length and cell width were determined using Cell^D (Olympus) to enable calculation of the FL ratio (flagella length over cell length) and LW (i.e. cell area) for each of the minimum 100 parasites that were evaluated per slide. These data were used to determine the percentage of metacyclic parasites using the kmeans cluster method (see poster of Cuypers B), being those parasites with a large FL ratio and small LW value. This revealed that strains isolated from MIL-relapsed patients showed a higher percentage of metacyclic parasites during stationary phase compared to strains isolated from MIL-cured patients, which is very similar to what was earlier observed for SSG-R and SSG-S L. donovani. We will discuss the implications of these findings and the additional questions they raise in relation to the high MIL treatment failure rates observed in the Indian subcontinent. This work was supported by the European Commission’s Seventh Framework Programme (Kaladrug-R project, grant222895).
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [255] P 387 - ANTIMONY SPECIATION IN MEGLUMINE ANTIMONIATE BY VOLTAMMETRY: A CHALLENGE FRÉZARD, F.1; SALAUN, P.2 1.DEPARTAMENTO DE FISIOLOGIA E BIOFÍSICA - ICB, UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL; 2.SCHOOL OF ENVIRONMENTAL SCIENCES, UNIVERSITY OF LIVERPOOL, LIVERPOOL, UNITED KINGDOM.
Keyword:glucantime; speciation; voltammetry
Abstract: Glucantime, a Meglumine antimoniate solution, is commonly used for the treatment of leishmaniasis, but the presence of residual Sb(III) is thought to be responsible for the toxic side effects observed within patients. Numerous analytical studies have thus focused on determining Sb(III) concentration in Glucantime but without reaching a consensus: results span over 3 orders of magnitude. Here, we present our voltammetric approach, which suggests that: 1- Sb(III) levels are higher than previously reported and, 2- acidic conditions have to be used very carefully to avoid oxidation of Sb(III). This latter point explains to a large extent previously reported results. Measurements were made at a vibrated gold microwire electrode (VGMWE) by stripping chronopotentiometry or stripping voltammetry. Methods were first developed for the detection of Sb(III) in acidic, neutral or alkaline conditions. These methods are: 1- sensitive (detection limits of 19 pM for 120s deposition time), stable (<6%, N = 100), 2- precise (5%, N = 5) and, 3- robust and so were used for the speciation of Sb(III) in Glucantime. In acidic conditions, the Sb(III) signal is not stable and decreased with time (probably due to Sb(III) oxidation) the loss rate increased with acidification, preventing reliable determination of Sb(III) at pH < 2.5. At pH 3 and above, the presence of various Sb(III) complexes was shown by reduction waves obtained by stripping scanned voltammetry (SSV). The presence of stronger Sb(III) complexes were possibly hidden by the reduction of Sb(V) at deposition potentials below -1.3 V. Calibration curves obtained after additions of Sb(III) in diluted solutions of Glucantime were always linear, suggesting that either ligands were saturated or complexation is
kinetically slow. Concentrations of Sb(III) determined in 1 mM HCl were amounting to 27-30 mg.mL-1, i.e ~ 30% of the total Sb (~ 85 mg.mL-1). The levels of Sb(III) reported here are much higher than previously reported. As an implication of these unexpected results, an alternative model can be proposed for the mode of action of pentavalent antimonials against leishmaniasis, in which pentavalent antimony complexes may act as molecular carrier of Sb(III) and release it specifically in the acidic intracelular compartment where Leishmania parasite resides. Supported by CNPq e FAPEMIG. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [256] P 388 - WITHANIA SOMNIFERA CHEMOTYPE NMITLI 101R AUGMENTS THE ANTILEISHMANIAL EFFICACY OF MILTEFOSINE, PAROMOMYCIN, AND AMPHOTERICIN B IN LEISHMANIA DONOVANI INFECTED HAMSTERS TRIPATHI, C.D.P.; KUSHAWAHA, P.K.; GUPTA, R.; KHARE, P.; BHATTACHARYA, S.M.; DUBE, A. CENTRAL DRUG RESEARCH INSTITUTE, LUCKNOW, INDIA.
Keyword:vl, visceral leishmaniasis; immunopotentiation; sld, soluble leishmania donovani antigen
Abstract: The characteristic feature of visceral leishmaniasis (VL), is immunosuppression modulating the T-cell responses towards Th2 type which is responsible for disease progression, thereby, immune system is unable to play its natural role to control the disease or augment the effects of the drugs. To circumvent immunosuppression, immunostimulation of the infected host presents a logical and viable alternative. The natural products have been a rich source of drugs and immunomodulators. Withania somnifera (WS), commonly known as Ashwagandha, is one of the most important medicinal plant in the traditional Indian medical systems for over 3000 years for various diseases and disorders. Immunomodulation using medicinal plants is critically being considered as an immunoprophylactic and/or adjunct approach to conventional treatment of various diseases. In the present study we demonstrated excellent immnunopotentiation activity of a chemotype of W. somnifera (Compound 101R) in twenty five day old Leishmania donovani infected hamsters at 30 mg/kg for 15 days either alone or in adjunct with the ED50 dose of standard antileishmanials- Miltefosine (10 mg/kg), Paromomycin (30 mg/kg) and Amphotericin B (0.5 mg/kg) . On days 30 and 60 p.t., the antileishmanial efficacy of the standard drugs were significantly enhanced when administered with 101R as observed through significant reduction in parasite burden (~98%). The mRNA expression of cytokines by real time PCR revealed a clear cut polarization of Th1 response in hamsters treated with NMITLI 101R+ antileishmanial drugs on day 30 & day 60 post treatment as displayed by increased expression of TNF- , IFN-y, IL-12 and increased inducible NO synthase mRNA transcript. Infected untreated hamsters, on the contrary, developed Th2 polarized response as revealed by enhanced production of TGF- , IL-4 and IL-10. The soluble Leishmania donovani antigen(SLD) stimulated lymphocytes of hamsters treated with combination of chemotype and standard drugs showed significant lymphocyte proliferation. Further, there was a considerable increase in reactive oxygen species (ROS) and nitric oxide production in peritoneal macrophages of hamsters as well as mouse macrophage cell line J774.1 as compared with the normal and infected control. This was further supported by a remarkable increase in antileishmanial IgG2 antibody levels. The results establish that this chemotype possess immunopotentiatory potential and warrants further investigations. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [258] P 389 - DEPOLARIZATION OF MITOCHONDRIAL MEMBRANE POTENTIAL AND UPREGULATION OF REACTIVE OXYGEN SPECIES CONTRIBUTE TO NITAZOXANIDE
INDUCED DEATH IN LEISHMANIA (L.) INFANTUM MESQUITA, J.T.1; ALEXANDRE, T.R.1; TANIWAKI, N.N.1; GALISTEO, A.J.2; TEMPONE, A.G.1 1.INSTITUTO ADOLFO LUTZ, SAO PAULO, SP, BRAZIL; 2.INSTITUTO DE MEDICINA TROPICAL, SAO PAULO, SP, BRAZIL.
Keyword:leishmania; nitazoxanide; mitochondria
Abstract: The search for new therapeutic targets for neglected diseases is an important tool to discover selective drugs. In addition, drug repurposing has been a cost-effective strategy which consists in finding new therapeutic purposes for drugs in clinical use. Visceral Leishmaniasis (VL) is neglected disease that victimizes underprivileged populations in developing countries; considering the limited and toxic therapy, novel treatments are needed. In this work, the lethal action of nitazoxanide (NTZ), a drug in clinical use for helmints and giardiasis, was investigated in Leishmania (L.) infantum. NTZ showed to be a selective compound, eliminating both stages of Leishmania, resulting in 50% inhibitory concentration (IC50) values of 43 µg/mL for promastigotes and 7 µg/mL for intracellular amastigotes. Considering the lack of mitochondrial dehydrogenases activity (MTT assay), NTZ showed a leishmanicidal effect after 24 hours. By using different fluorescent probes, NTZ showed a lethal effect in Leishmania, causing a rapid depolarization of the mitochondrial membrane potential and an up-regulation of reactive oxygen species (ROS), which might have contributed to an oxidative stress of the parasite. Flow cytometry analysis using the fluorescent probes annexin and propidium iodide, showed an exposure of phosphatidylserine induced by NTZ, suggesting an apoptosis-like death. By using the fluorescent vital dye SYTOX Green, NTZ showed no alteration of plasma membrane permeability, which was also corroborated by transmission electron microscopy studies. In addition, NTZ also induced important ultrastructural alterations, confirming the mitochondrial injury at initial incubation time and nuclear damages. These data suggest that NTZ affect the bioenergetic metabolism of Leishmania and if adequately used could be a potential drug candidate for VL. Supported by FAPESP and CNPq. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [259] P 390 - COMBINATION THERAPY STUDIES WITH NITAZOXANIDE AND AMPHOTERICIN B, GLUCANTIME®, MILTEFOSINE AND SITAMAQUINE AGAINST LEISHMANIA (L.) INFANTUM INTRACELLULAR AMASTIGOTES MESQUITA, J.T.; TEMPONE, A.G.; REIMÃO, J.Q. INSTITUTO ADOLFO LUTZ, SAO PAULO, SP, BRAZIL.
Keyword:leishmania; combination therapy; nitazoxanide
Abstract: Leishmaniasis is a neglected disease that affects poorest population mainly in developing countries, representing one of the major causes of mortality and morbidity. Therefore, efforts to find new chemotherapeutics for Leishmaniasis remain a priority. Previous reports demonstrated the in vitro and in vivo anti-leishmanial activity of nitazoxanide, an anti-protozoal agent used in the treatment of infectious diarrhea caused by Cryptosporidium parvum and Giardia lamblia. The present work was carried out to determine the effect of nitazoxanide in combination with amphotericin B, Glucantime®, miltefosine and sitamaquine on Leishmania (L.) infantum. Mouse peritoneal macrophages were infected with Leishmania (L.) infantum amastigotes in order to calculate the 50% inhibitory concentration (IC50) values. Drug interactions were assessed with a modified fixed ratio isobologram method and Fractional Inhibitory Concentrations (FIC), sum of FIC ( FIC) and overall mean FIC were calculated for each combination. The nature of interactions was classified according to the overall mean FIC. The combination between nitazoxanide and amphotericin B, Glucantime®, miltefosine and sitamaquine showed overall mean FIC values of 1.12, 0.82, 1.06 and 0.94, respectively, indicating indifferent interaction.
Graphical isobologram analysis showed that the combination of nitazoxanide and Glucantime® was the most promising, with FIC value of 0.67 in the proportion 2:3 nitazoxanide:Glucantime®). Considering the in vitro activity of nitazoxanide and the obtained results into in vitro combinations with current anti-leishmanial drugs, further in vivo studies may be considered to evaluate possible drug interactions in Visceral Leishmaniasis. Supported by FAPESP and CNPq. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [264] P 391 - SPLEEN ASPIRATE IS THE MOST SUITABLE SAMPLE FOR LEISHMANIA INFANTUM DNA DETECTION IN SYMPTOMATIC DOGS SOLCÀ, M.S.1; BASTOS, L.A.1; GUEDES, C.E.S.1; BORDONI, M.1; NASCIMENTO, E.G.2; OLIVEIRA, G.G.S.1; DOS-SANTOS, W.L.C.1; FRAGA, D.B.M.3; VERAS, P.S.T.4
1.CENTRO DE PESQUISA GONÇALO MONIZ - FIOCRUZ, SALVADOR, BA, BRAZIL; 2.CENTRO DE REFERÊNCIA EM DOENÇAS ENDÊMICAS PIRAJÁ DA SILVA, JEQUIÉ, BA, BRAZIL; 3.ESCOLA DE MEDICINA VETERINÁRIA, UNIVERSIDADE FEDERAL DA BAHIA; INCT-DT, SALVADOR, BA, BRAZIL; 4.CENTRO DE PESQUISA GONÇALO MONIZ - FIOCRUZ; INCT-DT, SALVADOR, BA, BRAZIL.
Keyword:canine visceral leishmaniasis; qpcr; spleen
Abstract: Canine visceral leishmaniasis (CVL) is a systemic disease that affects a variety of tissues with different degrees of parasitism. Several biological samples obtained by invasive and noninvasive procedures have been used for Leishmania infantum DNA detection in dogs. However, studies evaluating different tissues of infected dogs have shown variable and sometimes conflicting results. In the present study we aimed to determine which biological sample conferred the most accurate detection of parasite DNA, as well as compare the tissue parasite loads using qPCR. Stray dogs were captured in an endemic area for visceral leishmaniasis (VL) in Bahia, Brazil and euthanized. During necropsies, spleen aspirates (SP), blood samples (BL), lymph node fragments (LN), bone marrow aspirates (BM), and conjunctival swabs (CS) from either eyes were obtained. Diagnosis of CVL was performed by ELISA and SP culture. Samples’ DNA was extracted, and only those who fulfilled the selection criteria for DNA integrity were used: 51 for CS, SP, BL and LN, as well as 24 for BM. First, to determine the parasite DNA detection rates a standard curve for parasite load quantification was generated using a serial dilution of L. infantum DNA. Then, each value was normalized against the value from the host constitutive gene (18S rRNA). Differences among detection rates from each canine samples were evaluated using chi-square and differences among parasite loads using Friedman test (p < 0.05). All the dogs presented at least 1 clinical sign related to CVL. ELISA was able to detect antibody anti-Leishmania in 82% of the dogs, spleen culture detected the parasite in 37%, while qPCR detected parasite DNA in 100% of the animals, considering at least one of the samples evaluated. Positivity in qPCR was detected in 98% of the SP, 80% of BL, 55% of LN, 42% of BM, 37% in the left eye and 33% in the right eye. Using qPCR, among the samples analyzed, CS showed the lowest positivity rate. Parasite DNA was better detected in SP compared to all the other samples (p < 0.01). However SP, showed a higher parasite load only when compared to LN (p 0.05). In conclusion, the results indicate that in symptomatic dogs SP is the most suitable sample for parasite DNA detection. Furthermore, among the samples requiring a less invasive collection, BL was considered a valid alternative sample for L. infantum DNA detection. Supported by CNPq, CAPES and FAPESB.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [269] P 392 - PERIPHERAL BLOOD SMEAR DIAGNOSIS OF CANINE VISCERAL LEISHMANIASIS ABBOUD, L.C.S.1; AZEVEDO, L.1; GENESCA, M.A.M.2; GRAVINA, M.C.S.1 1.PREFEITURA DO RIO DE JANEIRO, RIO DE JANEIRO, RJ, BRAZIL; 2.MEDICA VETERINARIA AUTONOMA, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:leishmaniasais; diagnostic; canine
Abstract: An 8-year-old, male, medium-sized, mixed-breed dog was admitted to a local veterinary clinic with anorexia, pale mucous membranes, prostration, history of convulsive episodes, heart murmur, and no dermatological signs. Complementary tests revealed anemia, thrombocytopenia, hyperproteinemia, presence of microfilariae, AST (87 IU/l), increased levels of serum creatinine (8.4 mg/dl) and blood urea nitrogen (368 mg/dl), determining renal failure. In the routine performance of differential leukocyte count, the blood smear revealed the presence of Leishmania sp. amastigotes within neutrophils. Specific serological testing by indirect immunofluorescence assay (IFA) was then carried out, and the sample resulted positive with a 1:320 titration. The patient died 2 days later. A necropsy was then done, and imprints of spleen, liver, bone marrow and lymph nodes were made, all showing large amounts of Leishmania sp. amastigotes, thus confirming the presence of the disease. Given this scenario, leishmaniasis would not be diagnosed if the parasite was not detected in the blood smear. Therefore this technique is to be regarded as a valid one, and its importance should be recognized, although this is not a very common diagnostic approach. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [272] P 393 - AMPHIPHILIC ANTIMONIAL COMPLEX: IN VITRO ACTIVITY AND EFFICACY OF A TOPICAL FORMULATION IN MURINE CUTANEOUS LEISHMANIASIS MODEL ALMEIDA, G.G.1; FERNANDES, F.R.2; FERREIRA, W.A.3; VOGAS, G.P.1; BAHIA, A.P.C.O.2; FERREIRA, L.A.M.4; DEMICHELI, C.3; FRÉZARD, F.J.G.2; MELO, M.N.1
1.DEPARTAMENTO DE PARASITOLOGIA, INSTITUTO DE CIÊNCIAS BIOLÓGICAS, UFMG., BELO HORIZONTE, MG, BRAZIL; 2.DEPARTAMENTO DE FISIOLOGIA E BIOFÍSICA, INSTITUTO DE CIÊNCIAS BIOLÓGICAS, UFMG., BELO HORIZONTE, MG, BRAZIL; 3.DEPARTAMENTO DE QUÍMICA, INSTITUTO DE CIÊNCIAS EXATAS, UFMG., BELO HORIZONTE, MG, BRAZIL; 4.FACULDADE DE FARMÁCIA, UFMG., BELO HORIZONTE, MG, BRAZIL.
Keyword:topical treatment; tegumentary leishmaniasis; antimony
Abstract: Leishmaniasis is considered a major public health problem in 88 countries worldwide. The available drugs require long-term treatment and cause several side effects. It is considered a neglected disease by the World Health Organization, thus reinforcing the need for new effective and less toxic drugs. Efforts to achieve effective topical formulation for tegumentary leishmaniasis have been unsuccessful so far. Here, we investigated a strategy based on the increase of lipophilicity of existing antimonial drug, to improve the membrane permeation of antimony. Antimony(V) was found to bind to amphiphilic octanoil-metilglucamida surfactant (L8), forming an amphiphilic antimony complex. The aim of this study was to evaluate the in vitro activity of SbL8 complex and the in vivo efficacy of a topical formulation in a murine model of cutaneous leishmaniasis. In vitro assays using mice peritoneal macrophages showed that SbL8 complex was about 100 times more toxic (CC50 = 0.26 mM Sb) than Glucantime (CC50 = 26.31 mM Sb) and 6 times more toxic than L8 ligand alone (CC50 = 4.9 mM L8). There was no anti-amastigote activity of SbL8 after 72 h of exposure, at concentrations below the cytotoxic concentration (CC 50), in the model of murine peritoneal macrophages infected with L. amazonensis. In vivo studies used BALB/c mice infected with L. amazonensis and
treatment with a topical formulation containing 20 g% of SbL8 twice a day for 3 weeks. This assay resulted in the stabilization of the lesion size and a decrease in parasite burden, compared to non-treated control. The percutaneous permeation of the antimony from SbL8 formulation was also evaluated in the Franz diffusion cell model. Antimony permeation levels were about 0.008% and 4.3% after 8 h across intact and stripped skin, respectively. The present study shows that the topical formulation, despite the negative in vitro results, may present an anti-leishmania activity in vivo. Further studies are needed to elucidate the potential effectiveness of the SbL8 complex. Supported by FAPEMIG, CNPq and CAPES. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [293] P 394 - COMPARISON AMONG ENZYME-LINKED IMMUNOSORBENT ASSAY WITH TWO DISTINCT CONJUGATES AND INDIRECT FLUORESCENT ANTIBODY TEST FOR THE DIAGNOSIS OF FELINE VISCERAL LEISHMANIASIS ROSSI, C.N.1; SOBRINHO, L.S.V.2; NUNES, C.M.2; LIMA, V.M.F.2; TOMOKANE, T.Y.3; LAURENTI, M.D.3; MARCONDES, M.2 1.SCHOOL OF VETERINARY MEDICINE, UNIVERSITY OF SÃO PAULO, SÃO PAULO, SP, BRAZIL; 2.SCHOOL OF VETERINARY MEDICINE, SÃO PAULO STATE UNIVERSITY, ARACATUBA, SP, BRAZIL; 3.MEDICAL SCHOOL, UNIVERSITY OF SÃO PAULO, SÃO PAULO, SP, BRAZIL.
Keyword:leishmania infantum chagasi; cats; serology
Abstract: Blood, lymph node and bone marrow samples were collected from 200 cats, older than six months, from Araçatuba, São Paulo, Brazil, an endemic area for zoonotic visceral leishmaniasis (VL). Serum samples were used for L. infantum chagasi serological evaluation by three different techniques: enzyme-linked immunosorbent assay (ELISA) with two distinct conjugates (protein A – ELISA-A; anti-cat IgG – ELISA-IgG) and indirect fluorescent antibody test (IFAT). Lymph node and bone marrow samples were obtained by fine needle aspiration for direct parasitological diagnosis of leishmaniasis and for PCR. ELISA cut-off values (mean value plus 3 SD) were determined by analysis of serum samples from 30 healthy cats from a non-endemic area for leishmaniasis. IFAT cut-off value was 1:40. An overall of 9/200 (4.5%) cats were considered infected by direct parasitological examination and/or PCR. Amastigote forms of Leishmania sp were identified in lymphoid organs from eight animals. All of them were also positive by PCR. One cat was positive only by bone marrow PCR. The seroprevalence of leishmaniasis was 11.5% (23/200) by ELISA-IgG, 3.0% (6/200) by ELISA-A and 0.5% (1/200) by IFAT. Only three (33.33%) infected (parasitologically positive) cats were seroreagent; all of them by ELISA-IgG, 2/3 (66.67%) by ELISA-A, and 1/3 (33.33%) by IFAT. Twenty two cats were seropositive with negative parasitological results; 18/22 (81.8%) by ELISA-IgG, 2/22 (9.1%) by ELISA-A and 2/22 (9.1%) by both. A poor agreement was observed between IFAT and ELISAA (K = 0.2795), IFAT and ELISA-IgG (K = 0.0745) and ELISA-A and ELISA-IgG (K = 0.2397). Sensibility was 33.3%, 22.2% and 11.1%, while specificity was 89.5% 97.9% and 100% for ELISA-IgG, ELISA-A and IFAT, respectively. Although we increased ELISA serum and conjugate concentrations in comparison with those used in our laboratory to VL diagnosis in dogs, our results show that VL serological diagnosis in cats should be used with caution and, if possible, in combination with parasitologic diagnosis. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [296] P 395 - OVERTIME ANALYSIS OF DIFFERENT SEROLOGICAL TESTS IN VACCINATED DOGS FOR VISCERAL LEISHMANIASIS MARCONDES, M.1; IKEDA, F.A.1; LIMA, V.M.F.1; ARAUJO, M.F.L.2; HIRAMOTO, R.M.2; TOLEZANO, J.E.1; VIEIRA, R.F.C.3; BIONDO, A.W.4
1.SCHOOL OF VETERINARY MEDICINE, SÃO PAULO STATE UNIVERSITY, ARACATUBA, SP, BRAZIL; 2.INSTITUTO ADOLFO LUTZ, SÃO PAULO, SP, BRAZIL; 3.UNIVERSIDADE FEDERAL DA PARAÍBA, AREIA, PB, BRAZIL; 4.UNIVERSIDADE FEDERAL DO PARANÁ, CURITIBA, PR, BRAZIL.
Keyword:leishmania infantum chagasi; vaccine; diagnosis
Abstract: Development of vaccines against Canine Visceral Leishmaniasis (CVL) may provide a prophylactic barrier, but antibody response detected by standard diagnostic techniques may not differentiate vaccinated from naturally infected dogs. 18 healthy mongrel dogs were submitted to a complete 3-dose protocol of a commercially available vaccine against CVL (Leishmune®, Fort Dodge, USA), and evaluated in 5 different moments. At the time of each blood sampling dogs were monitored by bone marrow and lymph nodes smears obtained by needle aspiration biopsy and by a PCR protocol for the detection of Leishmania species. In order to avoid postvaccination infection, vaccinated dogs received individual deltamethrin-impregnated collars (Scalibor®). Serological detection of anti-Leishmania IgG antibodies was performed by an “Inhouse” ELISA and by 3 commercially available kits adopted by the Brazilian Ministry of Health (Bio-Manguinhos/FIOCRUZ): an ELISA kit, an IFAT kit and a Dual Path Plataform (DPP®) CVL rapid test. All samples were negative at M0 (prior to 1st dose of vaccine). In moment M1 (prior to 2nd dose of vaccine), 9 (50%) dogs were seropositive by the "in-house" ELISA, 1 (5.5%) by IFAT and none by the commercial ELISA or DPP®. In moment M2 (prior to 3rd dose of vaccine), 18 (100%) dogs were seropositive by the "in-house" ELISA, 15 (83.3%) by IFAT, 2 (11.1%) by the commercial ELISA and 1 (5.5%) by the DPP®. In moment M3 (90 days after the first dose of vaccine), 18 (100%) dogs were seropositive by the "in-house" ELISA, 15 (83.3%) by the commercial ELISA, 13 (72.2%) by IFAT and 6 (33.3%) by the DPP®. In moment M4 (180 days after the first dose of vaccine), 16 (88.8%) dogs were seropositive by the "in-house" ELISA, 6 (33.3%) by the commercial ELISA, 2 (11.1%) by the DPP® and 1 (5.55%) by IFAT. Our results have shown that vaccinated animals may not be differentiated overtime from naturally infected animals using different serological tests.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [298] P 396 - IMMUNOMODULATORY EFFECTS OF P-MAPA ON TLR2, ROS AND NITRIC OXIDE OF BLOOD MONONUCLEAR CELLS FROM DOGS WITH VISCERAL LEISHMANIASIS DE LIMA, V.M.F.; MELO, L.M.; SANCHES, L.C.; PEROSSO, J.; SILVA, K.L.O. UNIVERSIDADE DE ESTADUAL PAULISTA, ARAÇATUBA, SP, BRAZIL.
Keyword:immunomodulator; canine visceral leishmaniasis; tlr2
Abstract: Canine visceral leishmaniasis (CVL) is caused by a protozoan Leishmania chagasi is an important zoonotic disease. The parasite in dogs suppress cellular immune response, and the therapeutic arsenal against LVC is limited, the new immunomodulator aggregate fosfolinoleatopalmitoleate magnesium and ammonium protein (P-MAPA) improves immunocompetence when the immune system is impaired, but their dependence on toll like receptors (TLRs) and the mechanisms involved in immune response are still unclear. To investigate the in vitro action of P-MAPA, mononuclear cells were isolated from peripheral blood from healthy dogs and dogs infected with Leishmania spp and cultured in the presence of P-MAPA concentrations of 0, 50, 100 and 200 g/mL in a humid environment at 37°C with 5% CO2 for 24h. The in vitro observation revealed that the P-MAPA significantly increased the expression of TLR2, ROS and nitric oxide in mononuclear cells from healthy dogs and Leishmania spp infected dogs . These
findings suggest that P-MAPA has potential as a therapeutic drug in the treatment of canine visceral leishmaniasis. Financial Support: FAPESP
- CLINICAL LEISHMANIASIS [303] P 397 - QUANTITATIVE KDNA ASSESSMENT DURING TREATMENT OF MUCOSAL LEISHMANIASIS AS A POTENTIAL BIOMARKER OF OUTCOME JARA, M.1; VALENCIA, B.M.1; ALBA, M.1; ADAUI, V.1; AREVALO, J.1; LLANOS-CUENTAS, A.1; BOGGILD, A.K.2 1.INSTITUTO DE MEDICINA TROPICAL, LIMA, PERU; 2.UNIVERSITY OF TORONTO, TORONTO, CANADA.
Keyword:mucosal leishmaniasis; leishmania (viannia) braziliensis; therapy
Abstract: Mucosal leishmaniasis (ML) is a disfiguring clinical manifestion of infection with Leishmania (Viannia) spp. As there is no known biomarker of treatment outcome in ML, we evaluated the concentration of kinetoplast minicircle DNA (kDNA) by cytology brush quantitative PCR before, during, and after treatment of ML in Peruvian patients. ML lesions were sampled by cytology brushes for quantitative PCR at enrolment, days 14 and 21—28 of therapy, and 3-, 6-, or 12-mos after treatment. Parasite concentration in tissue was correlated to demographic, clinical, and parasitologic factors. Twenty patients have completed follow-up: 12 men and 8 women, with median age of 37 yrs (range 18—78 yrs). Fifteen patients were treated with sodium stibogluconate, and 5 with amphotericin B. Cure was achieved in 17 patients, while 2 patients failed multiple courses of therapy. Clinical outcome is unknown in 1 patient. Mean parasite load (PL) at enrolment was 85,614.8 ± 60,427.3 parasites per ug of tissue DNA (par/ug tDNA). Three patterns of quantifiable kDNA during therapy and follow-up emerged: pattern 1 (N=10) was characterized by a mean PL of 170,867 ± 117,482.6 at enrolment, with sequential decline in PL during and after therapy until kDNA was undetectable. Pattern 2 (N=4) was characterized by mean PL of 566.4 ± 306.4 at enrolment, with clearance of detectable kDNA by D14 of treatment, followed by an increased PL by D21-28 of treatment to 80.4 ± 32.1 par/ug tDNA. Pattern 3 (N=6) was characterized by mean PL of 226.7 ± 116.1 at enrolment, with clearance of detectable kDNA during treatment, followed by increased PL by 6-mos follow-up to 36.6 ± 13.1 par/ug tDNA. Both patients who failed treatment demonstrated Pattern 1. Patterns 2 and 3 were associated with granulomatous inflammation (p=0.02). Younger age (33.5 vs. 64 yrs, p=0.10) and shorter ML duration (20.5 vs. 48 mos, p=0.11) are potentially correlated to sequential clearance (pattern 1). Baseline PL, sex, exposure duration, lesion number, and ML location were not correlated to pattern of PL. We have demonstrated that the concentration of parasite kDNA in ML can be quantified by cytology brush sampling and quantitative PCR during and after treatment. Interim analysis demonstrates 3 distinct patterns of PL during and after treatment, which warrant further investigation. Histopathological granulomatous inflammation may predict rebound of PL during or after treatment, though the clinical significance of this rebound is presently unknown. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [305] P 398 - IMPROVEMENT IN CLINICAL SIGNS AND CELLULAR IMMUNITY OF DOGS WITH VISCERAL LEISHMANIASIS USING THE IMMUNOMODULATOR P-MAPA DE LIMA, V.M.F.; SANTIAGO, M.E.B.; NETO, L.S.; ALEXANDRE, E.C.; MUNARI, D.P.; ANDRADE, M.M.C.; SOMENZARI, M.A.; CIARLINI, P.C. UNESP, ARAÇATUBA, SP, BRAZIL.
Keyword:immunomodulator; dogs; cytokines
Abstract: This study investigated the immunotherapeutic potential of the protein aggregate magnesiumammonium phospholinoleate-palmitoleate anhydride immuno-modulator (P-MAPA) on canine visceral leishmaniasis. Twenty mongrel dogs presenting clinical symptoms compatible with leishmaniasis and diagnosis confirmed by the detection of anti-Leishmania antibodies were studied. Ten dogs received 15 doses of the immunomodulator (2.0 mg/kg) intramuscularly, and 10 received saline as a placebo. Skin and peripheral blood samples were collected following administration of the immunomodulator. The groups were followed to observe for clinical signals of remission; parasite load in the skin biopsies using real-time PCR, the cytokines IL-2, IL-10 and IFN-gamma in the supernatant of peripheral blood mononuclear cells stimulated in vitro with either total promastigote antigen or phytohemagglutinin measured by capture ELISA, and changes in CD4+ and CD8+ T cell subpopulations evaluated by flow cytometry. Comparison between the groups showed that treatment with the immunomodulator promoted improvement in clinical signs and a significant reduction in parasite load in the skin. In peripheral blood mononuclear cell cultures, supernatants showed a decrease in IL-10 levels and an increase in IL-2 and IFN-gamma. An increase in CD8+ T cells was observed in peripheral blood. In addition, the in vitro leishmanicidal action of P-MAPA was investigated using 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and no leishmanicidal activity was detected. These findings suggest that P-MAPA has potential as an immunotherapeutic drug in canine visceral leishmaniasis, since it assists in reestablishing partial immunocompetence of infected dogs. Financial Support: FAPESP - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [311] P 399 - EVALUATION OF A NOVEL HISTONE DEACETYLASES INHIBITOR AGAINST LEISHMANIA AMAZONENSIS VERÇOZA, B.R.F.1; HUBER, K.2; BRACHER, F.2; DE SOUZA, W.1; RODRIGUES, J.C.F.1
1.UNIVERSIDADE FEDERAL DO RIO DE JANEIRO, RIO DE JANEIRO, RJ, BRAZIL; 2.LUDWIG-MAXIMILIANS-UNIVERSITT, MNCHEN, GERMANY.
Keyword:chemotherapy; inhibitors of histone deacetylases; leishmania amazonensis
Abstract: Leishmaniasis is one of the most important neglected tropical diseases caused by protozoan parasites of the Leishmania genus. The treatment is based on the use of antimonials, miltefosine, amphotericin B and pentamidine. However, all these drugs cause several side effects for the patients. In this context, there is an urgent necessity to develop new compounds safer, more efficacious and accessible. Histone desacetilases inhibitors are new compounds studied against gastric tumors, inducing alterations on the gene expression that promoting apoptosis in the treated-cells. Thus, the aim of this work is study the effects of a novel histone desacetilases inhibitors, against Leishmania amazonensis promastigotes and intracellular amastigotes. The effects induced by TFMDI were evaluated using different techniques such as: growth curve, immunofluorescence microscopy, scanning and transmission electron microscopy, western blotting, and fluorimetry. For promastigotes, the IC50 value was 2 M. Against intracellular amastigotes, the effect was more pronounced after 48h of treatment and the IC50 value was around 3 M. Immunofluorescence, DIC and scanning electron microscopy revealed an alteration on the promastigote shape, that presented elongated and thinner, and an increase in the expression of acetylated tubulin. Western blotting using an anti-tubulin acetylated antibody
confirmed this increase. Furthermore, transmission electron microscopy revealed several ultrastuctural alterations, such as: 1) Mitochondrial swelling followed by the formation of many vesicles inside the matrix; 2) Presence of many lipid bodies randomly distributed through the cytoplasm; 3) Abnormal chromation condensation; and, 3) Formation of blebs on the plasma membrane. Fluorimetric analysis with Nile Red confirmed the increase in the number of lipid bodies. All these results together indicated that this compound is a promising molecule against leishmaniasis, however new studies are necessary to understand better this mechanism of action Financial supported by CNPq, CAPES and FAPERJ.
- OTHER SPECIAL TOPICS [340] P 400 - EVALUATION OF TWO NOVEL CULTURE MEDIA TO LEISHMANIA INFANTUM CULTIVATION VERSUS DIFFERENT CULTURE MEDIA CASTELLI, G.1; GALANTE, A.2; LO VERDE, V.2; MIGLIAZZO, A.1; REALE, S.2; LUPO, T.1; PIAZZA, M.1; VITALE, F.1; BRUNO, F.1 1.CENTRO REFERENZA NAZIONALE LEISHMANIOSI - ISTITUTO ZOOPROFILATTICO SPERIMENTALE DELLA SICILIA, PALERMO, ITALY; 2.AREA BIOLOGIA MOLECOLARE - ISTITUTO ZOOPROFILATTICO DELLA SICILIA, PALERMO, ITALY.
Keyword:culture medium; cell growth; leishmania
Abstract: The aim of this study is to improve the cultivation of Leishmania promastigotes without the use of common semi-solid culture medium, such as Evans’ modified Tobie’s medium (EMTM), liquid RPMI 1640 and Peptone-yeast extract medium (P-Y). Although EMTM medium permits the growth of a high number of parasites, it is technically difficult to prepare as it requires the use of fresh rabbit blood from animals breed on farms, while RPMI 1640 and Peptone-yeast extract medium show lower growth rate than the EMTM. There is, therefore, a need to develop new blood free and time saving culture systems. The authors propose new modified microbiological media, named RPMI-PY and Tobie-PY, to isolate Leishmania and cultivate parasites for research and diagnostic purposes. The authors have compared the classic culture media to the new media RPMI-PY and Tobie-PY which have been confirmed as achieving a better performance in terms of time and parasitic load with reference to the classic culture technique. Experimental data has been collected showing the higher growth rate of the parasite, registered during 24, 48, 72 hours cultivation and using the count on Bürker’s chamber. This was carried out at the National References Centre for Leishmaniasis (C.Re.Na.L.) where the isolation procedures are conducted daily from a number of different biological matrices.
- CELLULAR AND MOLECULAR BIOLOGY [344] P 401 - ANTIMONY RESISTANT BUT NOT SENSITIVE LEISHMANIA DONOVANI INDUCES HOST CELL AUTOPHAGY AND FAVOURS PARASITE GROWTH MUKHOPADHYAY, R.1; CHOWDHURY, S.1; MUKHERJEE, B.1; MUKHERJEE, S.1; SUNDAR, S.2; DUJARDIN, J.C.3; ROY, S.1 1.CSIR-INDIAN INSTITUTE OF CHEMICAL BIOLOGY, KOLKATA, INDIA; 2.INSTITUTE OF MEDICAL SCIENCES, BENARAS HINDU UNIVERSITY, VARANASI, INDIA; 3.INSTITUTE OF TROPICAL MEDICINE, ANTWERP, BELGIUM.
Keyword:autophagy; resistance; infection
Abstract: Autophagy, a major cellular pathway for the degradation of cytoplasmic macromolecules and organelles, is crucial for cell survival in response to starvation and for preventing intracellular accumulation of abnormal protein aggregates. Usually autophagic mechanism acts as an innate immune defense mechanism by entrapping invading pathogens and targeting the resulting vesicles for fusion with lysosomes. However, intracellular Leishmania donovani (LD) have evolved distinct mechanisms to survive and multiply within membrane-bound compartments. We have now shown that chemical induction of autophagy favours antimony resistant (SbR) LD infection whereas suppression of autophagic pathway hinders it. But antimony sensitive (SbS) LD parasites behave differently. Confocal microscopy confirmed the presence of autophagic vacuoles as it colocalises with the intracellular parasites. Cleavage of microtubule-associated protein 1A/1B light chain 3 I (LC3 I) to LC3 II further supports the notion that autophagy is indeed induced following SbR LD infection. In vivo experimental studies in mice revealed a transient activation of autophagic pathway followed by apoptosis. Involvement of LC3 I and Beclin1 in the intricate relationship between autophagy and SbR LD infection is currently under study. The presence of the characteristic double layered membrane also needs to be confirmed by electron microscopy. This study will provide insight into the mechanism by which SbR LD exploit host cell autophagy for more aggressive infection as compared to sensitive parasites, leading to apoptosis of the host cells which might aid in parasite egress. Developing means of selectively inhibiting autophagy in infected cells should therefore be viewed as a new window of opportunity in dealing with hard-to-eliminate intracellular drug resistant pathogens. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [351] P 402 - COMPARISON OF OCCURRENCE OF INFECTION WITH LEISHMANIA SPP IN LYMPH NODES AND BONE MARROW OF DOGS VIOL, M.A.; AQUINO, M.C.C.; OLIVEIRA, B.C.M.; LIMA, V.M.F.; PERRI, S.H.V.; BRESCIANI, K.D.S. UNIVERSIDADE ESTADUAL PAULISTA, UNESP, ARAÇATUBA, SP, BRAZIL.
Keyword:aspiration; amastigotes; leishmaniasis
Abstract: The diagnosis of leishmaniasis may be made by methods parasitological, immunological or molecular. The parasite can be identified in smears of lymphoid organs, cytological and histological examinations. The confirmation of suspected cases of infection with Leishmania spp. can be done through direct parasitological examination. This study evaluated the occurrence of Leishmania spp. by cytologic examination of lymph nodes and bone marrow of canines, as the objective of comparing these two tissues in which there is a greater presence of the parasite. A total of 49 dogs, 20 females and 29 males, 10 with and 39 without a breed standard racial determined were used for the study. Slides were stained with aspiration biopsies using the dye Fast Panótico. These were analyzed by light microscopy using oil immersion at 100X one. Through the McNemar test were observed amastigotes of Leishmania spp. in 81.6% (40/49) of samples of lymph nodes and 42.9% (21/49) of material containing bone marrow. Significant difference was noted between the lymphoid organs (p <0.05). The kappa coefficient was 0.2139 and is therefore considered weak. Through the results, it was concluded that this study had a higher occurrence of Leishmania spp. in lymph nodes than in bone marrow of dogs examined.
- OTHER SPECIAL TOPICS [357] P 403 - LEISHMANIA EUKARYOTIC INITIATION FACTOR 4A AS A DRUG DISCOVERY TARGET AGAINST VISCERAL LEISHMANIASIS HARIGUA, E.S.1; BOUVIER, G.2; BLONDEL, A.2; BARHOUMI, M.1; NILGES, M.2; GUIZANI, I.1 1.INSTITUT PASTEUR DE TUNIS, TUNIS, TUNISIA; 2.INSTITUT PASTEUR, PARIS, FRANCE.
Keyword:leishmanian eif4a; drug discovery; structural bioinformatics
Abstract: Leishmaniases are a complex group of parasitic diseases that annually affect 2 million people worldwide with 0.5 million cases of visceral leishmaniasis (VL) caused by parasites of the L. donovani species complex. VL is fatal if left untreated. Thus, novel control strategies including drugs and vaccines constitute research priorities. Our study aims at identifying and characterizing new anti-Leishmania molecules. We seek to deliver in silico-optimized molecules that will need to be tested on their target in the next step. Such molecules are expected to constitute lead compounds. Leishmania eukaryotic Initiation Factor (LeIF) has 53% identity with human eIF4A, a prototype of DEAD-box family that is considered as promising therapeutic target against cancers. Like other DEAD-box proteins, LeIF is both an RNA dependent ATPase and an ATP-dependent RNA helicase in vitro. It shows sequence diversity in its N-terminal half including substitutions and Leishmania-specific peptides, when compared to its orthologues. These N-terminal differences were proved deterministic in organism specific interactions. We modeled the 3D structures of LeiF in its substrate-bound and ligand-free conformations by comparative modeling, using 3D structures of human eIF4A and other DEAD-box proteins. Molecular dynamic simulations proved the stability of both models and served to identify two potential docking sites: one is located close to the LeIF specific N-terminus; the other is plausibly the LeiF RNA binding site and is adjacent to Ia and Ib motifs, known to be important in the interaction with RNA. Hence, we expect to identify selective inhibitors/stimulators, a difficult task due to the high similarity of LeiF and human eIF4A. Virtual screening of the CNRS library (43400 chemicals) on these two pockets, using multiple docking programs, generated docking poses for each compound with an estimated binding free energy (docking score). We developed a novel algorithm based on Self-Organizing Maps (SOM), which has the decisive advantage that multiple docking poses from various algorithms and target structures can be analyzed in a single framework. This allows us to select the most promising molecules out of the initial set (43400). 300 compounds were selected and will be tested experimentally for their effect on biochemical activities of the protein, and on in vitro parasite growth and macrophage infection. Most interesting molecules will be then optimized in silico. * E.S. Harigua is an International Unesco-L’Oréal fellow “For Women In Science program 2012” * The study received financial support from MESRS (LR00SP04 & LR11IPT04), Institut Pasteur Calmette & Yersin Programme, & the Institut Pasteur PTR (ID: 426).
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [367] P 404 - SUSCEPTIBILITY OF LEISHMANIA (VIANNIA) BRAZILIENSIS ISOLATES TO MILTEFOSINE ESPADA, C.R.1; CAPPELLAZZO, A.C.1; PEREIRA, L.I.A.2; RIBEIRO-DIAS, F.2; DORTA, M.L.2; ULIANA, S.R.B.1 1.UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL; 2.UNIVERSIDADE FEDERAL DE GOIÁS, GOIÂNIA, GO, BRAZIL.
Keyword:miltefosine; susceptibility; l. braziliensis
Abstract: Leishmaniasis is a widespread disease caused by protozoan parasites of the Leishmania genus. In Brazil, tegumentary leishmaniasis is mainly caused by Leishmania braziliensis and consists in ulcerative lesions that can develop into mucocutaneous or disseminated forms. Leishmaniasis chemotherapy in Brazil is still largely based on the use of meglumine antimoniate, a high cost drug that requires parenteral administration and results in several adverse effects. Furthermore, treatment success rates have been decreasing in recent years. Miltefosine is an alkylphosphocholine drug with antiprotozoal activity. It is the only oral agent used for the treatment of leishmaniasis and does not require any hospitalization for the treatment. Miltefosine is currently in use for visceral leishmaniasis in Asia and Europe. In India, where miltefosine is largely used for the treatment of visceral leishmaniasis, rates of 94% of cure have been described. However, some studies have shown that there are differences in the susceptibility among different species and strains. In this work, we evaluated the susceptibility to miltefosine in Leishmania braziliensis isolates from different regions of Brazil. Promastigotes were incubated in different concentrations of the drug and the EC50 were determined. Significant differences in the susceptibility among different isolates were detected. Our findings will contribute in the evaluation of miltefosine’s potential to be included in the chemotherapeutic arsenal in Brazil and also will contribute in the understanding of the mechanisms of action and resistance to this drug. Supported by FAPESP, CAPES and CNPq
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [370] P 405 - MOLECULAR CHARACTERIZATION OF LEISHMANIA (LEISHMANIA) AMAZONENSIS STRAINS AND SELECTED MUTANTS RESISTANT TO MILTEFOSINE COELHO, A.C.1; COSTA, C.H.N.2; ULIANA, S.R.B.1
1.UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL; 2.UNIVERSIDADE FEDERAL DO PIAUÍ, TERESINA, PI, BRAZIL.
Keyword:leishmania amazonensis; miltefosine; drug resistance
Abstract: The treatment of leishmaniasis relies on costly parenteral administered drugs with multiple undesirable effects. The main drugs available for the treatment of leishmaniasis in Brazil are pentavalent antimony, amphotericin B and pentamidine. However, clinical failure after treatment has been increasingly reported, especially for antimony. Recently, the effectiveness of miltefosine (MF) in the treatment of visceral leishmaniasis was described and the drug is currently in use mainly for visceral leishmaniasis in Asia and Europe as the first oral treatment of the disease. However, the knowledge about miltefosine’s mechanisms of action and resistance and the cellular targets in the parasite is limited. In this study, we evaluated the sensitivity of a L. (L.) amazonensis field isolated strain that was at least two-fold more resistant than the L. (L.) amazonensis reference strain available in the laboratory. Potential genes
associated with MF resistance in vitro, previously described in other Leishmania spp., were sequenced. We sequenced MT and Ros3 genes that are responsible for the translocation of phospholipids across the plasma membrane and the pyridoxal kinase (PK) gene involved in MF susceptibility. Nucleotide sequence analysis showed a restricted number of single nucleotide polymorphisms (SNPs) between these strains, with some non-synonymous SNPs, but not located in conserved motifs of MT and PK. No SNP was found in the Ros3 gene. We also selected MF resistant mutants using a stepwise selection protocol. MT, Ros3 and PK genes from these mutants are presently being analyzed. Aiming to identify and compare potential genes associated to the mechanism of MF resistance in the field and in vitro, our findings will contribute in the evaluation of MF’s potential to be included in the chemotherapeutic arsenal in Brazil. This study may also contribute in the understanding of the mechanisms of action and resistance of this drug and result in the identification of potential molecular markers related to MF resistance in L. (L.) amazonensis. Supported by FAPESP and CNPq.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [386] P 406 - 3’ BULKY SUBSTITUTIONS OF 6-BR-INDIRUBIN-3 -OXIME (6-BIO) PRIMARILY TARGETING CRK3, SHIFT SPECIFICITY TOWARDS GSK-3 EFSTATHIOU, A.1; SMIRLIS, D.1; MYRIANTHOPOULOS, V.2; GABORIAUD-KOLAR, N.2; VOUGOGIANNOPOULOU, K.2; MIKROS, E.2; SKALTSOUNIS, A.2; SOTERIADOU, K.1 1.HELLENIC PASTEUR INSTITUTE, ATHENS, GREECE; 2.UNIVERSITY OF ATHENS, ATHENS, GREECE.
Keyword:leishmania; gsk3; indirubins
Abstract: Leishmaniasis is considered one of the most serious parasitic infectious diseases. While no human vaccine is available and the current treatment has serious drawbacks, there is an urgent need to discover effective new targeted drugs. Excellent candidates for this purpose are the bisindole indirubins which target mammalian GSK3 and the CDKs but also their parasitic counterparts. Previously, we found that the indirubin analogue, 6-BIO had selectivity towards CRK3 over LdGSK3, a reverse selectivity from the respective mammalian kinases, while 5- e6had selectivity against LdGSK3. In our effort to identify selectivity determinants that could be exploited for designing analogues of enhanced specificity towards LdGSK3, we performed a comparative structural analysis of GSK3 and CRK3. Results showed that variations were located in the glycine-rich loop and in the ribose and phosphate binging sub-sites, thus inducing a differentiation of the hydration pattern of the otherwise highly similar ATP binding pockets, as determined by Szmap program. Given the well-known impact of protein desolvation on binding affinity, it was hypothesized that compounds that bind the kinases through specific interactions at that part could have a ifferential affinity. Therefore, we evaluated a new small 6substituted indirubin library with a bulky substitution at position 3', which was expected to bind adjacent to the aforementioned region of the pocket and overlap with the hydration sites of interest. The inhibitory activity of indirubins against L. donovani promastigotes and intracellular amastigotes was first evaluated. This screen identified 7 new indirubins that displayed potent antileishmanial activity (<1,5 ), and 2 with a promising selectivity index ( 9). Then the inhibitory activity against LdGSK3 and CRK3 of the most potent antileishmanials displaying a good electivity index was further assessed. Interestingly, a 3’ pyrrolidine and a 3’ piperazine analogue displayed enhanced electivity towards GSK3, over CRK3 with the latter analogue displaying more than 33-fold, confirming our initial hypothesis. The specificity of these analogues towards GSK3 was also evaluated by testing cell-cycle progression and parasitic cell-
death. Our results confirmed that these indirubins had a similar pattern observed with the inhibition of GSK3, but not CRK3. In conclusion, indirubins seem to be promising lead drug candidates for rational drug design against leishmaniasis. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [393] P 407 - TREATMENT OF CUTANEOUS LEISHMANIASIS USING A SUBCUTANEOUS IMPLANT OF PLGA MICROPARTICLES LOADED WITH AMPHOTERICIN B LIMA, W.P.1; GONDIM, F.C.1; FALCÃO, C.B.1; CERIZE, N.P.2; RÉ, M.I.3; BERGMANN, B.R.1 1.UFRJ, RIO DE JANEIRO, RJ, BRAZIL; 2.IPT, SÃO PAULO, SP, BRAZIL; 3.CENTRE RAPSODEE, ALBI, FRANCE.
Keyword:leishmaniase; nanotecnology; implant
Abstract: The conventional treatment of cutaneous leishmaniasis (CL) is based on multiple injections with antimonial or amphotericin B (AmB) which produce systemic toxicity. In this study, we aimed at developing a single-dose therapy consisting of a subcutaneous implant of poly-(lactide-coglycolide) PLGA microparticles (mps) loaded with amphotericin B (AmB/PLGA) for sustained drug release system. In vitro, L. amazonensis-GFP-infected macrophages were treated for 72 h with AmB/PLGA, or AmB and PLGA controls, and the parasite load and NO production was measured by fluorimetry and Griess method. In vivo, BALB/c mice were infected in the ear with L. amazonensis-GFP, and on day 26 of they received an intralesional injection with AmB/PLGA (50 µg /500 µg). Controls were liposomal AmB (Ambisome®,50 µg), free AmB (50 µg), empty PLGA (500 µg) and 10 µl of PBS. Lesion sizes were measured throughout infection with a dial calliper and the parasite loads on day 69 by fluorimetry. Chronic toxicity was assessed on day 69 by elevation of AST, ALT and creatinin serum levels. Local skin sensitivity was assessed by the Mouse Ear Swelling Test. Histopathological studies were done at various times up to day 120 after implant to monitor tissue inflammation and polymer degradation. The in vitro results showed that AmB/PLGA had higher anti-amastigote and NO activities than free AmB. In vivo, a single dose of AmB/PLGA implant, but not PLGA alone or liposomal AmB, effectively reduced the lesion growth and the parasite burden. No indicators of heart, liver and kidney toxicity were detectable in the serum. Likewise, the AmB/PLGA implant led to undetectable MEST response. A transient cellular infiltration peaking on day 7 was seen. These findings show that the subcutaneous implant of AmB/PLGA promoted a sustained drug release at the lesion site, with a durable and safe therapeutic effect, supporting its use for a single-dose localized treatment of CL. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [402] P 408 - MECHANISM OF ACTION, EFFICACY IN EXPERIMENTAL VISCERAL LEISHMANIASIS AND SUBACUTE TOXICITY OF LQB-118. CUNHA-JUNIOR, E.F.1; SABINO, K.C.C.; FAIÕES, V.S.1; VASCONCELOS, M.F.1; REMPEL, S.S.1; MARTINS, T.M.2; MARQUES, P.R.2; CANTO-CAVALHEIRO, M.M.1; DA SILVA, A.J.M.3; COSTA, P.R.R.3; ALMEIDA-AMARAL, E.E.1; TORRES-SANTOS, E.C.1 1.FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 2.UERJ, RIO DE JANEIRO, RJ, BRAZIL; 3.UFRJ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:chemotherapy; quinone; oxidative stress
Abstract: Aiming the development of new drugs, we designed and synthesized a new series of compounds via the molecular hybridization of naphthoquinones with pterocarpans. Previously we demonstrated that the pterocarpanquinone LQB-118, the more promising prototype, was effective in experimental cutaneous leishmaniasis via oral delivery, and that the mechanism of
action involves induction of oxidative stress with characteristic events of cell death via apoptosis in Leishmania amazonensis. In continuing this study, we evaluated the effect of LQB-118 on experimental visceral leishmaniasis, the subacute toxicity and the early events involved in triggering death in L. amazonensis. To evaluate the in vivo activity, BALB/c were infected with L. infantum and after one week the treatment was carried out for 23 consecutive days. The same treatment procedure was done in parallel experiments with non-infected Swiss mice to evaluate subacute toxicity. Treatment of the infected mice showed a dose dependent reduction of parasite load in the liver (67, 88 and 100%) and spleen (38, 83 and 99%) respectively at doses of 2.5, 5 and 10mg/kg per oral route. In the subacute toxicity evaluation, higher doses (10 and 50 mg/kg) did not induce variation in weight gain and in biochemical parameters of liver and kidney functions. Unexpectedly, a slight change in leukocytes count was observed, suggesting that additional studies are needed. To assess the primary effects of LQB-118, promastigotes were incubated with LQB-118 and antioxidants. The production of ROS was reversed, however, cell viability and mitochondrial membrane potential were not, suggesting that production of ROS is not the major cause of cell death. Interestingly, when promastigotes or intracellular amastigotes of L. amazonensis were incubated with LQB-118, an intense mitochondrial stress is produced in the early hours, evidenced by the strong reduction of Alamar Blue. Inhibitors of complex I, II or III failed to reverse this effect in promastigotes. Only complex IV inhibitor was able to reduce this phenomenon. Concomitantly to the increase in Alamar Blue reduction, we also observed an increase in ATP production in the early hours. After 48 h, mitochondrial activity fails, ceasing the ATP production. In conclusion, our findings indicate that LQB-118 acts on different clinical forms of leishmaniasis with no sign of toxicity at therapeutic dose and suggest the impairment of parasite mitochondrial activity. The in vivo activity and the understanding of the processes involved in early stages of the parasite death encourages the progression of this compound in the drug development pipeline. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [411] P 409 - EXPLOITING NOVEL NAPHTOQUINONE DERIVATIVES AS DRUG PROTOTYPES AGAINST LEISHMANIA (L.) INFANTUM: AN IN VITRO STUDY PINTO, É.G.1; SANTOS, I.O.2; DA ROCHA, D.R.2; FERREIRA, V.F.2; TEMPONE, A.G.1
1.DEPARTMENT OF PARASITOLOGY, INSTITUTO ADOLFO LUTZ, AV. DR. ARNALDO, 351, 8. ANDAR, 01246-000, SÃO PAULO, SP, BRAZIL; 2.UNIVERSIDADE FEDERAL FLUMINENSE, INSTITUTO DE QUÍMICA, DEPARTAMENTO DE QUÍMICA ORGÂNICA, 24020-150, NITERÓI, RJ, BRAZIL.
Keyword:leishmania; naphthoquinones; drugs
Abstract: Naphthoquinones (NTQ) represent a class of privileged structures with a broad spectrum against parasites, bacteria and fungi. Considering promising NTQ in clinical use as buparvaquone, parvaquone and atovaquone, the exploitation of this class as novel candidate prototypes against Leishmania could be an excellent strategy. In this work a series of nine phenylsulfanylmethyl[1,4]-naphthoquinones were synthesized and its in vitro activity was evaluated by different methods using promastigotes (MTT assay) and intracellular amastigotes of L. (L.) infantum (light microscopy counting). Cytotoxicity studies were performed using NCTC clone 929 cells and the viability was detected by the MTT assay. Among the nine synthetic compounds all compounds showed activity against promastigotes, with IC50 values ranging from 9 to 72 µM. Seven of them showed promising activity against intracellular amastigotes, with IC50 values ranging from 12 to 57 µM. Among the synthetic compounds, two (14 and 15) showed the highest selectivity, as no cytotoxicity could be detected to the highest concentration of 200 µM. The inclusion of chloro or fluor to the thiophenolic ring of phenylsulfanylmethyl-[1,4]naphthoquinones might have conferred the highest activity and lowest mammalian cytotoxicity, suggesting that electron withdrawing substituents could improve the antileishmanial effect. Supported by FAPESP.
- CLINICAL AND EXPERIMENTAL IMMUNOLOGY [419] P 410 - LOCAL INCREASE OF ARGINASE ACTIVITY IN LESIONS OF PATIENTS WITH CUTANEOUS LEISHMANIASIS IN ETHIOPIA KROPF, P.; ABEBE, T.; HAILU, A.; WOLDEYES, M.; MEKONEN, W.; BILCH, K.; CLOKE, T.; FRY, L.; CORWARE, K.; MODOLELL, M.; MUNDER, M.; TACCHINI-COTTIER, F.; MULLER, I. IMPERIAL COLLEGE LONDON, LONDON, UNITED KINGDOM.
Keyword:cutaneous leishmaniasis; arginase; t cells
Abstract: Cutaneous leishmaniasis is a vector-borne disease that is in Ethiopia mainly caused by the parasite Leishmania aethiopica. This neglected tropical disease is common in rural areas and causes serious morbidity. Persistent nonhealing cutaneous leishmaniasis has been associated with poor T cell mediated responses, however the underlying mechanisms are not well understood. We have recently shown in an experimental model of cutaneous leishmaniasis that arginase-induced L-arginine metabolism suppresses antigen-specific T cell responses at the site of pathology, but not in the periphery. To test whether these results translate to human disease, we recruited patients presenting with localized lesions of cutaneous leishmaniasis and assessed the levels of arginase activity in cells isolated from peripheral blood and from skin biopsies. Arginase activity was similar in peripheral blood mononuclear cells (PBMCs) from patients and healthy controls. In sharp contrast, arginase activity was significantly increased in lesion biopsies of patients with localized cutaneous leishmaniasis as compared with controls. Furthermore, we found that the expression levels of CD3 , CD4 and CD8 molecules were considerably lower at the site of pathology as compared to those observed in paired PBMCs. Our results suggest that increased arginase in lesions of patients with cutaneous leishmaniasis might play a role in the pathogenesis of the disease by impairing T cell effector functions.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [425] P 411 - INITIAL SCREENING FOR THE USE OF IGG1 ISOTYPE ON THE DIAGNOSIS BY FLOW CYTOMETRY FOR AMERICAN TEGUMENTARY LEISHMANIASIS IN PERNAMBUCO OLIVEIRA, B.C.; OLIVEIRA, A.P.; CASTRO, M.C.A.B.; ALMEIDA, A.F.; SOUZA, M.A.; ALMEIDA, T.M.; SILVA, A.C.; BRITO, M.E.F.; PEREIRA, V.R.A. CENTRO DE PESQUISAS AGGEU MAGALHÃES, RECIFE, PE, BRAZIL.
Keyword:flow cytometry; leishmania (viannia) braziliensis; igg1
Abstract: In Brazil, the responsible species for the most of the cases of American Tegumentary Leishmaniasis (ATL) is Leishmania (Viannia) braziliensis (L. [V] braziliensis), and in Pernambuco (PE) it predominates circulating until now. Although studies evaluate the hummoral immune response of ATL, the role of specific antibodies on the immunity against Leishmania is not completely clear. It is also known that there is no gold standard for diagnosis on ATL, and it is accomplished gathering clinic, epidemiologic and laboratorial factors. Taking the applicability of flow cytometry into consideration, a new approach was taken in the present study, used in the research on antibodies with better applicability than different conventional protocols of detection and development. Therefore, our objective aims the padronization of IgG1 isotype for use in flow cytometry with applicability on diagnostic and cure criterion of ATL. Dilutions of sera (1:64 to 1:8192) from individuals with confirmed active infection of
ATL (I) and individuals without clinic manifestations (NI) (residents of non-endemic areas of ATL in PE) were incubated with L. (V) braziliensis parasites. Then, they were incubated again with human anti-IgG1, using serial dilutions of 1:100 to 1:6400; and reincubated with streptavidin conjugated with phycoerythrin-PE on 1:400 dilution. To monitor unspecific bonds on the test, it was used an internal control of the reaction and the conjugate control was tested in serial dilutions. The preview of the results was accomplished on the flow cytometer FACScalibur using the software CellQuestPro, the results were expressed on levels of IgG1 reactivity, from the percentage of positive fluorescent parasites (PPFP). Through the statistics program MedCalc, it was possible to confirm that PPFP 20% indicates positive diagnosis for ATL. The results showed high reactivity on all seven dilutions of the tested conjugate, with PPFP 20% on I group for ATL. Considering the lowest level of PPFP to the NI group, where PPFP 20%, the dilution 1:1600 of human anti-IgG1 proved to be the best dilution, in order to guarantee the sensibility of the flow cytometry test as a diagnostic tool for ATL. Thereby, this study opens perspectives to the use of this and another subclasses of IgG, through flow cytometry, to detect patients with ATL on the state of Pernambuco; contributing to achieve a method with greater specificity and sensibility for diagnostic application. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [426] P 412 - OPPOSITE ROLES OF LIPOPHOSPHOGLYCANS AND GLYCOINOSITOLPHOSPHOLIPIDS FROM LEISHMANIA INFANTUM AND L. BRAZILIENSIS DURING THE INTERACTION WITH MACROPHAGES SOARES, R.P. CENTRO DE PESQUISAS RENÉ RACHOU/FIOCRUZ, BELO HORIZONTE, MG, BRAZIL.
Keyword:lipophosphoglycan; glycoinositolphospholipids; leishmania
Abstract: The cell surface of Leishmania is covered by a dense glycocalix composed mainly of lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs). Both molecules are involved in a wide range of functions during the interaction with the vertebrate and invertebrate hosts. It is not known if interspecies variations in the LPG and GIPLs are important during the interaction with macrophages. To investigate the issue of interspecies polymorphisms, two Leishmania species from the New World that express structural variations in the LPG and GIPLs were examined. In this context, the procyclic form of L. braziliensis LPG (strain M2903), is devoid of side chains and exhibits type II GIPLs. On the other hand, L. infantum LPG (strain BH46) has with up to three glucoses residues in the repeat units and exhibit type I GIPLs. Mice peritoneal macrophages from Balb/c, C57BL/6 and knock-out (TLR2 -/- e TLR4 -/-) were primed with IFN-gama and stimulated with purified LPG and GIPLs from both species. Nitric oxide and cytokine production and MAPKs (p38 and ERK) activation were evaluated. Macrophages stimulated with L. braziliensis LPG, had a higher TNF- , IL-1 , IL-6 e NO production than those stimulated with that of L. infantum. Furthermore, the LPGs from the two species resulted in differential kinetics of signaling via MAPK activation. L. infantum LPG exhibited a gradual activation profile, whereas L. braziliensis LPG showed a sharp but transient activation. Further the higher pro-inflammatory acativity of L. braziliensis was confirmed by its ability in activating NF-kB. On the other hand, the GIPLs from both species were able to inhibit macrophage activation even after stimulation with IFN-gamma and LPS. Also, GIPLs from both species failed to activate MAPKs. In conclusion, these data suggest that interspecies variations in Leishmania glycoconjugates were able to differentially modulate host’s innate immune system during initial steps of infection. The LPGs had a more pro-inflammatory role while GIPLs exhibited an inhibitory function in macrophages.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [427] P 413 - A NEW ABC TRANSPORTER IN LEISHMANIA IS INVOLVED IN RESISTANCE TO ANTIMONY MANZANO, J.I.; GARCÍA-HERNANDEZ, R.; CASTANYS, S.; GAMARRO, F. INSTITUTO DE PARASITOLOGÍA Y BIOMEDICINA LOPEZ–NEYRA, IPBLN-CSIC, GRANADA, SPAIN.
Keyword: abc transporter; antimony resistance; efflux thiol-conjugated complexes
Abstract: The family of ABC transporters in Leishmania contains an unclassified group of proteins termed as others that included 4 different transporters with no homology with other eukaryotic ABC proteins. We were interested in the functional characterization of this divergent and unclassified group of transporters specific of trypanosomatids. The group includes 4 different genes that we have named as LABCI1, LABCI2, LABCI3 and LABCI4; all coding for half-transporters with a variable topological disposition of the NBD and the TMD, but having in common the requirement to form homo/heterodimers to be functionally active proteins. We describe here the characterization of LABCI4, an ABC half-transporter bearing the highly conserved NBD at the COOH-terminus and the TMD at the NH2-terminus. We show that LABCI4 is involved in resistance to Pentostam, SbIII and to the metal ions AsIII and CdII, due to a reduction in their cellular accumulation as a consequence of an increased drug efflux and their ability to reduce the mitochondrial toxic effects of antimony by decreasing the ROS production, and maintaining higher values of both the mitochondrial electrochemical potential and total ATP levels versus controls. LABCI4 is a half-transporter that form homodimers as determined by coimmunoprecipitation assay. Subcellular localization studies under a confocal microscopy and a surface biotinylation assay using parasites overexpressing GFP-LABCI4 and FLAG-tagged LABCI4 parasites, suggest that the transporter presents a dual localization in both mitochondria and plasma membrane. Parasites overexpressing LABCI4 present an increased replication in mouse peritoneal macrophages versus control and mutant LABCI4K/M parasites. We have determined that LABCI4 have porphyrins as substrates and consequently the overexpression of LABCI4 confers resistance to some toxics porphyrins like zinc-protoporphyrin due to a lower accumulation by a significant efflux as determined using the fluorescent zinc-mesoporphyrin, a validated heme analog. Additionally, LABCI4 has a significant ability to efflux thiol-conjugated complexes and could be considered as a potential thiol-X-pump. Altogether, we have shown that this new ABC transporter is involved in drug sensitivity to antimonials and its clinical relevance remains to be determined. Supported by the Spanish Grants SAF2012-34267 (to F.G.), SAF2011-28102 (to S.C.) and by FEDER funds from the EU to F.G. and S.C.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [434] P 414 - ANTIPROLIFERATIVE AND ULTRASTRUCTURAL EFFECTS OF BTFDI, A HISTONE DESACETILASES INHIBITOR, IN LEISHMANIA AMAZONENSIS DE ARAUJO, C.N.1; VERÇOZA, B.R.F.1; BRACHER, F.2; DE SOUZA, W.1; RODRIGUES, J.C.F.1 1.UNIVERSIDADE FEDERAL DO RIO DE JANEIRO, RIO DE JANEIRO, RJ, BRAZIL; 2.LUDWIG-MAXIMILIANS-UNIVERSITT, MNCHEN, GERMANY.
Keyword:biochemistry; histone desacetilases inhibitor; btfdi
Abstract:
Leishmaniasis is one of the most important neglected tropical disease distributed around the world, and is caused by protozoan parasite of the Leishmania genus.In Brazil, endemic areas are mainly concentrated in the North and Northeast, however there is a significant increase in other regions more developed. In Brazil, Leishmania amazonensis is one important species responsible for cutaneous and diffuse cutaneous leishmaniasis, which is so difficulty to treat. The treatment is based on the use of pentavalent antimonials, miltefosine, amphotericin B and pentamidine. Therefore, there is a urgent necessity to develop new drugs more efficient and less toxic. Recently, histone deacetylases inhibitors have been studied as potential target for the treatment of cancer. These enzymes act directly on the packaging of the DNA, thus regulating the expression of various genes related to apoptotic cell death. Thus, the aim of our work is study the effects of a novel histone deacetylases inhibitor on Leishmania amazonensis. The present study focuses on the effect of BTFDI in promastigote forms. The IC50 value found was 2 M, with total inhibition of the growth after treatment with 4 M. DIC and Immunofluorescence microscopy with anti- -tubulin antibody showed that the promastigotes became swollen mainly in the nuclear region of the cell body after treatment with concentrations up 1 M. Scanning electron microscopy confirmed this alteration on the cell body. Furthermore, transmission electron microscopy was carried out to identify the organelles and structures affected by the treatment. Several alterations were observed, such as: mitochondrial swelling, presence of many lipid bodies, and changes in the chromatin condensation. These initial results obtained suggest that BTFDI is a potential new compound against Leishmania, which needs to be tested in intracellular amastigotes and in murine model of leishmaniasis, thus demonstrating its real efficacy. New experiments also have to be carry out to understand better this mechanism of action. Financial supported by CNPq, CAPES and FAPERJ.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [435] P 415 - EVALUATION OF THE PERFORMANCE OF AN AVIAN ENZYME CONJUGATE AND COMPARISON WITH A MAMMALIAN ENZYME CONJUGATE IN THE IMMUNODIAGNOSTICS OF CANINE VISCERAL LEISHMANIASIS SANTOS, F.N.1; SILVA, V.L.1; MOLINARO, C.M.2; DE SOUZA, C.S.F.3; FIGUEIREDO, F.B.4; MADEIRA, M.F.5; MOUTA CONFORT, E.5; CHAVES, S.A.M.6 1.SETOR DE IMUNODIAGNÓSTICO DO LABORATÓRIO DE PESQUISAS E SERVIÇOS EM SAÚDE PÚBLICA DCB-ENSP, FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 2.LABORATÓRIO DE TECNOLOGIA DIAGNÓSTICA, BIOMAGUINHOS- FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 3.LABORATÓRIO DE IMUNOMODULAÇÃO E PROTOZOOLOGIA, IOC- FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 4.LABORATÓRIO DE PESQUISA CLÍNICA EM DERMATOZOONOSES EM ANIMAIS DOMÉSTICOS, IPEC- FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 5.LABORATÓRIO DE VIGILÂNCIA EM LEISHMANIOSES, IPEC-FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 6.DEPARTAMENTO DE ENDEMIAS SAMUEL PESSOA,ENSP- FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:canine visceral leishmaniasis; elisa; igy production
Abstract: The identification of the canine reservoir in zoonotic visceral Leishmaniasis through immunodiagnostic is essential in controlling the canine disease and consequently the human disease. Thus, improvements are continuously being sought. Many researches have been made to improve the quality of antigens and diagnostic platforms. However, studies aiming at the improvement of secondary antibodies are scarce, because the use of immunoglobulin produced by mammals is consecrated. But its use involves operational issues such as low performance, laborious and costly purification methods and ethical issues due to invasive collection methods and euthanasia. In this context, the use of immunoglobulin from immunized chicken egg yolks (IgY) is more advantageous than IgG produced by mammals, allowing more sensitive, specific and less expensive tests. The goal of the present study was to assess the performance of an
enzyme conjugate produced in chicken in comparison with a commercial mammalian enzyme conjugate (IgG), using ELISA as diagnostic platform. To this end, chicken were immunized with purified canine IgG and the IgY was isolated with polyethylene glycol, purified through thiophilic adsorption and conjugated with horseradish peroxidase. ELISA accuracy parameters with IgY and IgG conjugates were assessed and compared in serum from dogs infected with L. (L.) infantum chagasi and non-infected, apparently healthy dogs using L. (L.) infantum chagasi raw extract as antigen. MedCalc 11.6.0.0 and SPSS 16.0 were used in the statistical analysis. IgY was conjugated to peroxidase enzyme without any damage to its structure, distinguishing between positive and negative samples in all conjugate dilutions and antigen concentrations tested. The ELISA using IgY showed 100% sensitivity and 100 % specificity. The comparison of the accuracy parameters of ELISA using IgY with those of ELISA using IgG showed identical values. Better repeatability was obtained using IgY, with intraclass correlation coefficient statistically higher when compared to IgG (p<0.0001), besides a larger grouping of positive and negative samples that was also verified when the correlation of duplicates of both observations was analyzed (p<0.0001). Thus, IgY linked to peroxidase proved to be an excellent conjugate that showed better performance than the conjugate produced by mammals. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [437] P 416 - COMPARISON OF REAL-TIME PCR AND AND KDNA PCR-HYBRIDIZATION FOR DIAGNOSIS OF VISCERAL LEISHMANIASIS IN SYMPTOMATIC AND ASYMPTOMATIC DOGS USING FOUR DIFFERENT CLINICAL SAMPLES FERREIRA, A.L.C.1; CARREGAL, V.M.1; LEITE, R.S.1; FERREIRA, S.A.2; MELO, M.N.2; ANDRADE, A.S.R.1 1.CDTN, BELO HORIZONTE, MG, BRAZIL; 2.UFMG - ICB, BELO HORIZONTE, MG, BRAZIL.
Keyword:diagnosis; canine visceral leishmaniasis; pcr
Abstract: Leishmania infantum = chagasi (L.infantum) is responsible for visceral leishmaniasis (VL) in Brazil and the dogs are the main domestic reservoir. Disease control is based on the elimination of infected animals and the use of a sensitive and specific diagnostic tests are necessary. The Brazilian VL control program emphasizes serologic surveys, mainly using the enzyme-linked immunosorbent assay (ELISA) and the immunofluorescence antibody test (IFAT), followed by the elimination of the seropositive dogs. However, these techniques present limitations in terms of sensitivity and specificity. Molecular methods like PCR associated to hybridization with DNA probes and the real-time PCR are alternatives in this scenario, since are very specific and sensitive. This study evaluated real-time PCR and PCR-hybridization for diagnosis of canine visceral leishmaniasis (CVL) among groups of symptomatic (S) and asymptomatic (A) dogs. The sensitivities of the methods were compared between different clinical specimens: conjunctival swab (CS), bone marrow (BM), skin biopsy (SB) and peripheral blood (B). A group of 60 mongrel dogs, all positive in the parasitological and serological tests, were divided equally in S and A groups. Specific primers addressed to the kDNA mini-circles from L. infantum were used for both molecular methods. The real-time PCR was performed using SYBR GREEN and the DNA probes for PCR-hybridization were labeled with 32P. The PCR hybridization showed the following positivities for the S group: CS 96.7% (29/30), BM 83.3% (25/30), SB 63.3% (19/30) and B 46.7% (14/30). For the A group the results were: CS 76.7% (23/30), BM 66.7% (20/30), SB 56.7% (17/30) and B 16.7% (5/30). In the S group the real time PCR allowed 100% of positivity for samples of CS, MB and SB, and of 90% (27/30) for B. In the A group the following results were obtained by real-time PCR: MB 100% positivity, CS 96.7% (29/30), SB 93.3% (28/30) and 83.3% (27/30) for B. The CS samples showed high sensitivity in S and A groups either by real-time PCR or PCR-hybridization. Because the CS is a noninvasive, painless and easily repeated sample it can be considered the most suitable for CVL diagnosis, mainly for routine epidemiological surveys.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [439] P 417 - LABCG2, A NEW ABC TRANSPORTER IMPLICATED IN THE INFECTIVITY AND PATHOGENICITY OF LEISHMANIA LEÓN-GUERRERO, D.; CAMPOS-SALINAS, J.; GONZÁLEZ-REY, E.; DELGADO, M.; CASTANYS, S.; PÉREZ-VICTORIA, J.M.; GAMARRO, F. INSTITUTO DE PARASITOLOGÍA Y BIOMEDICINA LOPEZ–NEYRA, IPBLN-CSIC, GRANADA, SPAIN.
Keyword:abc transporter; phospholipid translocation; infectivity
Abstract: In the present study, we show that LABCG2, a new ATP-binding cassette half-transporter (ABCG subfamily) from Leishmania, is involved in parasite virulence. Down-regulation of LABCG2 function upon expression of an inactive mutant version of this half-transporter (LABCG2K/M) is shown to reduce the translocation of short-chain analogues of phosphatidylserine. This dominant-negative phenotype is specific for the headgroup of the phospholipid, as the movement of phospholipid analogues of phosphatidylcholine, phosphatidylethanolamine or sphingomyelin is not affected. In addition, promastigotes expressing LABCG2K/M expose less endogenous PS in the stationary phase than control parasites. Transient exposure of PS at the outer leaflet of the plasma membrane is known to be one of the mechanisms used by Leishmania to infect macrophages and to silence their immune response. Stationary phase/metacyclic promastigotes expressing LABCG2K/Mare less infective for macrophage and show decreased pathogenesis in a mouse model of cutaneous leishmaniasis. Thus, mice infected with parasites expressing LABCG2K/M did not develop any lesion and showed significantly lower inflammation and parasite burden than mice infected with control parasites. Our results indicate that LABCG2 function is required for the externalization of PS in Leishmania promastigotes, a process that is involved in the virulence of the parasite. However, we cannot exclude the possibility that LABCG2 is implicated in the exposition of other virulence factors that remain to be elucidated. This work was supported by the Spanish SAF2012-34267 (to F.G.), SAF2011-28215 (to J.M.P.V.) and SAF2011-28102 (to S.C.) and by FEDER funds from the EU to F.G., S.C. and J.M.P.V.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [441] P 418 - MECHANISM OF ACTION OF RALOXIFENE AGAINST LEISHMANIA (LEISHMANIA) AMAZONENSIS REIMÃO, J.Q.1; TANIWAKI, N.N.2; ULIANA, S.R.B.1 1.DEPARTAMENTO DE PARASITOLOGIA, INSTITUTO DE CIÊNCIAS BIOMÉDICAS, UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL; 2.UNIDADE DE MICROSCOPIA ELETRÔNICA, INSTITUTO ADOLFO LUTZ, SÃO PAULO, SP, BRAZIL.
Keyword:raloxifene; autophagy; mechanism of action
Abstract: The activity of the selective estrogen receptor modulator raloxifene (RAL) against promastigotes and intracellular amastigotes of Leishmania spp. has been previously demonstrated. In the present study, we report the ultrastructural effects induced by RAL on Leishmania promastigotes. Transmission electron microscopy of treated promastigotes indicated the presence of structures resembling autophagosomes. Promastigotes treated with RAL for 14 hours presented no damage to the plasma membrane while severe destruction of the cytoplasm was observed. Membrane integrity in RAL-treated parasites was confirmed by staining with
propidium iodide. Additionally, RAL-treated promastigotes presented cytoplasmic vacuoles positively labeled with monodansylcadaverine (MDC), a marker of autophagy. The ultrastructural study also indicated severe mitochondria damage, with disruption of its internal membranes, marked swelling and loss of the matrix content, which could correlate with apoptotic-like processes. To evaluated the hypothesis that parasite death occur by a mixed mechanism, involving autophagic and apoptosis-like death, annexin V exposure on RAL-treated parasites is under investigation by flow cytometry. Our data demonstrate for the first time that RAL induces autophagy in Leishmania promastigotes. We suggest that the presence of autophagic vacuoles could be related to degradation of damaged organelles induced by the drug treatment or to an unsuccessful attempt of parasites to cope with the stress caused by RAL action. This work was supported by FAPESP and CNPq. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [443] P 419 - COMPARATIVE ANALYSIS OF THE PARASITE BURDEN IN INFECTIONS BY LEISHMANIA (LEISHMANIA) AMAZONENSIS DETERMINED BY LUCIFERASE DETECTION AND CLASSICAL METHODS REIMÃO, J.Q.; TRINCONI, C.M.; OLIVEIRA, J.C.; YOKOYAMA-YASUNAKA, J.K.U.; MIGUEL, D.C.; KALIL, S.P.; ULIANA, S.R.B. DEPARTAMENTO DE PARASITOLOGIA, INSTITUTO DE CIÊNCIAS BIOMÉDICAS, UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL.
Keyword:luciferase; amphotericin b; leishmania amazonensis
Abstract: Experimental protocols used in the search for new therapeutic options to treat leishmaniasis involve the use of mouse models to validate drug candidates. Evaluation of efficacy depends on the determination of the parasite burden. The standard methods to quantify parasite loads are mainly cell culture-based. These methods are technically demanding and time consuming. Recently, the use of in vivo imaging systems was proposed as a method to overcome these problems, allowing parasite detection in living organisms. We report the use of recombinant lines of Leishmania (L.) amazonensis expressing the luciferase gene (La-LUC) in the BALB/c model. This study was performed to compare luciferase detection in this model with classical parasite quantification methods, such as limiting dilution. Bioluminescence generated by LaLUC was shown to correlate with the number of promastigotes and amastigotes in vitro. The clinical pattern of lesion was also similar when the WT strain was compared to La-LUC. LaLUC promastigotes and intracellular amastigotes were equally sensitive to amphotericin B (AmB) as the wild type (WT) parasites, as shown by the half maximal effective concentration (EC50) for both strains. La-LUC amastigotes recovered from the tail of infected mice 13 weeks post-infection were luminescent confirming the maintenance of the transfected gene even after long periods in vivo. The half maximal effective dose (ED50) of AmB was determined in LaLUC infected mice through quantification of bioluminescence in vivo and ex vivo, by limiting dilution and using clinical parameters. There was agreement in the ED50 determined by all methods. Quantification of bioluminescence in vivo and/or ex vivo was elected as the best tool for determining parasite burden to assess drug efficacy in infected mice. The generation of L. (L.) amazonensis, L. (V.) braziliensis and L. (L.) infantum mutants expressing the reporter LUC gene integrated into the parasite genome is under development. This work was supported by FAPESP, CAPES and CNPq. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [453] P 420 - ANTI-LEISHMANIAL EFFECT AND IMPROVEMENT OF AMPHOTERICIN B LEISHMANICIDAL ACTIVITY OF RESVERATROL.
FERREIRA, C.1; SOARES, D.C.1; CUNHA DO NASCIMENTO, M.T.1; DA SILVA, L.H.P.2; SARAIVA, E.M.1 1.UFRJ, RIO DE JANEIRO, RJ, BRAZIL; 2.UFRRJ, SEROPEDICA, RJ, BRAZIL.
Keyword:resveratrol; amphotericin b; leishmania amazonensis
Abstract: Resveratrol (Rv) is a polyphenol present in red grapes and red wine, with biological activities such as anti-inflammatory, anti-cancer, anti-oxidant and even leishmanicidal. Our aim is to evaluate the effect of Rv, alone and in association with Amphotericin B (AMB) against Leishmania amazonensis. Our results demonstrate that Rv presents leishmanicidal activity for promastigotes of L. amazonensis with IC50 of 27 M, and 42 M for intracellular amastigotes. Isobolographic analysis demonstrated that the association of Rv with AMB (AMB-Rv), showed a synergic effect for promastigotes (13 M Rv + 0.01 M AMB) as well as amastigotes (15 M Rv + 0.0002 M AMB). In the AMB-Rv association, the concentration used of AMB was 11 and 45 times lower than the IC50 for promastigotes and amastigotes, respectively. Analyzing Rv effect on promastigotes we observed, after 48 hs treatment, an irregular number of nuclei and flagella. Rv induced a 11 folds increase in the percentage of cells in the subG0/G1 phase of the cell cycle. Damage to parasite mitochondria was assessed by the XTT text and mitochondrial membrane potential detection kit (JC-1). Thus, Rv decreased 37% the mitochondrial dehydrogenases activity and, the synergic AMB-Rv association, decreased 22% this activity as measured by XTT. The mitochondrial potential was reduced in 83% and 70% after treatment of promastigotes with Rv and AMB-Rv, respectively. Trying to elucidate the mechanisms involved in Rv and AMB-Rv leishmanicidal effects nitric oxide (NO), arginase and cytokines production were evaluated in infected and non-infected macrophages. Rv reduced around 8 times and AMB-Rv reduced 44 times NO production in infected macrophages treated with IFN- . NO production by Rv and AMB-Rv was also observed in non-infected macrophages activated by IFN- . Interestingly, Rv decreases 3 folds the arginase activity in non-infected macrophages stimulated with IL-4. In non-infected macrophages activated by IFN- , Rv and AMB-Rv induced increase of TGF- production 17.5 and 24 folds, respectively, and both decreased 6 folds the TNF- production. Similar effects were observed in infected macrophages. Rv and AMB were not toxic for murine peritoneal macrophages until 500 M and 5 M, respectively. Our results demonstrate the anti-L.amazonensis effect of Rv and its capacity to potentiate AMB leishmanicidal effect, pointing them as possible substances for further in vivo studies of drug association therapy. Supported by FAPERJ, CNPq, CAPES. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [457] P 421 - LEISHMANICIDAL ACTIVITY OF PIPER HISPIDUM (PIPERACEAE) VASCONCELOS, M.F.1; ALMEIDA, T.C.1; SPONTON, A.2; CORTEZ, D.A.G.2; CANTOCAVALHEIRO, M.M.D.1; TORRES-SANTOS, E.C.1 1.LABORATÓRIO DE BIOQUÍMICA DE TRIPANOSOMATÍDEOS, INSTITUTO OSWALDO CRUZ/FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 2.UNIVERSIDADE ESTADUAL DE MARINGÁ, MARINGÁ, PR, BRAZIL.
Keyword:phytotherapy; piperaceae; chemotherapy
Abstract: Leishmaniasis is a group of neglected diseases that affect approximately 12 million people in 98 countries. The drugs used to control these diseases are associated with low efficacy, high toxicity, difficulties of administration, high costs and increasing resistance. The search for new alternatives of treatment is a priority. Piper hispidum known as jaborandi is a shrub with cylindrical stem that is distributed throughout the Americas, represented by five genera and approximately 460 species. It has been previously studied for its antimalarial and antifungal properties. Many other Piper species have been studied for their leishmanicidal properties and
chalcones were shown to be the main active compounds. This study aimed to evaluate the leishmanicidal activity of P. hispidum. The leaves were collected, dried, macerated and subjected to different extraction procedures. Leishmania amazonensis promastigotes were cultured in the presence of several concentrations of the 7 extracts up to 200 g/mL for 72 hours and quantified colorimetrically by MTT assay. Under these conditions, 5 extracts were active, with IC50 less than 50 g/mL. The activity was also evaluated in intracellular amastigotes. The infectivity index was determined by light microscopy and the extracts showed a significant antiamastigote activity, with IC50 less than 20 g/mL. The ethyl acetate: dichloromethane (1:1) extract and the dichloromethane extract, showed IC50 of 5,6 g/mL and 6,4 g/mL, respectively. To evaluate the toxicity, murine macrophages were incubated with the extracts for 72 hours. The effect on the viability of the macrophages was quantified by MTT and the LD50 values were more than 30 g/mL, indicating a selectivity index more than 5-fold. In addition to above results, further procedures will be made to elucidate the structures of the compounds involved. Thus, Piper hispidum, abundant in Brazil, has a therapeutic potential and may become a new phytotherapeutic antileishmanial agent. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [458] P 422 - PREVALENCE OF CANINE LEISHMANIA (L.) INFANTUM CHAGASIINFECTION BY USING PCR ASSAY OF POPLITEAL LYMPH NODE ASPIRATION IN AN ENDEMIC AREA OF VISCERAL LEISHMANIASIS IN AMAZONIAN BRAZIL LIMA, J.V.F.1; CARNEIRO, L.A.1; LIMA, L.R.1; JESUS, R.S.1; SCHWANKE, K.2; ISHIKAWA, E.A.2; GOMES, C.C.3; LAURENT, M.D.3; CORBETT, C.P.3; SILVEIRA, F.T.1; RAMOS, P.K.1 1.INSTITUTO EVANDRO CHAGAS (SVS, MS), ANANINDEUA, PA, BRAZIL; 2.NÚCLEO DE MEDICINA TROPICAL (NMT) UFPA, BELÉM, PA, BRAZIL; 3.LABORATÓRIO DE PATOLOGIA EM DOENÇA INFECCIOSA (LIM-50) - USP, SÃO PAULO, SP, BRAZIL.
Keyword:canine infection; l. (l.) infantum chagasi; pcr
Abstract: Background: Dog has been incriminated as the principal source of Leishmania (L.) infantum chagasi-infection for the vector, Lutzomyia longipalpis, in endemic area of visceral leishmaniasis in Brazil. This was a cross-sectional study to analyze the prevalence of canine L. (L.) i. chagasi-infection in a cohort of 273 mongrel dogs, living in an endemic area in Barcarena municipality, Pará State, Brazil, by using the PCR assay of popliteal lymph node aspiration. Methods: Dogs were clinically classified as symptomatic (48) and asymptomatic (225); they were of both genders (122/44,7% female and 151/55,3% male), and different ages (50/18,3% <1 year old, 214/78,4% >1 and <7 years old and, 9/3,3% >7 years old). The DNA extraction of samples of popliteal lymph node aspiration was based in the phenol-chloroform followed by the classical PCR using the Taq DNA polimerase and the oligos D1 = 5’ – CCA GTT TCC CGC CCC G – 3’ and D2 = 5’ – GGG GGT GTA AAA TAG – 3’. Results: The PCR assay was positive in 47 popliteal lymph node aspiration samples revealing a canine L. (L.) i. chagasi-infection prevalence of 17,2%. The infection-frequency, however, was higher (P<0.05) in symptomatic dogs 29,2% (14/48) than in asymptomatic ones 14,6% (33/225), whereas it was similar between male 14,5% (22/151) and female 20,5% (25/122) animals, as well as among the three age-groups dogs: 18% (9/50) <1 year old, 16,8% (36/214) >1 and <7 years old and, 22,2% (2/9) >7 years old. Conclusion: These results strong suggest that PCR assay of popliteal lymph node aspiration might be useful for confirming the prevalence of canine L. (L.) i. chagasi-infection as well as the symptomatic dogs.
- CLINICAL LEISHMANIASIS [463] P 423 - SENSITIVITY OF LEISHMANIA (VIANNIA) BRAZILIENSIS PROMASTIGOTES ISOLATED FROM DIFFERENT FORMS OF AMERICAN TEGUMENTARY LEISHMANIASIS TO MEGLUMINE ANTIMONIATE (GLUCANTIME) IN VITRO SILVA, S.1; GIUDICE, Â.1; QUEIROZ, A.1; GUIMARÃES, L.H.1; MACHADO, P.R.L.1; SALGADO, K.1; LAGO, E.1; WILSON, M.E.2; CARVALHO, E.M.1; SCHRIEFER, A.1 1.UNIVERSIDADE FEDERAL DA BAHIA, SALVADOR, BA, BRAZIL; 2.UNIVERSITY OF IOWA, IOWA CITY, UNITED STATES.
Keyword:leishmania braziliensis; antimony; refractoriness
Abstract: The first line treatment of American tegumentary leishmaniasis (ATL) is based on antimony derivatives. Refractoriness to antimony is frequent among ATL patients. Leishmania (Viannia) braziliensis is the major cause of ATL in Brazil. L. (V.) braziliensis causes three forms of leishmaniasis in Bahia: localized cutaneous (CL), and the more severe and difficult to treat mucosal (ML) and disseminated leishmaniasis (DL). In the current study we enrolled 92 cases of ATL (67 CL, 7 ML, 18 DL) from the region endemic for L. (V.) braziliensis named Corte de Pedra, in the state of Bahia/Brazil, which rendered positive parasite cultures. We evaluated: (1) association between the L.(V) braziliensis genotypes and patients response to meglumine antimoniate (Glucantime®); (2) geographic distribution of Glucantime refractoriness within the affected region; and (3) resistance of L. (V.) braziliensis to Glucantime® in vitro. The parasites were genotyped by sequencing of a single locus found to be polymorphic (starting in position 3074 of chromosome 24) for that population of L. (V.) braziliensis. For evaluation of geographic distribution of responsive and refractory cases, the geographic coordinates of patients living sites were acquired by GPS and compared by the Cusick and Edwards test. The coordinates of the patients living sites were also plotted onto a satellite photograph of the region for visual inspection, using a geographic information system. We found that refractoriness is wide spread in the region, almost evenly, but could not detect association between parasite genotype and response to treatment. Sensitivity to antimony was evaluated exposing L.(V) braziliensis promastigotes to increasing concentrations of meglumine antimoniate (Glucantime®) in vitro. Parasites from ML and DL patients were significantly more resistant to antimony than those isolated from CL cases. Thus in vitro resistance to antimony paralleled what is commonly observed in the field, where ML and DL patients tend to respond more poorly to Glucantime® than CL individuals. Although we did not find associations between parasite strain and treatment response in these ATL patients, we used a single locus in the genotyping, which certainly limited our discrimination hability. Future studies should retest this association employing more loci polymorphic among the L. (V.) braziliensis of Corte de Pedra. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [467] P 424 - PCR ASSAY FOR THE MOLECULAR DIAGNOSIS OF THE CLINICALIMMUNOLOGICAL SPECTRUM OF HUMAN LEISHMANIA (L.) INFANTUM CHAGASIINFECTION IN AMAZONIAN BRAZIL SILVA, I.D.R.1; CORRÊA, Z.J.1; LIMA, J.V.F.1; CAMPOS, M.B.1; LIMA, L.V.1; JESUS, R.S.1; ISHIKAWA, E.A.2; GOMES, C.C.3; LAURENT, M.D.3; CORBETT, C.P.3; SILVEIRA, F.T.1; RAMOS, P.K.1
1.INSTITUTO EVANDRO CHAGAS (SVS, MS), ANANINDEUA, PA, BRAZIL; 2.NÚCLEO DE MEDICINA TROPICAL (NMT), UNIVERSIDADE FEDERAL DO PARÁ, BELÉM, PARÁ, BRASIL, BELÉM, PA, BRAZIL; 3.UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL.
Keyword:human infection; l. (l.) infantum chagasi; pcr
Abstract: Background: The clinical-immunological spectrum of human L. (L.) i. chagasi-infection has been defined in five profiles with basis of IFAT-IgG and DTH reactions: Asymptomatic infection (AI), Symptomatic infection (SI=AVL), Subclinical resistant infection (SRI), Subclinical oligosymptomatic infection (SOI) and, Indeterminate initial infection (III). The PCR assay was used to investigate the specific molecular diagnosis of these profiles. Methods: There were examined 129 peripheral blood samples from individuals of both genders, aged one year-old and older, from endemic area of American visceral leishmaniasis (AVL) in Bujarú municipality, Pará state, Brazil, with the following profiles: 64 infected individuals (32 AI, 3 SI=AVL prior treated, 11 SRI, 3 SOI and, 15 III) and 65 IFAT(-)/DTH(-) non-infected (control group). DNA extraction of peripheral blood samples was based in the phenolchloroform followed by the classical PCR using the Taq DNA polimerase and the oligos D1 = 5’ – CCA GTT TCC CGC CCC G – 3’ and D2 = 5’ – GGG GGT GTA AAA TAG – 3’. Results: The frequency of PCR reactivity in the infected group (64) was 9,3% (3/32) in AI, 27,3% (3/11) in SRI, 66,6% (2/3) in SOI, 13,3% (2/15) in III and, 33,3% (1/3) in SI (=AVL treated patients) profiles. In the non-infected control group (65), the PCR reactivity was 10,7% (7/65). Conclusion: These results reveal low PCR reactivity in AI and SRI profiles with activated cellular immune response (DTH+) and, in contrast, high reactivity in SOI and SI profiles with depressed cellular immune response (DTH-). The PCR reactivity in the III profile (13,3%) and in the control group (10,7%) seem indicate a very recent infection.
- CLINICAL LEISHMANIASIS [472] P 425 - LONG TERM FOLLOW-UP TO EVALUATE EFFICACY OF MEGLUMINE ANTIMONIATE PLUS ALLOPURINOL VERSUS MILTEFOSINE PLUS ALLOPURINOL IN THE TREATMENT OF LEISHMANIASIS IN LEISHMANIA INFANTUM NATURALLY INFECTED DOGS. MANNA, L.L.M. UNIVERSITY OF NAPLES, NAPLES, ITALY.
Keyword:leishmania infantum; follow up post-therapy; dog
Abstract: Visceral leishmaniasis is a life-threatening disease of medical, social and economic importance in endemic areas. It is an opportunistic infection in immunocompromised patients including HIV-positive subjects. Dogs are the main reservoir of Leishmania infantum. The aim of this study was to evaluate the efficacy of meglumine antimoniate plus allopurinol versus miltefosine plus allopurinol . The study included 18 leishmaniotic dogs divided in two groups : 9 leishmaniotic dogs were treated with a combination of meglumine antimoniate (100 mg/kg/die/30die, SC) plus allopurinol (10 mg/kg/die, PO) ; other 9 leishmaniotic dogs were treated with a combination of miltefosine (2mg/Kg/day/30 die/ PO) plus allopurinol 10 mg/kg/die PO). After the combined therapy, allopurinol was continued at the same dose until the end of the study (6 years).
Efficacy was measured by real-time PCR assay on lymph node aspirates collected at baseline and every three months for 24 months and every 4 months for the next 4 years. At each assessment, the dogs were examined for disease signs and a clinical score was recorded. Our results show that both combined therapy: meglumine antimoniate plus allopurinol or miltefosine plus allopurinol promotes a similar clinical improvement which is accompanied by a reduction of the parasitic load in lymph nodes in both dog groups in the first 3 months of treatment A relapse of clinical condition which is accompanied by an increase of the parasitic load in the lymph nodes was observed in 4/9 dogs treated with miltefosine plus allopurinol. Only 1 dog in the group treated with meglumine antimoniate plus allopurinol. Comparison of efficacy of meglumine antimoniate plus allopurinol versus miltefosine plus allopurinol showed that meglumine antimoniate plus allopurinol combination should be considered as the first-line treatment for leishmaniasis in dogs. - CLINICAL LEISHMANIASIS [476] P 426 - THERAPY AND VISCERAL LEISMANIASIS RESISTANCE IN MONTENEGRO DUPANOVIC, B.; ANDRIC, B.; TERZIC, D. CLINIC FOR INFECTIOUS DISEASES, PODGORICA, BOSNIA AND HERZEGOVINA.
Keyword:leismania donovany; therapy; resistance
Abstract: Features that have parasites or create resistance to the effects of drugs antiparazitarnih given in therapeutic doses is a stumbling block in the treatment of visceral leismaniasis Aim: The aim of this study was to analyze the antimicrobial susceptibility of the strains visceral leismaniasis Methods: Retrospective-prospective study included 77 patients of both sexes, different age cohort (35 children and 44 adults) with clinical, laboratory and microbiological confirmed and described disease caused Leismania donovany (L. D) who were treated at the Clinical Center of Montenegro from 1990 to 2010 year. Results: The average age of the patients was 23.71 Âą 20.6 years. Were males 58.9%, females 41.1% of patients. The most common outcome is cure diseases recorded 59 (76.6%) died, 5 (6.5%) patients, and relapse / resistance to disease was 13 (16.9) patients. In the first line of therapy used with meglumine antimony (Glucantime) which was treated with 70 (90.9%) patients had developed resistance to seven (9.1%) patients during treatment and relapse 5 (6.5%). Miltefosine was treated with 1 patients who had a relapse, and amphotericin B (Ambisome) 6 (7.8%) patients. All patients were treated with amphotericin B as first choice were healed. Lethality had patients: who came in the late stage of the disease 2 (2.6%), association with hematological disease, 2 (2.6%) and 1 (1.3%) patient who was resistant to all three drugs available . Patients
with recurrent / resistance were treated with amphotericin B, of which one (1.3%) became resistant to this drug Conclusion: The most favorable outcome of the disease was in the cases of visceral leismaniasis treated by amphotericin B. Antimicrobial resistance in L. D meglumine antimony, miltefosine and amphotericin B presents the problem of further therapy choice. The available treatment options for visceral leishmaniasis have problems relating to efficacy, adverse effects and cost, making treatment a complex issue. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [477] P 427 - EVALUATION OF THE MECHANISM OF ACTION AND THERAPEUTIC POTENTIAL OF NOVEL TETRAZOLES FAIÕES, V.S.1; DOS SANTOS, M.S.2; BERNARDINO, A.M.R.3; LEON, L.1; DO CANTO CAVALHEIRO, M.M.1; DOS SANTOS, E.C.T.1
1.FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 2.UNIFEI, ITAJUBÁ, MG, BRAZIL; 3.UFF, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:tetrazoles; l. amazonensis; chemotherapy
Abstract: Leishmaniasis is caused by protozoa of the genus Leishmania and constitutes a serious public health problem. The drugs used in the clinic are toxic and often ineffective. Thus, a rational search for new therapeutic alternatives becomes necessary. In this study we investigated the anti-Leishmania amazonensis (MHOM/BR/77/LTB0016) activity of nine tetrazoles synthetic derivatives. Among all derivatives, the compound 5 - [5-amino-1-(4'-methoxyphenyl)-1Hpyrazol-4-yl]-1H-tetrazole (MSN20) showed activity against promastigotes, with IC50 of 74 µM and it was also active on intracellular amastigotes, with IC50 of 22 µM. This compound presented low toxicity on murine peritoneal macrophages (LD50 = 210 µM). Quantification of nitrite in the supernatants of infected macrophages treated with MSN20 revealed no effect on the NO production, while in non-infected macrophages stimulated with LPS (5 g/mL) and IFN(1ng/mL) the compound showed a dose-dependent reduction of the nitrite. The MSN20 was also evaluated in the murine model of cutaneous leishmaniasis and was able to control the development of lesions and reduce the parasite burden significantly when administered orally. As the azole antifungal agents are known inhibitors of the biosynthesis of sterols, the sterol composition of promastigotes treated with MSN20 was analyzed by thin layer chromatography. No changes were observed in the sterol profile, even at concentrations above the IC50. As some tetrazoles with antifungal activity induces oxidative stress, oxidative metabolism was also investigated. The prototype MSN20 did not significantly alter the production of reactive oxygen species (ROS), or the mitochondrial membrane potential ( m) in promastigotes of L. amazonensis. Thus, MSN20 is a new tetrazole prototype, orally active in murine leishmaniasis, with a distinct mechanism of action from other azoles that remains to be elucidated. - CLINICAL LEISHMANIASIS [482] P 428 - CUTANEOUS LEISHMANIASIS CAUSED BY LEISHMANIA (L.) CHAGASI IN A PATIENT USING ADALIMUMAB. AQUINO, T.A.1; RODRIGUES, A.M.S.1; CARDOSO, R.M.1; GRAZIANI, D.2; GODINHO, N.D.V.2; DE LIMA, B.D.2; DA MOTTA, J.O.C.1; RIBEIRO SAMPAIO, R.N.1 1.HOSPITAL UNIVERSITÁRIO DE BRASÍLIA, BRASILIA, DF, BRAZIL; 2.UNIVERSIDADE DE BRASÍLIA, BRASILIA, DF, BRAZIL.
Keyword:leishmaniasis; l. chagasi; adalimumab
Abstract: INTRODUCTION: The use of biological anti-TNF alpha drugs for the treatment of several autoimmune diseases has become increasingly common since its introduction in the 90s. This type of therapy is associated with opportunistic infections, especially those that granuloma formation plays an important role in host defense. In the Americas, Leishmania chagasi is the most commonly involved species in cases of visceral leishmaniasis and has rarely been linked to cutaneous manifestations. We report a case of cutaneous leishmaniasis caused by L. chagasi in a patient using a TNF alpha inhibitor. CASE REPORT: We report the case of a 38-year-old male patient with the diagnosis of ankylosing spondylitis and psoriasis treated with methotrexate and adalimumab. After 4 months of treatment he developed 3 ulcers on his left arm. Leishmanin test and culture in NNN medium were positive for Leishmania. PCR-RFLP of the internal transcribed spacer 1 (ITS1) region digested with the restriction enzyme HaeIII identified L. chagasi. Physical examination did not reveal hepatoesplenomegaly and serology for human immunodeficiency virus was negative. Histopathological examination showed lymphohistiocytic dermal infiltrate with well formed granulomas. The patient was treated with intravenous N-methyl-glucamine (20mgSbV/kg/day) for thirty days and imunossupressors were discontinued. He evolved with complete healing of the lesions. DISCUSSION: Patients using anti-TNF medications are likely to develop more severe and atypical forms of leishmaniasis, since granuloma formation is inhibited. The presence of multiple cutaneous ulcers caused by L. chagasi without visceralization in a patient treated with adalimumab represents an uncommon clinical picture. The paradoxal hitopatological finding of well formed granuloma in an imunossupressed patient is also unexpected. Similar cases in Spain suggest that the formation of granulomas in patients using adalimumab could be related to the induction of Th17 immune response and activation of Toll-like receptor-9 by Leishmania infection. Cases of cutaneous leishmaniasis by L. (L.) chagasi are extremely rare in Brazil and the role of anti-TNF in mucocutaneous disease susceptibility by viscerotropic species of Leishmania is unclear. Given the expanding use of biological anti-TNF drugs in endemic countries such as Brazil, physicians must be attentive to the appearance of skin lesions in patients taking these medications. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [484] P 429 - DIAZEPAM EFFECT ON LEISHMANIA MEXICANA PROMASTIGOTES AND INTRACELLULAR AMASTIGOTES AND TRYPANOSOMA CRUZI EPIMASTIGOTES DAGGER, F.; CASTRO, N.; VALDIVIESO, E. INSTITUTO DE BIOLOGĂ?A EXPERIMENTAL/UNIVERSIDAD CENTRAL DE VENEZUELA, CARACAS, VENEZUELA.
Keyword:diazepam; ultrastructure; antiproliferative effect
Abstract: It has been reported that the benzodiazepine Diazepam, a mild sedative, causes actin diminution and disorganization in chicken embryo fibroblasts and in other systems, the reduction of myosin content per cell and its synthesis. In the unicellular algae Dunaliella, the drug blocks the basal body division and separation during prophase. In our studies with trypanosomatid parasites, it was found, using an MTT assay, that Diazepam inhibits Leishmania mexicana promastigotes proliferation in a dose dependent manner, with an IC50 of 27,98 Âľg/ml inducing strong cell morphology changes in 24 hours. Diazepam blocks basal body replication and separation
inducing cells with one kinetoplast and one nucleus. Ultrastructural studies evidence a possible action of Diazepam on cytoskeletal elements situated in the zone between the basal body and the mitochondrial membrane limiting the kinetoplast. The presence in this zone of an actin like protein in the alga Dunaliella and intermediate like filament in Leishmania has been reported. The mitochondrial membrane potential is not affected by the drug, as shown by rhodamine 123 labeling, in spite of ultrastructural alterations observed in its morphology. Diazepam diminishes the percentage of macrophage infection in 35% at 45 µg/ml and 15% of the macrophage infectivity at 90 µg/ml. The drug also inhibits completely cell differentiation from the amastigote to the promastigote form. Diazepam induces small morphological changes on the T. cruzi strain Y epimastigotes and the IC50 determined was 44.05 µg/ml, showing less sensitivity to the drug than Leishmania promastigotes. Surprisingly, Vero cells generally used for experimental Trypanosoma infection studies showed an IC50 of 55µg/ml, very close to the one obtained for T. cruzi epimastigotes. The results obtained with Leishmania parasites encouraged us to continue experimental studies with Diazepam in the murine system. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [486] P 430 - LUCIFERASE-EXPRESSING LEISHMANIA INFANTUM ALLOWS THE MONITORING OF AMASTIGOTE POPULATION SIZE, IN VIVO, EX VIVO AND IN VITRO MICHEL, G.1; FERRUA, B.1; LANG, T.1; MADDUGODA, M.P.2; MUNRO, P.3; POMARES, C.1; LEMICHEZ, E.3; MARTY, P.1 1.UNIVERSITY, NICE SOPHIA-ANTIPOLIS, FRANCE; 2.INSTITUT PASTEUR, PARIS, FRANCE; 3.INSERM U1065, NICE SOPHIA-ANTIPOLIS, FRANCE.
Keyword:leishmania infantum; luciferase; visceral leishmaniasis treatment, miltefosine
Abstract: Here we engineered transgenic Leishmania infantum that express luciferase, the objectives being to more easily monitor in real time their establishment either in BALB/c mice—the liver and spleen being mainly studied—or in vitro. Whatever stationary phase L. infantum promastigotes population—wild type or engineered to express luciferase—the parasite burden was similar in the liver and the spleen at day 30 post the intravenous inoculation of BALB/c mice. Imaging of L. infantum hosting BALB/C mice provided sensitivity in the range of 20,000 to 40,000 amastigotes/mg tissue, two tissues—liver and spleen—being monitored. Once sampled and processed ex vivo for their luciferin-dependent bioluminescence the threshold sensitivity was shown to range from 1,000 to 6,000 amastigotes/mg tissue. This model further proved to be valuable for in vivo measurement of the efficiency of drugs such as miltefosine and may, therefore, additionally be used to evaluate vaccine-induced protection. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [488] P 431 - EFFECTS IN VITRO OF PHOTODYNAMIC THERAPY ON PROMASTIGOTES OF LEISHMANIA AMAZONENSIS AURELIANO, D.P.1; SOARES, S.C.2; LAUTELA, J.A.2; RIBEIRO, M.S.3 1.CENTRO DE LASERS E APLICAÇÕES/INSTITUTO DE PESQUISAS ENERGÉTICAS E NUCLEARES/USP, SÃO PAULO, SP, BRAZIL; 2.LABORATÓRIO DE SOROEPIDEMIOLOGIA (LIM38 – HCFMUSP)/INSTITUTO DE MEDICINA TROPICAL DE SÃO PAULO/USP, SÃO PAULO, SP, BRAZIL; 3.CENTRO DE LASER E APLICAÇÕES/INSTITUTO DE PESQUISAS ENERGÉTICAS E NUCLEARES/USP, SÃO PAULO, SP, BRAZIL.
Keyword:pdt; leishmania amazonensis; parasite
Abstract: Leishmania spp. is an important parasite that causes several neglected diseases such as cutaneous leishmaniasis. There are only few effective treatment options. Among them, the most effective treatment is the chemotherapy that involves systemic treatments with antiparasitic drugs that cause a variety of side effects including an overall toxic effect on the organism. It is also possible an infection recurrence and a resistance to the treatment. New treatment approaches have been developing during the last two decades and the Photodynamic Therapy (PDT) reveals promissory results to treat cancer, fungal and bacterial infections and parasite diseases using different photosensitizers. PDT does not show side effects and also resistance treatment. The aim of this study is to evaluate the action of PDT in the cell viability of Leishmania amazonensis promastigotes. For that infective-stage L. amazonensis metacyclic promastigotes were isolated from stationary cultures and in the 96 well plates was incubated with 107 parasites with differences concentrations (50 µM, 100 µM, 250 µM, 500 µM) of methylene blue (MB) photosensitizer. The samples were incubated during 10 minutes with the MB. After that the samples were irradiated using two conditions of LED irradiation with power of 100 mW and 260 mW during one minute. To evaluate the cell viability the colorimetric assay MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was performed. The Anova One Way test was performed to analysis the statistical significance (P <0.05). We observed statistical differences among the values of cell viability using different concentrations of MB photosensitizer. The 250 uM MB concentration is the smallest promastigotes cell viability in both power LED tested. No statistically significant difference was found between groups using different irradiation condition at the 250 uM MB concentration. We conclude that PDT is able to reduce cell viability of L. amazonensis promastigostes around 40% in the parameters analyzed. However these are preliminary studies and need further analysis to explain better this promising effect of PDT on parasites. Financial Support: CNPq, FAPESP, LIM-38 (HC-FMUSP)
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [489] P 432 - EFFECTS OF THE COMBINATION THERAPY BETWEEN ALQUILPHOSPHOLIPIDS AND DINITROANILINES AGAINST LEISHMANIA AMAZONENSIS DA CRUZ, N.C.A.1; GODINHO, J.L.P.2; TAKIYA, C.M.2; DE SOUZA, W.3; RODRIGUES, J.C.F.4 1.POLO AVANÇADO DE XERÉM, UNIVERSIDADE FEDERAL DO RIO DE JANEIRO, RIO DE JANEIRO, RJ, BRAZIL; 2.INSTITUTO DE BIOFÍSICA CARLOS CHAGAS FILHO, UNIVERSIDADE FEDERAL DO RIO DE JANEIRO, BRASIL., RIO DE JANEIRO, RJ, BRAZIL; 3.UFRJ,IBCCF,IMETRO,INBEB, RIO DE JANEIRO, RJ, BRAZIL; 4.POLO AVANÇADO DE XERÉM,UFRJ,IBCCF,IMETRO,INBEB, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:trifluralin; miltefosine; glucantime
Abstract: Protozoan parasites of the Leishmania genus are able to cause leishmaniasis around the world. In Brazil, Leishmania amazonensis is responsible for cutaneous and diffuse cutaneous leishmaniasis. The current chemotherapy is based on pentavalent antimonials, amphotericin B, miltefosine and pentamidine. Nowadays, miltefosine has been implemented as oral treatment for visceral and cutaneous leishmaniasis in several countries. Trifluralin, is a dinitroaniline that displays important effects in some protozoan parasites, and its main action is specific against parasite tubulin. In this work, we evaluated the effect of miltefosine and trifluralin alone or in combination against L.
amazonensis in vitro and in vivo. For promastigotes in vitro, the IC50 values found were around 25 µM and higher than 20 µM for miltefosine and trifluralin, respectively. Both compounds, when tested alone against intracellular amastigote, showed IC50 values higher than 50 µM. Microscopy techniques showed alterations on the shape of promastigotes and the presence of lipid bodies. The first combination assay was done in Balb/C-infected mice with L. amazonensis. After the development of the lesions at the basis of the tail, the mice were divided in different groups: Glucantime group (50 mg/kg/ day), Trifluralin groups (30, 50 mg/kg/day), Miltefosine-treated groups (20; 30; 40; 50 mg/kg/day), and combination of Miltefosine/Trifluralin [(20 x 30 mg/kg/day); (20 x 50 mg/kg/day). Miltefosine and trifluralin were administrated by oral gavage, while Glucantime by intraperitoneal route, during 21 days of treatment. The efficacy was evaluated by measuring the size of the lesions, presence of parasite in lesions stained with Giemsa and histopathological analysis. The results obtained with miltefosine showed a significant dose-dependent decrease in the lesion size, which was not observed after treatment with trifluraline alone. Combination of trifluralin with miltefosine resulted in an effect similar to those obtained for miltefosine alone. Histophatological analysis showed an important immune response in the groups treated with miltefosine alone or in combination. Reduction of the number of foam macrophages and resident inflammatory cells was observed. Thus, this study suggests that miltefosine alone or in combination with trifluralin could be an alternative treatment for cutaneous leishmaniasis caused by L. amazonensis. Financial supported by CNPq, CAPES, and FAPERJ. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [494] P 433 - THERAPEUTIC POTENTIAL OF BIOCELLULOSE MEMBRANES CONTAINING SODIUM DIETHYLDITHILCARBAMATE IN CUTANEOUS LEISHMANIASIS CAUSED BY LEISHMANIA BRAZILIENSIS CELES, F.S.1; BARUD, H.S.2; TROVATTI, E.2; SANTOS, D.M.3; RIBEIRO, S.J.L.2; BORGES, V.M.1; DE OLIVEIRA, C.I.1 1.CENTRO DE PESQUISAS GONÇALO MONIZ, FIOCRUZ, SALVADOR, BA, BRAZIL; 2.INSTITUTO DE QUÍMICA DE ARARAQUARA, UNIVERSIDADE ESTADUAL PAULISTA, ARARAQUARA, SP, BRAZIL; 3.UNIVERSIDADE FEDERAL DE SERGIPE, LAGARTO, SE, BRAZIL.
Keyword:bacterial cellulose; diethyldithiocarbamate; cutaneous leishmaniasis
Abstract: Chemotherapy remains the primary tool for treatment and control of human leishmaniasis. Since currently available drugs present serious problems regarding side-effects, variable efficacy, and cost, affordable and less toxic drugs are urgently needed. Among these is sodium diethyldithiocarbamate (DETC), a super-oxide dismutase inhibitor that induces Leishmania killing in human macrophages in vitro and in vivo, employing a murine model of infection. Herein, we tested the efficacy of biocellulose or bacterial cellulose (BC) membrane containing DETC as a topical treatment for cutaneous leishmaniasis (CL) caused by Leishmania braziliensis. BC is a nontoxic and biocompatible polymer conventionally used as temporary skin substitute or support for dermal drug release. Never dried BC membranes were impregnated with 3.5, 35 and 350ug/cm2 of DETC and DETC loaded BC membranes were characterized by scanning electron microscopy (SEM) and Fourier Transform Infrared (FTIR) spectroscopy. SEM micrograph showed BC nanofibers covered by DETC. FTIR spectra present both DETC and BC typical bands, confirming the incorporation of the drug into the BC membranes. In vitro experiments performed with bone-marrow derived macrophages (BMDM) showed that BCDETC membranes (3.5 and 35ug/cm2) do no exert toxic effects as evaluated by Trypan Blue exclusion. Incubation of BC-DETC membranes with BMDM infected with L. braziliensis led to a significant decrease in the number of infected cells as well as in the number of amastigotes, after 24 and 48 hours. This result was associated with inhibition of super-oxide dismutase activity paralleled by an increase in superoxide production. Preliminary in vivo experiments
showed that application of BC-DETC membranes at the lesion site also led to a significant decrease in the parasite load detected at the draining lymph nodes of BALB/c mice infected with L. braziliensis. These results suggest the use of BC-DETC membranes as a new therapeutic option for topical treatment of CL caused by L. braziliensis. Financial Support: CNPq and FIOCRUZ
- CELLULAR AND MOLECULAR BIOLOGY [497] P 434 - MOLECULAR DIAGNOSTIC FOR DETECTION OF LEISHMANIA INFANTUM: A COMPARATIVE STUDY OF THREE DIFFERENT METHODS IN SKIN AND SPLEEN FROM NATURALLY INFECTED DOGS. REIS, L.E.S.1; BOUILLET, L.E.M.2; ROATT, B.M.3; KER, H.G.4; RESENDE, D.M.4; CARNEIRO, C.M.4; GIUNCHETTI, R.C.5; MARQUES, M.J.6; CARNEIRO, M.5; COURA-VITAL, W.3; REIS, A.B.3 1.UNIVERSIDADE FEDERAL DE OURO PRETO ; INCT-DT, CNPQ BRASIL, OURO PRETO, MG, BRAZIL; 2.UNIVERSIDADE FEDERAL DE OURO PRETO, OURO PRETO, MG, BRAZIL; 3.UNIVERSIDADE FEDERAL DE OURO PRETO ; INCT-DT, CNPQ, BRASIL, OURO PRETO - MG - BRASIL, OURO PRETO, MG, BRAZIL; 4.UNIVERSIDADE FEDERAL DE OURO PRETO, OURO PRETO - MG - BRASIL, OURO PRETO, MG, BRAZIL; 5.UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE MG - BRASIL, BELO HORIZONTE, MG, BRAZIL; 6.UNIVERSIDADE FEDERAL DE ALFENAS, ALFENAS - MG - BRASIL, ALFENAS, MG, BRAZIL.
Keyword:molecular diagnostic; leishmania; canine visceral leishmaniasis
Abstract: The detection of Leishmania DNA based on polymerase chain reaction (PCR) and its variations represent alternatives for CVL diagnosis with highly sensitive, specific and versatile methods. These molecular techniques enable several types of canine clinical samples as spleen, lymph node, urine, conjunctival swabs and skin biopsies to be used for CVL diagnosis. In this context, there is no consensus about the best method and biological samples to be used in the molecular diagnostic of leishmaniasis in large-scale in an endemic area. The aim of this work was to compare three different techniques of molecular diagnosis [conventional PCR (cPCR), seminested PCR (snPCR) and quantitative PCR (qPCR)] in samples of skin and spleen of 37 seropositive dogs (IFAT and ELISA) and with positive parasitologic (detection of amastigote forms and/or bone marrow culture). The molecular analyzes were performed using primers directed to the conserved region — L150/L152, LINR4/LIN17/LIN19 and DNA polymerase gene (DNA pol ) from L. infantum, used in the cPCR, snPCR and qPCR, respectively. Sensitivity, positive predictive values (PPV), negative predictive values (NPV) and agreement for each test were estimated using parasitological methods as the gold standard. In skin samples, the sensitivity of cPCR was 89.2% (CI 75.3–95.7), snPCR was 86.5% (CI 72.0–94.1) and qPCR was 97.3% (CI 86.2–99.5). PPV for cPCR was 36.0%, snPCR was 35.3% and qPCR was 38.1%. NPV for cPCR was 93.6%, snPCR was 92.1% and qPCR was 98.3%. The agreement between the molecular tests in skin and parasitological methods of cPCR was 89.2%, snPCR was 86.5% and qPCR was 97.3%. In spleen samples, the sensitivity of cPCR was 81.1% (CI 65.8-90.5), snPCR was 94.6% (CI 82.3- 98.5) and qPCR was 100.0% (CI 90.6-100.0). PPV for cPCR was 33.9%, snPCR was 37.4% and qPCR was 38.7%. NPV for cPCR was 89.3%, snPCR was 96.7% and qPCR was 100.0%. The agreement between the molecular tests in spleen and parasitological methods of cPCR was 81.1%, snPCR was 94.6% and qPCR was 100.0%. In the present study we demonstrated that qPCR method was the best technique in skin and spleen showing the highest sensitivity, PPV and NPV in comparison to other molecular techniques. In skin samples, PCR demonstrated to be more advantageous compared with the snPCR, and the opposite was observed in samples of spleen. However, the snPCR has the disadvantage of using two successive amplifications, increasing the time and risk of contamination. Supported by: CNPq, CAPES, FAPEMIG and UFOP.
- CLINICAL LEISHMANIASIS [507] P 435 - THE USE OF LIPOSOMAL AMPHOTERICIN B IN THE TREATMENT OF PACIENTS WITH MUCOSAL LEISHMANIASIS. ANALYSIS OF A CASE SERIES SANTOS, C.R.O.1; AMATO, V.S.2; DE CAMARGO, R.A.1; TUON, F.F.3; NICODEMO, A.C.1 1.DEPARTMENT OF PARASITIC AND INFECTIOUS DISEASES, HOSPITAL DAS CLÍNICAS DA UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL; 2.DIVISION OF PARASITIC AND INFECTIOUS DISEASES, HOSPITAL DAS CLÍNICAS DA UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL; 3.DIVISION OF INFECTIOUS DISEASES, HOSPITAL DE CLINICAS DA UNIVERSIDADE FEDERAL DO PARANÁ, PARANÁ, PR, BRAZIL.
Keyword:mucosal leishmaniasis; liposomal amphotericin; treatment
Abstract: BACKGROUND: Leishmaniasis is an anthropozoonosis that constitutes a serious public health problem in Brazil and remains a neglected disease. Studies assessing the efficacy and safety of using liposomal amphotericin B (LAFB) in the treatment of patients with mucosal leishmaniasis (ML) are very limited in the literature and involve a very small number of patients. An optimal dose regimen has not yet been defined. OBJECTIVE: To report the experience of our service using LAFB in treating patients with ML. METHODS: We performed a retrospective and descriptive analysis from records of patients diagnosed with LM enrolled in the leishmaniasis clinic of the Clinical Hospital of the University of São Paulo and that were treated with LAFB in the period from January 2000 to April 2012. RESULTS: There were 16 patients treated with LAFB. The mean length of hospital stay was 26 days with a median of 21 days and the mean LAFB usage time was 13.7 days with a median of 12 days. The mean daily dose of LAFB was 2.5 mg / kg / day. Infusion-related adverse effects occurred in 73.34% (11/15) of patients and the most common were phlebitis, back pain and tremor or chills. Systemic adverse effects occurred in 66.67% (10/15) of the cases. Electrolyte disturbances occurred in 46.67%, with hypokalemia representing 20% (3/15) and hypomagnesemia 40% (6/15) of the cases. Acute renal failure occurred in 33.34% (5/15) of the patients, where 80% (4/5) had baseline clearance of less than or equal to 60ml/min. Complete healing of the lesion occurred in 87.5% (14/16) with two treatment failures. The recurrence rate among those who completed the treatment, was 14.3% (2/14). The mean cumulative doses among those who achieved complete healing as an outcome, that is excluding the two therapeutic failures, were 2265.7 mg and 33.2 mg / kg, with a median of 2245 mg and 30.3 mg / kg. CONCLUSIONS: This study describes the largest number of cases so far reported on the use of LAFB in ML patients. According to our study, a LAFB cumulative dose of at least 30 to 35 mg / kg could achieve 100% effectiveness. This dose range is smaller than that suggested by the World Health Organization, what could reduce the cost of treatment. However, more studies are needed to improve our knowledge about the treatment of LM with LAFB. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [508] P 436 - PREVALENCE OF INFECTION BY LEISHMANIA CHAGASI USING ELISA (RK39 AND CRUDE) AND THE MONTENEGRO SKIN TEST IN THE ISLAND OF SÃO LUÍS - MA NASCIMENTO, M.D.D.S.B.1; BEZERRA, G.F.B.1; NETO DA SILVA, M.A.C.1; DE SOUSA, M.D.G.2; RIBEIRO, Y.J.S.1; COSTA, E.M.1; CASTELO BRANCO, R.C.1; MUNIZ, C.R.1 1.UNIVERSIDADE FEDERAL DO MARANHÃO, SÃO LUÍS, MA, BRAZIL; 2.UNIVERSIDADE ESTADUAL DO MARANHÃO, CAXIAS, MA, BRAZIL.
Keyword:visceral leishmaniasis; infection; prevalence
Abstract: Introduction: A population of less than fifteen years was studied from January 1994 to January 1995 in a town called San José Village, Paço do Lumiar, in the island of São Luís, Maranhão, suburban low-income and endemic for visceral leishmaniasis (VL), in order to know the prevalence of infection of Leishmania (L.) chagasi. Material and Methods: We studied the prevalence of infection through the skin tests of leishmanina (MST) and enzyme-linked immunosorbent assay (ELISA rK39 and CRUDE), and the presence of risk factors in the population. The enzyme immunoassay was done using antigens "crude" and rK-39. Results: 1320 children comprised predominantly male (52.9%) and the age group 5-9 years (33.4%). The prevalence of infection was 63.2% by MST. The prevalence by serology by ELISA-rk39 and ELISA-CRUDE was respectively 25.0% and 26.7%. In univariate analysis were found to sociodemographic characteristics of population and housing-related infection by L. (L.) chagasi, and the factors associated with progression to symptomatic forms of LV. Variables such as age (1-9 years), duration of stay in the locality (1-3 years); inadequate housing, the shift in location to collect water in community well, were the risk factors most strongly associated with infection. In the multivariate analysis was the option of three hierarchical models. The variables age (p=0.003) and seropositivity for antigen rK-39 (p=0.000003) were associated with clinical progression of this infection. Seropositivity for antigen "crude" is marginally associated with progression of infection (p=0.0628). The positivity in intradermal is a protective factor (p = 0.0146). Seropositivity antigen rK-39 (p=0.00001) and the absence of reaction to leishmanina (p=0.0301) were independently associated with progression to symptomatic forms of the disease. Conclusion: The high rate of infection with L. chagasi observed in the study location suggest the presence of asymptomatic children under 15 years of age. Control measures should be implemented in the study area, seeking not only the vector control, but also implementing public health measures aimed at improving overall living conditions of this population. - OTHER SPECIAL TOPICS [510] P 437 - THE IMPACT OF MALNUTRITION ON ARGINASE METABOLISM IN EXPERIMENTAL VISCERAL LEISHMANIASIS. CORWARE, K.D.; YARDLEY, V.; MULLER, I.; KROPF, P. IMPERIAL COLLEGE LONDON, LONDON, UNITED KINGDOM.
Keyword:visceral leishmaniasis ; malnutrition; arginase
Abstract: Malnutrition is a major health problem in resource-poor countries and is characterised by inadequate dietary intake of energy and micronutrients. Malnutrition has been clearly identified as a causal factor in increasing the severity of diseases. Leishmaniasis is a parasitic disease caused by intracellular flagellate protozoa that infects mammalian hosts via the bite of infected female sand flies. There are over 20 species of Leishmania spp. that are pathogenic to humans. Manifestation of disease is dependent on factors such as the strain that infects and the immune response of the host. The disease Leishmaniaisis can be broadly categorised into cutaneous and visceral leishmaniasis; the latter is a systemic disease that is fatal if left untreated. Visceral leishmaniasis presents with fever, weight loss and characteristically with hepatosplenomegaly due to the preferential targeting of the strains L. donovani or L. Infantum to the spleen and liver. The mechanisms by which malnutrition increases disease susceptibility are unclear and therefore our aim was to investigate the metabolic pathways that are affected by malnutrition and impact on parasite survival.
To test the impact of malnutrition on the development of visceral leishmaniasis we used L. infantum infected mice fed on a protein deficient diet. Our results show that protein-energy malnourished mice have increased parasite load and increased arginase activity. This effect is reversed by re-feeding with a normal protein diet.
- CELLULAR AND MOLECULAR BIOLOGY [511] P 438 - QUANTITATIVE ASSESSMENT OF PARASITE LOAD IN CUTANEOUS AND MUCOSAL LEISHMANIASIS USING DIFFERENT SAMPLING METHODS PARRADO, R.1; JARA, M.2; VALENCIA, B.2; ALBA, M.2; TORRICO, M.C.1; ANGULO, C.1; AREVALO, J.2; LLANOS-CUENTAS, A.2; VAN DER AUWERA, G.3; DUJARDIN, J.3; GARCIA, A.L.1; ADAUI, V.2 1.IIBISMED, FACULTAD DE MEDICINA, UNIVERSIDAD MAYOR DE SAN SIMON, COCHABAMBA, BOLIVIA; 2.INSTITUTO DE MEDICINA TROPICAL ALEXANDER VON HUMBOLDT, UNIVERSIDAD PERUANA CAYETANO HEREDIA, LIMA, PERU; 3.INSTITUTE OF TROPICAL MEDICINE, ANTWERP, BELGIUM.
Keyword:american tegumentary leishmaniasis; parasite load; quantitative pcr
Abstract: Cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) are the main clinical outcomes of infection with Leishmania (Viannia) species in the New World. The scarcity of parasites within CL and ML lesions, evidenced in histopathological studies, implies the need of a sensitive and accurate molecular tool for assessment of the parasite load in clinical samples. Little data exist on the clinical utility of quantitative PCR (qPCR) for parasite load assessment in human tegumentary leishmaniasis. Our preliminary findings in Peru, using qPCR on biopsy samples, indicated lower parasite abundance in ML lesions than in CL ones. We applied a standardised qPCR assay targeting the kinetoplast minicircle DNA (kDNA) specific to the Leishmania (Viannia) complex to evaluate the parasite load in tissue lesions from Peruvian and Bolivian patients with confirmed CL or ML. Lesions were sampled at enrolment for two specimens each: biopsy and scraping in Peru, or aspirate and scraping in Bolivia. Our sample in Peru consisted of 30 patients with a total of 30 lesions (14 CL and 16 ML). Median parasite load in biopsy samples was 10,425 parasites per µg of tissue DNA (par/µg tDNA) in CL lesions and 36.5 in ML (p=0.0001). Among scrapings, the median parasite load in CL lesions was 21,530 par/µg tDNA compared to 15.6 in ML (p<0.0001). Parasite load measurements on both biopsies and scrapings were highly correlated (Spearman’s rho=0.89, p<0.0001). In Bolivia, we enrolled 21 patients with a total of 21 lesions (9 CL and 12 ML). Among aspirate samples, the median parasite load was 950 par/µg tDNA in CL lesions and 896.8 in ML lesions (p=0.5). Among scrapings, the median parasite load was 1,683 par/µg tDNA in CL lesions and 3,488 in ML lesions (p=0.8). Parasite load measurements on both aspirates and scrapings showed a good correlation (Spearman’s rho=0.56, p=0.008). We have demonstrated that the parasite load can be quantified by qPCR with high sensitivity and accuracy on both invasive clinical specimens (biopsies) and less invasive clinical specimens (dermal scrapings, aspirates). Interim analysis indicates that the parasite load differs according to CL and ML in Peru. In remarkable contrast, there was no significant difference between these patient groups in Bolivia as for the parasite load quantified. The results will be discussed in relation to demographic, clinical and parasitologic parameters. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [514] P 439 - THE MILTEFOSINE COMBINED WITH PENTOXIFYLLINE SEEMS EFFECTIVE IN THE TREATMENT OF AMERICAN CUTANEOUS LEISHMANIASIS IN MICE C57BL6
GREGGIANIN, G.F.; AMORIM, A.A.S.; RIBEIRO, J.B.P.; GARCIA, R.D.; RIBEIRO SAMPAIO, R.N. UNIVERSIDADE DE BRASILIA, BRASILIA, DF, BRAZIL.
Keyword: american cutaneous leishmaniasis; miltefosine; pentoxifylline
Abstract: American Cutaneous Leishmaniasis (ACL) is an infectious and non-contagious disease caused by different species of protozoa of genus Leishmania. Therapeutic arsenal against ACL is still very restricted, and the pentavalent antimonials remain the drugs of choice for more than fifty years. These drugs have high toxicity and recurrence rates, which shows the need to find new therapeutic alternatives. Pentoxifylline (PXF), a xanthine derivative, has been investigated in the treatment of ACL by its protector effect against that high toxicity. This study aims to compare the treatment of ACL with miltefosine and its association with PXF in an animal model. Thirty C57BL6 isogenic mice and parasites of Leishmania (L.) amazonensis sub-species were used. The animals were inoculated in the right footpad with 3 x 106 metacyclic promastigote parasites, and after fourteen days were divided into three groups, and then treated for ten days: (M) oral treatment with miltefosine (200mg/kg/day); (M+P) oral treatment with miltefosine (200mg/kg/day) with PXF (8mg/kg/day); and (C) no treatment. Then it was performed a lymph aspiration of infected paws for culturing and smears, as well as measurement of diameter of the paws. After, the paws were dissected and triturated to culture in plates of 96 pools in successive dilutions, and then submitted to colorimetric assay with MTT. Obtained cultures after twelve days of treatment beginning showed positivity in 100% of animals of (C) control group (5 of 5), demonstrating infectivity of parasites and success of infection. Animals of (M) group showed positivity of 60% (3 of 5), and animals of (M+P) group showed positivity in 20% (1 of 5) of performed cultures, demonstrating a higher cure rate for the (M+P) group. Analysis of measurement of paws indicated average reduction of diameter in 14.05% for (M) group, 17.38% for (M+P) group, and in (C) group ocurred a average increase of diameter of paws in 0.1%. The results indicate a higher cure rate and average reduction of diameter of paws for (M+P) group, suggesting that this association has greater efficacy in treatment of ACL in mice. Immunomodulatory effects of PXF might be involved in a more effective immune response. PXF is also nephroprotective related to toxicity of pentavalent antimonials, with possibility of exerting similar effect with respect to toxicity of miltefosine. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [521] P 440 - EVALUATION OF THE ANTILEISHMANIAL ACTIVITY OF THE ISOLATED COMPOUNDS FROM WISSADULA PERIPLOCIFOLIA (MALVACEAE). SANTOS, M.S.1; TELES, Y.C.F.2; GOMES, R.A.2; SILVA, D.A.2; QUEIROZ, A.C.1; DIAS, T.L.M.F.1; MELO, G.M.A.1; DA SILVA, A.E.1; AGRA, M.F.2; SOUZA, M.F.V.2; ALEXANDRE MOREIRA, M.S.1 1.UFAL, MACEIÓ, AL, BRAZIL; 2.UFPB, JOÃO PESSOA, PB, BRAZIL.
Keyword:antileishmanial activity; wissadula periplocifolia; leishmaniasis
Abstract: Leishmaniasis is caused by the protozoan Leishmania species. This disease affects more than 12 million people around the world, but current treatments are unsatisfactory. Therefore treatments for leishmaniasis with fewer side effects and a lower cost than present drugs are needed. The objectives of this study were to evaluate the citotoxicity and the antileishmanial activity of the isolated compounds from Wissadula periplocifolia (Malvaceae): phaeophytin a, 132-hydroxi(132-S)-phaeophytin a, phaeophytin b, acacetin, 7,4 -Di-O-methylisoscutellarein, 5,7-dihydroxi-8,4’-dimethoxyflavone and tiliroside. The colorimetric assay tetrazolium dye (MTT Bromide 3-4.5-dimethylthiazol-2-yl-2,5-dephenyltetrazolium) was realized to determine the
citotoxicity in J774 cells. To evaluation of the antileishmanial activity were utilized promastigotes of the Leishmania braziliensis, and as a parameter were used, the maximum leishmanicidal effect and IC50 value (i.e. the sample concentration causing 50% reduction in survival/viability of the parasites). The results showed that only phaeophytin a, phaeophytin b and 7,4 -Di-O-methylisoscutellarein demonstrated cytotoxicity to host cell in the concentration of 50 M, and phaeophytin a showed deleterious effects to J774 cells in the concentration of 10 M. It was observed that the compounds phaeophytin a, 132-hydroxi-(132-S)- phaeophytin a, phaeophytin b, acacetin and 7,4 -Di-O-methylisoscutellarein exhibited antileishmanial activity against promastigotes of Leishmania braziliensis, with maximal effect greater than 50%, especially 132-hydroxi-(132-S)- phaeophytin a and acacetin, which showed maximum effect of 70.8 ± 5.7% and 75.2 ± 2.4%, respectively. However, all compounds tested were lower effective and potent to dead promastigotes than the therapeutically used antileishmanial drug, pentamidine, which presented maximum effect of 94.8 ± 0.2% and IC50 of 1.3 ± 0.4 M. Morever, compounds phaeophytin a, 132-hydroxi-(132-S)- phaeophytin a, phaeophytin b, acacetin and 7,4 -Di-O-methylisoscutellarein demonstrated equivalent potency, with IC50 values of 77.2 ± 3.9, 72.7 ± 6.3, 89.0 ± 1.9, 49.5 ± 4.0 and 78.1 ± 9.5, respectively. Thus compounds isolated from Wissadula periplocifolia (Malvaceae) were identified as new leishmanicidal drug prototypes candidates. - CELLULAR AND MOLECULAR BIOLOGY [535] P 441 - LEISHMANIA (VIANNIA) BRAZILIENSIS IN NATURALLY INFECTED DOGS: A SEROLOGICAL AND MOLECULAR STUDY MARQUEZ, E.S.1; THOMAZ-SOCCOL, V.2; RIBEIRO, M.2; CASTRO, E.A.2; COUBEU, S.B.2; NABUT, L.B.3; NAVARRO, I.T.3 1.UNIVERSIDADE ESTADUAL DO NORTE DO PARANÁ, BANDEIRANTES, PR, BRAZIL; 2.UNIVERSIDADE FEDERAL DO PARANÁ, CURITIBA, PR, BRAZIL; 3.UNIVERSIDADE ESTADUAL DE LONDRINA, LONDRINA, PR, BRAZIL.
Keyword:leishmania (viannia) braziliensis; dogs; diagnosis
Abstract: This study aimed to determine the infection rate of Leishmania (Viannia) braziliensis in dogs and evaluate the sensitivity and specificity of several detection techniques. To this end, we used different methods of diagnosis, serological techniques (indirect immunofluorescence - IFI; enzyme linked immunosorbent assay - ELISA), and molecular methods (Polymerase Chain Reaction - PCR). Among 411 dogs studied, 186 (45.3%) presented evidence of anti-Leishmania antibodies, with titers equal to or greater than 1:40 by IFI, 155 dogs (37.7%) were positive for Leishmania by ELISA and 160 (38.9%) by PCR. The parasite was detected using various techniques in 14.4% (59/411) of dogs. Several factors were likely involved in the variability in sensitivity and specificity among the different methods of diagnosis analyzed, including the type of sample used, time of infection and cross-reactions. Detection of the parasite in the bloodstream demonstrates its presence in locations other than the original lesion, indicating hematogenous dissemination. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [536] P 442 - FUNCTIONAL ANALYSIS OF TRYPAREDOXIN PEROXIDASE IN ANTIMONYRESISTANT AND -SUSCEPTIBLE LEISHMANIA BRAZILIENSIS LINES ANDRADE, J.M.; MURTA, S.M.F. CENTRO DE PESQUISAS RENÉ RACHOU-FIOCRUZ, BELO HORIZONTE, MG, BRAZIL.
Keyword:l. braziliensis; antimonial resistance; tryparedoxin peroxidase
Abstract: Tryparedoxin peroxidase (TxP) participates of antioxidant defense by metabolizing hydrogen peroxide in water molecules. Literature data have reported that drug-resistant parasites may increase the levels of TxP along with other enzymes, protecting them against oxidative stress. In this study, we analyzed the TxP mRNA levels and protein expression levels in lines of L. braziliensis susceptible and resistant to potassium antimonyl tartrate (SbIII). This SbIII-resistant (LbSbR) line exhibits index of resistance to SbIII 20-fold higher than its susceptible counterpart (LbWTS). The level of TxP mRNA, determined by northern blot and real time RT-PCR (qRTPCR), was 2-fold higher in the SbIII-resistant L. braziliensis line, than in susceptible pair. We determined whether the increase in mRNA levels was due to gene amplification using Southern blot analysis. We found that the number TxP gene copies was similar between both lines analyzed. Western blotting assays using polyclonal antibody against the TxP recombinant protein from T. cruzi, showed that TxP protein is 4-fold more expressed in LbSbR than in susceptible LbWTS line. Functional analysis of TxP was performed to determine whether overexpression of TxP in the SbIII-susceptible and -resistant L. braziliensis lines would change the susceptibility of transfected parasites to antimony SbIII. After transfection, the parasites were submitted to PCR and western blot assays for confirmation of integration of construct pIR1BSD-LbTxP. Western blotting analysis showed that the level of TxP protein expression was 2 to 4-fold higher in transfected clones than in the non-transfected parental lines. IC 50 analysis showed that clones from SbIII-susceptible L. braziliensis line (LbWTS-TxP), that overexpress the TxP protein, are 2-fold more resistant to SbIII compared to its parental nontransfected line. Moreover, in vitro assays revealed that these clones transfected with TxP (LbWTS-TxP) also are more tolerant to exogenous hydrogen peroxide than its parental LbWTS line. On the other hand, clones from SbIII-resistant line (LbSbR-TxP) after TxP transfection became very susceptible to SbIII. In conclusion, our functional anaysis results suggest that the enzyme tryparedoxin peroxidase may be involved in the antimony-resistance phenotype in L. braziliensis. Financial Support: FAPEMIG, CNPq, CPqRR and UNICEF/UNDP/World Bank/WHO/TDR. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [540] P 443 - SEPARATION OF TWO POLYPEPTIDES WITH SPHINGOSINE KINASE (SK) ACTIVITY FROM LEISHMANIA MEXICANA, WITH STRUCTURAL SIMILARITIES TO HUMAN SK2 FERMIN, Z.1; GAMERO, M.D.1; GIL-ZAMBRANO, G.C.1; NARVAEZ, M.L.A.2; RINCON, M.G.1; HERMOSO, T.2 1.LABORATORIO DE INMUNOLOGIA CELULAR, INSTITUTO DE BIOMEDICINA, UNIVERSIDAD CENTRAL DE VENEZUELA, CARACAS, VENEZUELA; 2.SECCION DE BIOQUIMICA DE PARASITOS, INSTITUTO DE MEDICINA TROPICAL, UNIVERSIDAD CENTRAL DE VENEZUELA, CARACAS, VENEZUELA.
Keyword:sphingosine kinase ; lipid metabolism; leishmania mexicana
Abstract: A sphingosine kinase (SK) activity has been recently identified by our group in the parasite Leishmania mexicana. SK enzymes have been reported in a wide number of organisms and are responsible of the production of the second messenger sphingosine-1-phosphate (S1P), by phosphorylating the membrane sphingolipid sphingosine. S1P is involved in the regulation of several processes, like Ca2+ homeostasis, cell growth, cell adhesion, apoptosis, neuronal excitation, and cytoskeletal architecture. The aim of this study was to explore the existence of more than one SK activity in Leishmania mexicana, and to evaluate the recognition of the molecules responsible these activities by polyclonal antibododies, produced in our laboratory, against the human SK1 and SK2 enzymes. For that purpose, Isa Brown hens were immunized,
intramuscularly, with the human recombinant enzymes SK1 and SK2 (hSK1 and hSK2) using Freund´s adjuvant. After purification of IgY antibodies from egg yolks, and validation of the recognition of the human recombinant enzymes by western blot, the antibodies produced were tested for the detection of potential SK enzymes on Leishmania mexicana promastigote lysates, using the same technique. Six bands of 164, 150, 99, 79, 64, and 36 kDa were recognized by the anti-hSK1, and three bands of 245, 108 and 54 kDa by the anti-hSK2. The last two bands recognized by the anti-hSK2 coincided with two peaks of SK activity separated from the promastigote lysate soluble fraction by gel filtration cromatography. We found variations in the intensity of these bands during the promastigote growth in culture, which may indicate changes in the level of expression of the L. mexicana enzymes during the progression toward the stationary phase. Additionally, by 2D western blot, were detected three spots with molecular weights close to those of the bands observed in 1D western blots, of approximately pI:9.1, 4.6 and 5.6, respectively. These results indicate the presence of at least two polypeptides with SK activity in Leishmania mexicana, with structural similarities to hSK2, which expression varies during the promastigote cell cycle. Leishmania SKs may serve as target for developing drugs against these parasites. - CELLULAR AND MOLECULAR BIOLOGY [543] P 444 - DIAGNOSIS OF CUTANEOUS LEISHMANIASIS BY INDIRECT IMMUNOFLUORESCENCE, CELL CULTURE AND THE POLYMERASE CHAIN REACTION IN DOGS NATURALLY INFECTED IN THE STATE OF PARANÁ, BRAZIL. MARQUEZ, E.S.1; NAVARRO, I.T.2; NABUT, L.B.2; CASTRO, E.A.3; COUBEU, S.B.3; RIBEIRO, M.3; FORDELLONE, M.R.C.1; THOMAZ-SOCCOL, V.3 1.UNIVERSIDADE ESTADUAL DO NORTE DO PARANÁ, BANDEIRANTES, PR, BRAZIL; 2.UNIVERSIDADE ESTADUAL DE LONDRINA, LONDRINA, PR, BRAZIL; 3.UNIVERSIDADE FEDERAL DO PARANÁ, CURITIBA, PR, BRAZIL.
Keyword:american cutaneous leishmaniasis; dog; pcr
Abstract: American cutaneous leishmaniosis (ACL) is an endemic disease in the State of Paraná, constituting a public health problem as in many other states in Brazil. The most widely used methods of diagnosis, serology and culture, have limitations. The polymerase chain reaction (PCR) has been widely used in the diagnosis of Leishmania. This study aimed to evaluate different methods for the diagnosis of ACL by indirect immunofluorescence (IIF) in sera, cell culture of skin lesions and PCR of peripheral blood. Samples from 23 dogs with suggestive lesions of leishmaniosis were analyzed and divided in four groups according to positive or negative results in cell culture and in IIF. The first group consisted of 15 animals that were positive in both culture and IIF, from which seven animals (46.6%) obtained positive results by PCR. The second group consisted of two animals that were IIF positive and negative in culture, from which two (50%) were positive by PCR. The third group consisted of two animals with negative results in IIF and positive in culture, and revealed 100% negativity by PCR. The last group, four animals that had negative results by IIF and culture, presented two (50%) positive results by PCR. This study demonstrated that a positive PCR in peripheral blood samples proves the hematogenous spread of the parasite. The low percentage of PCR positivity in blood found in dogs with proven positive results by IIF and cultivation indicates that this technique should not be used as an isolated standard methodology. The findings of positive PCR results in blood samples of dogs that were negative by IIF and cultivation proved that PCR is a method that should be used in conjunction with the other techniques in order to increase the chances of defining truly ACL infected animals.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [544] P 445 - ANALYSIS OF GROWTH INHIBITION AND ULTRASTRUCTURE ALTERATION OF LEISHMANIA AMAZONENSIS PROMASTIGOTES TREATED WITH LIPOSOMAL FORMULATIONS OF CURCUMA SP. RODRIGUES, I.A.1; VERMELHO, A.B.1; ALMEIDA, L.1; AMARAL, A.C.F.2; SILVA, J.R.A.3; FERREIRA, J.L.P.2; RAMOS, A.S.2; ROSA, M.D.S.S.1 1.UNIVERSIDADE FEDERAL DO RIO DE JANEIRO, RIO DE JANEIRO, RJ, BRAZIL; 2.FUNDAÇÃO OSWALDO CRUZ, RIO DE JANEIRO, RJ, BRAZIL; 3.UNIVERSIDADE FEDERAL DO AMAZONAS, MANAUS, AM, BRAZIL.
Keyword:leishmania amazonensis; liposome formulations; curcuminoids
Abstract: The chemotherapy currently available for leishmaniasis is not effective presenting high toxicity and serious side effects. Therefore, new chemotherapeutic agents are urgently needed. Curcuminoids isolated from Curcuma species have a long history as antimicrobial agents, including against Leishmania. However, new strategies for drug delivery could enhance the efficacy of those compounds. In this study, lipid-based drug delivery systems (liposomes) have been investigated for the antileishmanial activity improvement of Curcuma sp. extract and curcuminoid-rich fraction. Promastigote forms of Leishmania amazonensis were treated with different concentrations of the extract and curcuminoids fraction and their respective liposomal formulations in order to evaluate growth inhibition and the minimal inhibitory concentration (MIC). In addition, cellular alterations of treated promastigotes were investigated under transmission and scanning electron microscopies. Curcuma extract and curcuminoid-rich fraction loaded liposomes presented lower MIC, 12.5 and 5.5 µg / ml, respectively, when compared to non-encapsulated herbal drugs. Parasite growth inhibition demonstrated to be dosedependent. Important morphological changes as rounded body and presence of several roles on plasmatic membrane could be seen on L. amazonensis promastigotes after treatment with sub inhibitory concentration (subMIC) of curcuminoids fraction. In addition, mitochondrial swelling and rarefied cytoplasm could be noted under transmission electron microscopy. In conclusion, drug delivery systems are currently under development to minimize drug degradation, loss and to increase drug bioavailability. In that sense, the extract of Curcuma and their metabolites incorporated in formulations could represent good strategy for the development of new antileishmanial agents. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [555] P 446 - IMMUNOSTIMULATORY ABILITY OF POLY-ICLC AGAINST EXPERIMENTAL INFECTION WITH LEISHMANIA BRAZILIENSIS SANTOS, D.M.1; CELES, F.S.1; BOZZA, M.T.2; SALAZAR, A.M.3; ANDRADE, B.B.4; DE OLIVEIRA, C.I.1
1.CENTRO DE PESQUISAS GONÇALO MONIZ, SALVADOR, BA, BRAZIL; 2.INSTITUTO DE MICROBIOLOGIA, UFRJ, RIO DE JANEIRO, RJ, BRAZIL; 3.ONCOVIR INC., WASHINGTON, UNITED STATES; 4.NATIONAL INSTITUTES OF HEALTH, BETHESDA, UNITED STATES.
Keyword:poly-iclc; intranasal; leishmania braziliensis
Abstract: Poly-ICLC, a stabilized version of Poly-IC currently used in clinical trials for the treatment of cancer and HIV, induces expression of IFN-stimulated genes (ISG) and production of type I IFN and pro-inflammatory cytokines, following binding to cytosolic and/or endosomal innate immune receptors. Poly-ICLC treatment was shown to be effective in reducing the parasite burden in hamsters experimentally inoculated with L. donovani, when used in combination with anti-Leishmania therapy. In the present study, we test the potential immunostimulatory capacity of Poly-ICLC against L. braziliensis infection. In vitro infection of thyoglycolate-elicited
BALB/c macrophages with L. braziliensis followed by Poly-ICLC treatment led to a significant reduction in the number of infected cells and in the parasite load per cell when compared to untreated infected cells. This effect was associated with an increase in cytokine, TNF- , IL-6 and IFN- and CXCL-10 production and with simultaneously enhanced production of reactive oxygen species (ROS). We next assessed if the enhancement in parasite killing induced by PolyIC involves directly the binding of type I IFN to the IFN- / receptor (IFNAR). 129/Sv IFNAR/- macrophages infected with L. braziliensis followed by treatment with Poly-ICLC displayed a significant increase in parasite burden, demonstrating the importance of signaling through IFNAR in controlling L. braziliensis infection and replication. We then analyzed the in vivo effect of Poly-ICLC treatment in BALB/c mice intradermally infected with L. braziliensis. At four weeks after infection, mice were treated with Poly-ICLC, intranasally, twice a week. PolyICLC treatment resulted in an acceleration of wound healing, which was associated with an increase in IFN- and TNF- production by draining lymph node cells. We conclude that PolyICLC is able to stimulate innate immune responses, promoting L. braziliensis killing in vitro and improving the clinical resolution of the disease lesions. Studies are in progress to better characterize the in vivo effects of PolyICLC, aiming at understanding its potential for immunotherapy of cutaneous leishmaniasis. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [560] P 447 - GROWTH FACTOR SIGNALING PATHWAYS CONVERGE WITH STAT6 TO REGULATE PATHOLOGIC ARGINASE EXPRESSION IN EXPERIMENTAL VISCERAL LEISHMANIASIS OSORIO, E.Y.1; TRAVI, B.L.1; DA-CRUZ, A.M.2; SALDARRIAGA, O.A.1; MELBY, P.C.1 1.DEPARTMENT OF INTERNAL MEDICINE, DIVISION OF INFECTIOUS DISEASES, UNIVERSITY OF TEXAS MEDICAL BRANCH, GALVESTON, UNITED STATES; 2.LABORATÓRIO INTERDISCIPLINAR DE PESQUISAS MÉDICAS, INSTITUTO OSWALDO CRUZ-FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:visceral leishmaniasis; hamster, growth factor; stat-6
Abstract: Host arginase 1 (Arg1) expression contributes to the pathogenesis of tegumentary and visceral leishmaniasis (VL) in animal models of the disease. Previously we found that Arg1 expression was dependent on STAT6 activation and was amplified by, but did not require, IL-4. To further explore the mechanisms involved in Arg1 regulation, we screened a chemical library of kinase inhibitors and found that inhibitors of growth factor signaling reduced Arg1 expression in splenic macrophages from hamsters infected with Leishmania (Leihmania) donovani by 5080%. To evaluate the significance of growth factor signaling in VL we use a phospho-antibody array and confirmed the results by Western blot. We found that the Fibroblast Growth Factor Receptor 1 (FGFR-1) and Insulin-like Growth Factor 1 Receptor (IGF-1R) and downstream signaling proteins p-ERK, p-p38, and p-CREB, were activated in splenic macrophages isolated from hamsters infected with L. donovani (SM ) for 15 days (fold change > 2 reference to uninfected). The expression of p-FGFR-1, FGF-2, p-STAT3, p-STAT6 and p-IGF-1R (p-AKT, p-GSK3- ) was also increased at 28 days post-infection, however, p-ERK, p-p38, and p-CREB were down regulated, suggesting inhibition of cell differentiation and apoptosis and promotion of cell survival at this time later point. Recombinant FGF-2 and IGF-1 increased the expression of Arg1 in hamster bone marrow-derived macrophages (BMM ) and fibroblasts (p<0.05). Inhibition of FGFR-1 and IGF-1R with chemical inhibitors decreased Arg1 expression and showed anti-Leishmania activity in both in vitro infected BMM and SM s. Collectively, these data indicate that these receptors influence parasite survival and promote a pathological immune response. Because STAT6 has a critical role in the transcriptional regulation of Arg1 we evaluated its role in the FGFR-1 and IGF-1R-mediated Arg1 expression. p-STAT-6 was induced in infected BMM s stimulated with both FGF-2 and IGF-1 and the transcriptional activity of STAT6 was activated in a luciferase reporter assay (p<0.05). Furthermore, siRNAi knockdown
of STAT6 in hamster BMM stimulated with FGF-2 and IGF-1, abrogated the induction of Arg-1, confirming that STAT6 was also required for growth factor induced Arg1 transcription. The results suggest that the FGFR/IGF-1R signaling pathways converge with STAT-6 to regulate Arg1 expression and contribute to the accumulation of disease promoting macrophages in VL. Support: UTMB, CAPES, CNPq. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [561] P 448 - CANINE VENEREAL TRANSMISSIBLE TUMOR: A POTENTIAL ALTERNATIVE ROUTE OF LEISHMANIA CHAGASI INFECTION FERRARI, H.F.1; CARREIRA, V.S.2; MACKENZIE, C.D.3; LANGHOR, I.3; MONTEZZO, L.C.4; RUI LUVIZOTTO, M.C.1
1.UNESP, ARAÇATUBA, SP, BRAZIL; 2.UNIVERRSITY OF CINCINNATI, CINCINNATI, UNITED STATES; 3.MICHIGAN STATE UNIVERSITY, EAST LANSING, UNITED STATES; 4.INSTITUTO DE PATOLOGIA, ANDRADINA, SP, BRAZIL.
Keyword:visceral leishmaniasis; tvt ; canine
Abstract: Visceral leishmaniasis is an anthropozoonotic disease with worldwide distribution, caused by Leishmania chagasi, which is endemic in the region of Araçatuba, in the state of Sao Paulo Brazil. Canine transmissible venereal tumor (TVT) is an exquisite sexually transmissible neoplasm of histiocytic origin, that isreported to represent the oldest known mammalian somatic cell in continuous propagation. In veterinary medicine, leishmaniasis has been previously described in association with dogs bearing canine transmissible venereal tumor as well as within the neoplasm tissue per se. The possibility that reminiscent phagocytic capabilities of the histiocytic TVT cells play a role in the parasitic invasion has been speculated. Since this tumor is transmissible via implantation of intact cells, it has been hypothesized that the presence of amastigotes in neoplastic TVT cells can play an alternative mode of transmission of canine leishmaniasis (CL) where these diseases co-exist. A 10 year-old, mixed breed intact female dog was submitted to the Veterinary Teaching Hospital with a three months history of a 4,0 x 3,0 cm vaginal vestibule mass. Cytology examination of imprint smears revealed abundant typical TVT cells admixed with degenerate neutrophils. Standard chemotherapy protocol with Vincristine Sulphate 0,5 mg/m2 IV once weekly was performed. After the fourth chemotherapy session and with a reminiscent 3,0 x 2,0 cm mass an additional cytology sample and biopsy was performed. Cytology smears presented predominantly keratinized epithelial cells admixed with neutrophils, lymphocytes, plasma cells and mixed bacteria. Few amastigotes could be observed within macrophages cytoplasm or free in the smear. Following a punch biopsy of the remaining mass, histopathology findings included extensive mixed inflammatory infiltrate within the subepithelial stroma and abundant macrophages that presented loaded with amastigote. Immunohistochemistry confirmed Leishmania spp within macrophages and dispersed through the neoplastic tissue. Despite the chemotherapy which temporarily compromised the immune system, the dog described herein was healthy, presenting no clinical signs of CL. Finally, TVT are moist lesions that could provide an attractive feeding site to the female sand fly vector and thus constitute the infection site. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [563] P 449 - CLONING, OVEREXPRESSION, PURIFICATION, CRYSTALLIZATION AND DATA PROCESSING OF NUCLEOSIDE DIPHOSPHATASE KINASE (NDK) FROM LEISHMANIA AMAZONENSIS. MISHRA, A.K.1; SINGH, S.P.1; KOLLI, B.K.2; BANSAL, M.3; CHANG, K.P.2; PRATAP, J.V.1
1.MOLECULAR AND STRUCTURAL BIOLOGY DIVISION, CSIR-CENTRAL DRUG RESEARCH INSTITUTE, LUCKNOW, INDIA; 2.CHICAGO MEDICAL SCHOOL, ROSALIND FRANKLIN UNIVERSITY OF MEDICINE AND SCIENCE, NORTH CHICAGO, UNITED STATES; 3.CENTRE FOR BIOTECHNOLOGY, ANNA UNIVERSITY, CHENNAI, INDIA.
Keyword:nucleoside diphosphate kinase; leishmania, extracellular atp; macrophages, multifunctional enzyme
Abstract: Nucleoside diphosphate kinases (NDKs) are ubiquitous enzymes that play crucial roles in different organisms such as regulation of gene expression in mammalian cells, bacterial pathogenesis, participation in the purine salvage pathways of trypanosomatid protozoa and parasite housekeeping functions, by modulating the levels of NTPs. NDK is a multifunctional enzyme in Leishmania infection. Accumulated extra-cellular ATP (eATP) triggers the death of macrophages that are the exclusive and true host of these intracellular parasites. Leishmania releases NDK at the time of infection and beyond to prevent ATP mediated cytolysis of their host cells by decreasing eATP, which may accumulate at the site of inflammation for modulation of immune cells. NDKs act via a ping-pong mechanism in which a histidine residue is phosphorylated by the transfer of the terminal phosphate group from ATP. These functions indicate that NDK is of potential interest for developing specific therapeutic strategies against leishmaniasis and structural characterization of a leishmanial NDK initiated to better understand the molecular mechanism. NDK from L. amazonensis was cloned in pET28a vector, overexpressed in E.coli BL21(DE3)pLysS cells and purified by affinity and size exclusion chromatography. The size exclusion profile shows that NDK exists as a trimer in solution. Preliminary crystallization trials with a protein concentration of 10 mg/ml and in the presence of different nucleotides using standard screens resulted in needle shaped crystals from some conditions that did not diffract. These conditions were further explored by altering pH, protein, precipitant and salt concentrations, resulting crystals that diffracted to ~5Å. Seeding approaches were initiated and in parallel, newer conditions explored. Good diffraction quality crystals were obtained from a condition containing 50 mM MgCl2, 100 mM Bis-Tris pH 6.2 and 18% PEGMME-500 as the precipitant. These crystals diffracted to ~2.5Å resolution at the ESRF beamline BM14, Grenoble, France. Preliminary processing of one data set suggests that the crystal belongs to the hexagonal space group P6322 with unit cell parameters a = b = 115.27Å, c = 62.54Å and = = 90°, = 120°. Packing calculations suggest the presence of a monomer in the asymmetric unit. Structure solution using the homologous enzyme from L. major as the search model has been successful and further refinement is in progress. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [570] P 450 - THE EFFICACY OF AEROSOL TREATMENT WITH NON-IONIC SURFACTANT VESICLES CONTAINING AMPHOTERICIN B IN RODENT MODELS OF LEISHMANIASIS AND PULMONARY ASPERGILLOSIS INFECTION. CARTER, K.C.1; ALSAADI, M.1; ITALIA, J.1; MULLEN, A.B.1; KUMAR, M.R.1; CANDLISH, A.A.; WILLIAMS, R.M.1; SHAW, C.1; GAWHARI, F.1; COOMBS, G.H.1; WEISE, M.H.1; THOMSON, A.1; PUIG, M.1; WALLACE, J.1; SHARP, A.2; WHEELER, L.1; WARN, P.2 1.UNIVERSITY OF STRATHCLYDE, GLASGOW, UNITED KINGDOM; 2.UNIVERSITY OF MANCHESTER, MANCHESTER, UNITED KINGDOM.
Keyword:drug delivery; amphotericin b; inhalation
Abstract: Delivery systems can help to improve the efficacy of drugs by altering their targeting to tissues or their retention within tissues. We have shown that non-ionic surfactant vesicles (NIV) can improve the delivery of a number of drugs given by the parental route but non-invasive ways of delivering drugs are required. In this study the ability NIV to deliver amphotericin B (AMB) after treatment by inhalation was determined using murine models of leishmaniasis and a rat
model of invasive pulmonary aspergillosis. AMB levels were determined in the tissue of treated animals using a HPLC assay. Treatment with AMB-NIV resulted in higher AMB levels in the lungs and skin (p < 0.05) but significantly lower plasma levels (p < 0.05) in animals compared to results for animals given AMB solution. Treatment with AMB-NIV resulted in a significant reduction in fungal lung burdens (p < 0.05) compared to treatment with the carrier alone. Treatment with AMB-NIV but not AMB solution significantly suppressed L donovani liver parasite burdens (p < 0.05) but did not inhibit the growth of cutaneous L. major lesions. The results of this study indicate that aerosolised NIV enhanced pulmonary and hepatic delivery whilst minimising systemic exposure and toxicity. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [571] P 451 - COMPARISON OF PARASITE FITNESS IN WILD-TYPE AND PAROMOMYCINRESISTANT AMASTIGOTES OF LEISHMANIA DONOVANI MAES, L.1; SARAH, H.1; GAELLE, B.1; ANNELIES, M.1; JEAN-CLAUDE, D.2; PAUL, C.1 1.UNIVERSITY OF ANTWERP, ANTWERP, BELGIUM; 2.INSTITUTE OF TROPICAL MEDICINE, ANTWERP, BELGIUM.
Keyword:paromomycin; resistance induction; fitness
Abstract: Paromomycine (PMM) is a relatively recent drug on the market aiming to alleviate the current first-line antimonial (Sb) treatment which is gradually losing efficacy because of spreading drug resistance. By subjecting intracellular L. donovani amastigotes to increasing PMM concentrations in vitro, amastigotes with a stable 4-fold higher tolerability could be obtained within two selection cycles. Since Sb-resistance has been associated by increased parasite fitness (Vanaerschot et al. Crit Rev Microbiol. 2012 Sep 6), it was useful to check whether the same would also apply to PMM resistant strains. In this study, a PMM resistant (R) and its wild-type (WT) parent strain were compared for parasite fitness, defined as: (1) parasite growth; (2) infectivity and (3) virulence. To compare both strains at the intracellular amastigote stage, metacyclogenesis is a major factor contributing to parasite infectivity. In a first phase, the optimal moment was selected at which most promastigotes in the in vitro culture were metacyclic. To accurately define this time point, 4 different criteria were used: (1) flow cytometry for a rough estimation of the time point at which metacyclogenesis starts, (2) morphological evaluation adopting a flagel / cell body length ratio of >2, (3) evaluation of infectivity by exposing primary mouse macrophages with promastigotes of different time points at a fixed 15/1 infection ratio and (4) promastigote resistance to complement-mediated lysis. Preliminary results indicate no difference in promastigote growth and metacyclogenesis between WT and PMM-R parasites. Results will be presented on the comparison of both strains for intracellular amastigote growth, promastigote infectivity and in vivo virulence in hamsters. This work was supported by the European Commission (Kaladrug-R, EC-FP7-222895)
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [576] P 452 - MACROPHAGE-PRODUCED REACTIVE OXYGEN SPECIES AND APOPTOSIS: POSSIBLE TARGETS FOR ANTILEISHMANIAL DRUGS? COS, P.; DESCHACHT, M.; HENDRICKX, S.; BOULET, G.; DELPUTTE, P.; MAES, L. UNIVERSITY OF ANTWERP, ANTWERP, BELGIUM.
Keyword:antileishmanial; free radicals; apoptosis
Abstract: This study investigated the effect of antileishmanial drugs SbV and SbIII, miltefosine (MIL) and the experimental compound PX-6518 on two major cellular processes of the macrophage: a/ the production of free radicals and b/ the induction of apoptosis. Free radicals and apoptotic characteristics were detected in (un)-treated and (non)-infected (L. infantum and L. donovani strains) primary peritoneal mouse macrophages, RAW 264.7 macrophages and U937 monocytes, using Electron Paramagnetic Resonance (EPR) for direct intracellular detection of superoxide (O2 -) and nitric oxide (NO) (Deschacht M. et al. Free Radic Res 44, 763-772 (2010)). Characteristics of early and late apoptosis were identified after dual staining with FITClabeled annexin-V and propidium iodide. Caspase-3 cleavage was determined using western blotting. All studied Leishmania strains induced a similar increase in free radical production in macrophages. O2 - production was significantly elevated during phagocytosis of the stationary phase promastigote. Conversely, NO levels increased later in the infection and none of the strains induced capsase-3 cleavage. In contrast to infected cells, treatment of uninfected cells with MIL, Sb or PX-6518 induced NO production in macrophages. MIL decreased slightly the O2 - response in infected cells. Only SbIII led to induction of phosphatidylserine externalization and caspase-3 cleavage in uninfected macrophages. Caspase-3 cleavage was also observed in RAW 264.7 cells upon SbIII treatment. In conclusion, the listed antileishmanial drugs induce NO production in macrophages, which becomes inhibited by the parasite. Induction of apoptosis seems to be important for the mechanism-of-action of SbIII.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [577] P 453 - IN VITRO DRUG SUSCEPTIBILITY OF LEISHMANIA STRAINS ISOLATED IN PORTUGAL FROM HUMAN AND CANINE CASES MAIA, C.1; NUNES, M.2; MARQUES, M.2; HENRIQUES, S.2; ROLÃO, N.2; CAMPINO, L.3
1.UEI PARASITOL MÉDICA/CMDT/INSTITUTO HIGIENE MEDICINA TROPICAL-IHMT/ UNIV NOVA LISBOA-UNL. FMV/ULHT, LISBOA, PORTUGAL; 2.UEI PARASITOLOGIA MÉDICA/CMDT/INSTITUTO HIGIENE MEDICINA TROPICAL-IHMT/ UNIVERSIDADE NOVA LISBOA-UNL, LISBOA, PORTUGAL; 3.UEI PARASITOLOGIA MÉDICA/IHMT/UNL. DEP. CIÊNCIAS BIOMED E MED/UNIVERSIDADE DO ALGARVE, LISBOA, PORTUGAL.
Keyword:leishmania infantum; in vitro susceptibility; human and canine treatment
Abstract: Visceral leishmaniasis (VL) caused by parasites of Leishmania donovani complex is a severe human disease which often leads to death if left untreated. Domestic dogs are considered the main reservoir hosts for zoonotic human visceral infection caused by L. infantum. In the absence of effective prophylactic measures, the only feasible way to control and treat leishmaniasis is through the use of affordable medications. In Portugal, as in other countries from SouthernEurope, VL was initially a paediatric disease but in the last years the number of cases in children has decreased with an increase of infection in adults with an incidence rate of 0.294/100000 inhabitants. Overall seroprevalence of canine leishmaniasis (CanL) estimated in mainland Portugal was 6.31%, in 2009. To know the drug susceptibility of human and canine Leishmania strains from Lisbon-Portugal, a study on a panel of strains was conducted by testing the susceptibility of promastigote forms and intracellular amastigotes to the common drugs used in CanL and human VL therapy, namely meglumine antimoniate, amphotericin B, miltefosine and allopurinol. A high heterogeneity of susceptibilities was obtained to each drug. In addition, a low susceptibility of the parasites isolated from a refractory human case to treatment was observed and canine strains were less susceptible to the main drugs used in CanL. These results highlight the importance of combined therapy to reduce/avoid the emergence and spread of
acquired drug resistance, a factor that requires consideration where there is an extensive treatment of the domestic canine host using the same few available anti-Leishmania drugs for human VL. Acknowledgements: C. Maia (SFRH/BPD/44082/2008) holds a fellowship from Fundação para a Ciência e a Tecnologia, Ministério da Ciência, Tecnologia e Ensino Superior, Portugal. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [583] P 454 - PARASITE LOAD IN PEREPHERAL BLOOD IS POSITIVILY CORRELATE WITH SPLENIC SCORE IN VISCERAL LEISHMANIASIS PATIENTS. SUDARSHAN, M.; SUNDAR, S. BANARAS HINDU UNIVERSITY, VARANASI, INDIA.
Keyword:leishmaniasis; qpcr; splenic score
Abstract: Early diagnosis of VL is very important for the elimination of it from endemic region. Ideally, a Diagnostic test should make the distinction between acute disease,cure status and asymptomatic infection. Microscopy is gold standard diagnosis method for Visceral Leishmaniasis but is risky as require spleen puncture. rk 39 strip test is well to diagnose VL but it is immune response based tool who show positivity after therapy, therefore there is need to evaluate any technique which can see Leishmania infection as well as quantitate their presence as active VL.qPCR is atechnique now in a use for Leishmania infection detection.This study was done on 100 VL patients comes for treatment in KAMARC hospital, muzaffarpur. Disease status is confirm by their clinical symptoms and positive splenic score. qPCR was done on DNA isolated from peripheral blood collected in citrate blood. Kinetoplast specific primer is used for standard curve based absolute quantification in qPCR. Parasitemia level is range from 5 to 75,000 Leishmania parasite DNA/ml of blood and correlate with their grading of splenic score. Correlation is positive with r2 value of 0.6 Therefore qPCR will be helpful to come out from doing such a risky invasive diagnosis of VL. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [584] P 455 - ANTI LEISHMANIAL ACTIVITY OF PLANT EXTRACTS DERIVED FROM MEDICINAL PLANTS IN PALESTINE HAMARSHEH, D.O.O.1; AZMI, K.2; AMRO, A.3; NASSEREDIN, A.2; SAWALHA, K.1; ALRIMAWI, F.4; ABDEEN, Z.2 1.DEPARTMENT OF LIFE SCIENCES, AL-QUDS UNIVERSITY, JERUSALEM, ISRAEL; 2.ANAHRI, AL-QUDS UNIVERSITY, JERUSALEM, ISRAEL; 3.FACULTY OF PHARMACY, AL-QUDS UNIVERSITY, JERUSALEM, ISRAEL; 4.DEPARTMENT OF CHEMISTRY, AL-QUDS UNIVERSITY, JERUSALEM, ISRAEL.
Keyword:leishmani tropica; plant extracts; palestine
Abstract: Leishmaniasis is a vector born parasitic disease, the clinical presentation ranging from mild coetaneous to life threatening visceral disease. The disease is caused by different species of Leishmania parasites and transmitted by correspondent sand fly species. Leishmania tropica is the causative agent of coetaneous leishmaniasis in different countries in the Mediterranean basin. Extracts of 60 plant species distributed in Palestine were screened against Leishmania tropica promastigotes using microplate Alamar blue bio assay. Four plants showed leishmanicidal activity at 63 g/ml concentrations. This study supports that selecting plants by ethnobotanical criteria may enhance the possibility of finding new candidate drugs against Leishmania parasites
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [586] P 456 - EVALUATION OF THE RESISTANCE PROFILE OF LEISHMANIA CHAGASI ISOLATED FROM ANTIMONY TREATMENT REFRACTORY PATIENTS SANTOS, M.L.B.1; OLIVEIRA, F.A.1; SANTOS, P.L.1; SERAFIN, L.C.1; CARVALHO, S.S.1; LEOPOLDO, P.T.G.1; JESUS, A.R.2; ALMEIDA, R.P.2; DE MOURA, T.R.1 1.UNIVERSIDADE FEDERAL DE SERGIPE, ARACAJU, SE, BRAZIL; 2.INSTITUTO DE INVESTIGAÇÃO EM IMUNOLOGIA, SÃO PAULO, SP, BRAZIL.
Keyword:l. (l.) chagasi; viability; resistance to antimony
Abstract: Introduction: Macrophage activation by Th1 cells is most important mechanism against Leishmania. This activation leads to release nitric oxide(NO), a toxic product for Leishmania parasites. Pentavalent antimonials (SbV) is known to reinforce the killing mechanisms of macrophages. This work, we evaluated if L. chagasi (Lc) isolated from refractory to antimony treatment patients show cross-resistance to SbIII, NO, liposomal amphotericin b(L-Amb) and also modulate macrophage infection. Methods and Results: Four L. chagasi isolates from antimony treatment refractory patients and two from antimony treatment responsive patients were used in this study. In vitro susceptibility nitric oxide(NO)was evaluated by exposing the parasites to different concentrations of NaNO2(NO donor)and the viability was determined by alamar blue™ method. The Lc isolates from refractory patients were considered resistant to NO, showing an average of 81%survival at NaNO2 8mM, while isolates from patients responsive were considered susceptible to NO, showing an average of 53%,p<0.005. Promastigotes were submitted to increasing concentrations of antimony trivalente(SbIII)and the viability was determined by cell count. The SbIII 50%inhibitory concentration (IC50) for the isolates from refractory patients were highly resistant to SbIII with an average IC50 741+32 compared with isolates from sensitive patients, IC50 381+100,p<0.01.Moreover, parasites were exposed(LAmb)and inhibition growth was determined by the colorimetric method alamar Blue™,we observed in isolates from patients refractory elevated IC50 comparede to isolates from patients responsive,33+9 and 16+3, p<0.05. To evaluate the susceptibility of amastigotes to SbV and the macrophages killing mechanism, macrophages were infected with isolates from patients refractory in the presence of LPS/IFN- or SbV,at 24,48 and 72hours. Isolates from refractory patients were more infective than isolates from patients responsive to treatment, independently of these stimuli and in all times post infection. Although isolates from refractory patients produced more nitrite than those from responsive, the rate nitrite/amastigotes was six times lower in the macrophage infected by isolates from refractory patients. Conclusion: Taken together, these results suggest that in isolates from refractory patients there is cross resistance between SbIII, NO,L-Amb and, also, these parasites were able to down modulate the macrophage killing mechanisms. - OTHER SPECIAL TOPICS [590] P 457 - RESULTS OF A STEP-WISE TREATMENT APPROACH IN CUTANEOUS LEISHMANIASIS MORIZOT, G.1; KENDJO, E.2; MOURI, O.2; THELLIER, M.2; RAVEL, C.3; CAUMES, E.4; LACHAUD, L.3; BUFFET, P.A.2
1.UNITE D’IMMUNOLOGIE MOLECULAIRE DES PARASITES, INSTITUT PASTEUR, PARIS, FRANCE; 2.SERVICE DE PARASITOLOGIE-MYCOLOGIE, HOPITAL PITIE-SALPETRIERE, PARIS, FRANCE; 3.FRENCH REFERENCE CENTRE ON LEISHMANIASIS, MONTPELLIER, FRANCE; 4.SERVICE DE MALADIES INFECTIEUSES ET TROPICALES, HOPITAL PITIESALPETRIERE, PARIS, FRANCE.
Keyword:cutaneous leishmaniasis; step-wise treatment ; recommendation
Abstract:
Abstract Background: Cutaneous leishmaniasis (CL) is a stigmatizing but not life-threatening disease. The WHO recommends the use of potentially toxic therapy only when lesions cannot be treated locally or when local therapy has been tried and failed. Methods: To determine the feasibility and results of this “local treatment first” approach we analyzed the results of a centralized referral treatment program in 135 patients with parasitologically proven CL. Results: Infections involved 10 Leishmania species and were contracted in 29 different countries. Sixty two percent (84/135) of patients were initially treated without systemic therapy. Twenty-three of 25 (92%) evaluable patients treated with simple wound care and 37 of 47 (79%) evaluable patients treated with local anti-leishmanial therapy were cured by D42-60. Systemic adverse events were observed in 15 patients, all receiving systemic therapy with antimony, liposomal amphotericin B, or miltefosine. Conclusion: The recommended step-wise approach favoring local therapy therefore resulted in at least 44% of patients being cured without exposure to the risk of systemic adverse events. The impact of this approach will further increase when effective antileishmanial ointments become widely available.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [591] P 458 - LEISHMANIASIS IN SURINAME: QUANTITATIVE REAL TIME PCR AS A TOOL TO MONITOR PARASITE NUMBERS IN BIOPSIES DURING TREATMENT KENT, A.D.1; HU, R.2; ADAMS, E.3; MANS, D.1; SCHALLIG, H.3 1.ANTON DE KOM UNIVERSITY OF SURINAME, PARAMARIBO, SURINAME; 2.DERMATOLOGY SERVICE, MINISTRY OF HEALTH, PARAMARIBO, SURINAME; 3.ROYAL TROPICAL INSTITUTE, BIOMEDICAL RESEARCH, PARASITOLOGY UNIT, AMSTERDAM, NETHERLANDS.
Keyword:cutaneous leishmaniasis; quantitative real time pcr; treatment monitoring
Abstract: Background Cutaneous leishmaniasis (CL) is caused by a protozoan flagellate of the genus Leishmania, which is transmitted by the bite of an infected female Phlebotomine sand fly. The clinical forms of CL range from single skin ulcers to more disseminated forms with disfiguring scar formation. In Suriname, diagnosis is still done by microscopy; however this method lacks sensitivity and cannot be used as a tool to monitor treatment. Aim This study assesses the feasibility of quantitative real time PCR (qPCR) to detect and quantify Leishmania parasites in skin biopsies. Methods Biopsies of 2 mm were taken before treatment, 6 weeks and 12 weeks after treatment and the qPCR assay measured the parasite load in those skin biopsies. The results of the qPCR were compared to the clinical outcome (healed, relapse and treatment failure).
Results So far, 78 patients have been recruited. The parasitaemia before treatment ranged from 1 x102 108 parasites per biopsy. Sixty one patients (78%), the parasite load declined at week 6 and week 12 to 0-1x103 per biopsy. These patients were clinically healed. Eight patients (10.3%) had a relapse, as indicated by the decrease in parasite load to 1 x 102 – 103 in weeks 6, but the increase above 1 x 105 in week 12. In 9 patients (11.5%), the parasitaemia did not decrease either in week 6 or 12. Clinically, these patients had failed the therapy. Conclusion qPCR is a useful tool for the diagnostics and monitor the course of the treatment.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [600] P 459 - EFFECTS OF GOLD COMPLEXES WITH BIOISOSTERES OF RESVERATROL ON SEVERAL LEISHMANIA SPECIES MACHADO, P.A.; MOTA, V.Z.; DE CARVALHO, G.S.; CUIN, A.; DA SILVA, A.D.; COIMBRA, E.S. UNIVERSIDADE FEDERAL DE JUIZ DE FORA, JUIZ DE FORA, MG, BRAZIL.
Keyword:leishmania; gold complexes ; bioisosteres of resveratrol
Abstract: Leishmania cause a spectrum of diseases, ranging from a cutaneous ulceration to a fatal visceral disease. Leishmaniasis is endemic in 98 countries, over 350 million people are at risk and 2 million de new cases arise every year. The treatment requires the administration of toxic and poorly tolerated drugs, thus, there is an urgent need for effective drugs to replace or supplement those in current use. In rational trials we analyzed the antileishmanial activity of three new bioisosteres of resveratrol and four new gold complexes with bioisosteres of resveratrol. Compounds were tested against promastigote and amastigote forms of L. amazonensis, L. braziliensis and L. major. Their citotoxicity against mammalian cells also were assayed. Viability of the promastigote forms and mammalian cells was checked using the MTT colorimetric method, after three days incubation period. Antiamastigote activity was determined by counting the number of intracellular parasites after 72 hours of treatment and the supernatant was also collected for measure of the nitrite, a by-product of nitric oxide (NO), by the Griess method. Only the gold complexes with bioisosteres of resveratrol were active in L. amazonensis, L. braziliensis and L. major promastigotes (IC50 ranging from 1.29 to 54.01 uM), thus this compounds were tested in amastigotes and all were active (IC50 ranging from 3.16 to 25.95 uM). L. braziliensis was the most sensitive specie to the action of the compounds. Two compounds showed toxicity in macrophages, but all showed significant selectivity for the parasite. None of compounds induced significant nitrite production in the culture medium in all concentration used compared to untreated controls. These results show a good in vitro leishmanial activity of gold complexes with bioisosteres of resveratrol and the leishmanicidal effect observed on amastigote forms is unlikely due to NO production by macrophages. Supported by FAPEMIG, CNPq and UFJF. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [601] P 460 - A 10-DAY REGIMEN OF OLEYLPHOSPHOCHOLINE CAN INDUCE STERILE CURE IN EXPERIMENTAL L. INFANTUM LEISHMANIASIS IN HAMSTERS.
FORTIN, A.1; KUYPERS, K.2; FEIJENS, P.2; VERMONT, M.2; COS, P.2; JANSEN, H.3; MAES, L.2
1.DEPARTMENT OF BIOCHEMISTRY, MCGILL UNIVERSITY, MONTREAL, CANADA; 2.LABORATORY FOR MICROBIOLOGY, PARASITOLOGY AND HYGIENE, UNIVERSITY OF ANTWERP, WILRIJK-ANTWERP, BELGIUM; 3.DAFRA PHARMA RESEARCH & DEVELOPMENT, TURNHOUT, BELGIUM.
Keyword:drug development; visceral leishmaniasis; hamster model
Abstract: The alkylphospholipid oleylphosphocholine (OlPC) is a structural analogue of miltefosine (MIL) and represents a potential therapeutic alternative for the treatment of leishmaniasis. OlPC has good oral bioavailability and is long-acting with a mean elimination half-life of about 70 hrs in rodents. In a comparative study with MIL, OlPC showed an in vitro activity similar to MIL but a superior in vivo efficacy in the hamster model of L. infantum (Fortin et al. 2012). Dosetitration comparing aqueous (OlPC/H2O) and liposomal formulations of OlPC administered orally revealed that more than 95% of the parasite burden could be eliminated from the target organs liver, spleen and bone marrow using a 100 mg/kg total dose given as a single or over a multiple administration (5 days) regimen. The goal of the present study was to evaluate if oral OlPC could lead to complete parasitological cure in hamsters infected with L. infantum adopting a 10-day dosing regimen at 20 mg/kg, in which aqueous (OlPC/H2O) and liposomal formulations of OlPC were compared to an aqueous formulation of MIL. To evaluate possible recrudescence of the infection following initial efficacy, amastigote burdens were determined in target organs on 10 and 40 days post-treatment (dpt). When measured 10 dpt, parasite burdens after OlPC/H2O were reduced by 100%, 99.7% and 99.5% in liver, spleen and bone marrow compared to vehicle-treated controls. No recrudescence was seen 40 dpt with parasite reductions of 100%, 100% and 98.8% in the same organs. MIL treatment led to initial reduction of parasite burdens but with subsequent recrudescence, going from 98.2% at 10 dpt to 74.9% at 40 dpt in the liver, from 99.5% to 97.8% in the spleen, and from 89.9% to 78.3% in the bone marrow. More importantly, the 10-day regimen with OlPC-liposomes at 20 mg/kg resulted in 100% reduction of parasite burdens in all target organs on both 10 dpt and 40 dpt, suggesting full parasitological cure with absence of recrudescence. In summary, this study confirms that OlPC has a higher efficacy compared to MIL and represents a promising new candidate to shorten and simplify current case management of visceral leishmaniasis. Further studies to assess the full potential of OlPC as a drug candidate are underway. This research was funded by Agentschap voor Innovatie door Wetenschap en Technologie (project no.110402)
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [609] P 461 - CHARGE DELOCALIZED CATIONS AS TARGETING MOTIVES FOR MITOCHONDRIA.INHIBITION OF SUCCINATE DEHYDROGENASE (COMPLEX II) OF THE LEISHMANIA DONOVANI RESPIRATORY CHAIN BY DIPHENYL BISPHOSPHONIUM SURROGATES SARAIVA CORRÊA, D.1; ABENGOZAR, M.A.1; HEALY, A.2; TALADRIZ, A.2; LOPEZ-SANCHEZ, A.1; FLORES LOPEZ, E.J.2; TEMPONE, A.G.3; BARBAS, C.4; DARDONVILLE, C.2; RIVAS, L.1 1.CENTRO DE INVESTIGACIONES BIOLÓGICAS (CSIC), MADRID, SPAIN; 2.INSTITUTO DE QUÍMICA MÉDICA, IQM-CSIC, MADRID, SPAIN; 3.DEPARTMENT OF PARASITOLOGY, INSTITUTO ADOLFO LUTZ,, SAO PAULO, SC, BRAZIL; 4.DEPARTMENT OF ANALYTICAL CHEMISTRY. CEMBIO SAN PABLO CEU UNIVERSITY, BOADILLA(MADRID), SPAIN.
Keyword:drug targeting; respiratory chain; mitochondria
Abstract: Intracellular targeting of drugs into mitochondrion frequently profits from the high electrochemical potential of this organelle to accumulate drugs with delocalized cations in the matrix or innermembrane in the mitochondria. . This study evaluates the leishmanicidal potential of 48 bisphosphonium surrogates derived from 4,4´- bis ((tri-n-pentylphosphonium)methyl) benzophenone. From the initial set of 48 compounds, 4 were selected according to their EC50s and LC50s at low µM concentrations, both on promastigotes (L. donovani) and on axenic amastigotes (L. pifanoi); as well as to their selectivity index (SI) ranging from 20 – 49. Those compounds with lower or nil number of phosphonium structures showed a crippled leishmanicidal activity. The four selected compounds induce a rapid and dose-dependent drop in intracellular ATP, measured in real time on living parasites expressing a recombinant cytoplasmic form of luciferase, but even at 15µM (ca 10 X EC50s), none of these compounds induced entrance of vital dyes, ruling out plasma membrane permeabilization as the origin of this effect. By contrast, these four compounds induced depolarization of the mitochondrion, with faulty Rhodamine 123 accumulation, and swelling of the organelle in absence of apparent damage to the plasma membrane, as observed in transmission electron microscopy. Altogether, inhibition of mitochondrial ATP synthesis by these compounds was inferred. In a step ahead, using, digitonin-permeabilized parasites in a Clark O2 electrode, the site of activity of these compounds was mapped into the complex II (succinate dehydrogenase), and confirmed by inhibition of its enzymatic activity. Finally, all of them induced apoptosis of the parasite with increase of the sub-G1 population.
Although we can not discard additional targets for these drugs, inhibition of the mitochondrial ATP synthesis, an essential process for parasite viability was a major target for diphenyl bisphosphonium. Furthermore, this activity is highly dependent on the presence of phosphonium grups as pharmacophore to improve mitochondrial localization of leishmanicidal drugs. Supported by projects: EU (HEALTH-2007-223414), ISCIII-FEDER RD 06/0021/0006, RD12/0018/0007 and ISCIII PI09-01928, PI12-02706 to LR MICINN (SAF2009-10399) to CD, D.S.C is an Airbus Military-Força aerea brasileira fellow.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [610] P 462 - APPARENT CLINICAL CURE IN HAMSTERS EXPERIMENTALLY INFECTED WITH L. (L.) AMAZONENSIS AND TREATED WITH AMIODARONE AND GLUCANTIME ANVERSA, L.; MARTINS, T.Y.; TIBURCIO, M.G.S.; BATISTA, L.R.; HERNÁNDEZ, C.G.; GIRALDO, L.E.R. UNIVERSIDADE FEDERAL DO TRIÂNGULO MINEIRO, UBERABA, MG, BRAZIL.
Keyword:amiodarone; glucantime; l (l.) amazonensis
Abstract: Amiodarone has been the target of recent studies investigating its appliance as an antitrypanosomatide agent. The present study aimed to assess the therapeutic role of amiodarone used singly or compound with glucantime in the treatment of experimental cutaneous leishmaniasis in hamsters (Mesocricetus auratus). A total of 60 animals were divided into four groups submitted to different treatments (Amiodarone / Glucantime / Amiodarone + Glucantime/ Control group). The animals were infected with L. (L.) amazonensis and the inoculum was 1 x 106 realized intradermally on one of its hind legs. After 20 days post inoculums (with apparent lesion) treatment started, lasting 20 days consecutively, being amiodarone administered orally (by gavage) and glucantime intraperitoneally. The effectiveness of treatment was assessed by measuring the skin lesion, performed every ten days throughout the study, and after euthanasia by means of parasitologic examination (imprint) and molecular (PCR) methods. The results evidenced that glucantime used isolated is partially effective in control the lesion development, whereas animals treated with this drug presented significantly lower lesion as compared to control group (untreated). The treatment with amiodarone singly did not demonstrated significant difference when compared to control group. However, animals treated with the combination of both drugs (amiodarone and glucantime) exhibited the best results, presenting the smallest lesions at the end of treatment and, moreover, in some cases were observed the extinction of the lesion. However, no animal presented parasitological cure, as established by PCR and imprint methods. Accordingly, it is suggested that the apparent clinical cure does not always correspond to parasitological cure. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [622] P 463 - IN VITRO EVALUATION OF THE LEISHMANICIDAL EFFECT OF SELECTED PEROXIDES CORTES, S.1; ALBUQUERQUE, A.2; CABRAL, L.3; CRISTIANO, M.L.3; CAMPINO, L.4 1.UEI PARASITOLOGIA MÉDICA/CMDT/INSTITUTO HIGIENE MEDICINA TROPICAL/UNIVERSIDADE NOVA LISBOA, LISBOA, PORTUGAL; 2.UEIPM//IHMT-UNL, LISBOA, PORTUGAL; 3.CCMAR/DEPARTAMENTO DE QUÍMICA E FARMÁCIA/UNIVERSIDADE DO ALGARVE, FARO, PORTUGAL; 4.UEIPM/IHMT-UNL, DEP.CIENCIAS BIOMED. E MED.UNIVERSIDADE DO ALGARVE, LISBOA, PORTUGAL.
Keyword:peroxides; l. infantum; chemotherapy
Abstract: Leishmania infantum is the causative agent of human and canine leishmaniasis in the Mediterranean basin. It affects canines and is an opportunistic and emerging disease in humans, therefore representing a very important health problem. WHO considers treatment of leishmaniasis as a research priority The few available drugs currently recommended for leishmaniasis treatment have high cost, severe toxic side effects and declining efficacy, since selection of Leishmania resistance has emerged, namely in hyper-endemic regions Thus, it is benefic the search for new, efficient and safe drugs for the treatment of this disease. Trioxanes and trioxolanes are peroxidic compounds, and some of them have demonstrated efficacy against other eukaryote parasites such as Plasmodium spp., at nanomolar concentrations, and Perkinsus spp., at micromolar concentrations. We now report unprecedented information concerning the in vitro leishmanicidal effect of selected peroxides. Synthetic compounds used in this investigation were obtained in good yields, fully characterized and their leishmanicidal activity was assayed in promastigote forms of a Leishmania infantum strain (MHOM/PT/88/IMT151). Parasites were cultured in RPMI medium supplemented with 10% heat-inactivated fetal calf serum, at 24 ºC ± 1 ºC. Different concentrations of each compound were added to the promastigote culture and after 48 hours of incubation, parasites viability was evaluated by MTT assay and activity of each compound was
expressed as % reduction of parasite burdens compared to the untreated controls. Inhibitory concentrations (IC50) were calculated. Amphotericin B was use as control drug. Preliminary results showed that all compounds are effective against L. infantum promastigotes (similar activity to control drug), with one of them exhibiting a comparatively higher activity. Additional studies are ongoing to determine if this class can be considered as a source of molecules with antiproliferative activity against Leishmania, bearing potential to be developed as effective agents for leishmaniasis’ chemotherapy. This work was supported by funding from the research project PTDC/CVT/ 112371/2009 from Fundação para a Ciência e a Tecnologia (FCT), Ministério da Ciência, Tecnologia e Ensino Superior, Portugal. S. Cortes holds a fellowship from FCT (SFRH/BPD/44450/2008). L. Cabral holds a fellowship from CCMAR/CIMAR. S. Cortes and A. Albuquerque contributed equally for this work. scortes@ihmt.unl.pt - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [634] P 464 - BUPARVAQUONE (HYDROXYNAPHTHOQUINONE) INHIBITS CYTOCROME C REDUCTASE (COMPLEX III) IN LEISHMANIA INFANTUM PROMASTIGOTES SARAIVA CORRÊA, D.1; LÓPEZ-SÁNCHEZ, A.1; ABENGOZAR, M.A.1; TEMPONE, A.G.2; RIVAS, L.1 1.CENTRO DE INVESTIGACIONES BIOLÓGICAS (CSIC), MADRID, SPAIN; 2.DEPARTMENT OF PARASITOLOGY, INSTITUTO ADOLFO LUTZ, SAO PAULO, SP, BRAZIL.
Keyword:buparvaquone ; mitochondria ; mechanism of action
Abstract: Buparvaquone (BPQ), a second-generation hydroxynaphthoquinone, is a standard drug for treatment against theileriosis. BPQ is in current evaluation for its implementation as a new drug against Leishmania; in in vitro studies EC50s for BPQ range between 0.03 and 0.8 µM, whereas controversy underlies results in animal models, in canine leishmaniasis BPQ caused only a transitory reduction in parasite burden (Vexenat,1998, Vet. Parasitol, 77,71) but new data on its administration as liposomal formulations showed a significant efficacy in hamsters infected with L. chagasi (Reimao, 2007, Exp Parasitol , 130:195). Furthermore, preliminary data pointed towards the bioenergetic metabolism as a target for buparvaquone, granting a further study on its mechanism of action (Luque-Ortega, 2001 Antimicrob. Agent Chemother. 45:1121). BPQ caused a significant drop in the intracellular level of ATP in Leishmania, associated to inhibition of the respiratory rate of the parasite and mitochondrial depolarization, as assessed by Rhodamine 123 accumulation. In a step ahead using polarographic methods in digitonin-permeabilized parasites, inhibition of the respiratory chain at the level of cytochrome c reductase (Complex III) was mapped. Additionally, BPQ induced mitochondrial swelling as observed in electron microscopy on treated parasites. Altogether, BPQ resulted as a new promising scaffold for development of new leishmanicidal drugs targeting mitochondrial ATP synthesis, an essential process for the parasite. Projects: EU (HEALTH-2007-223414), ISCIII-FEDER RD 06/0021/0006, RD12/0018/0007 and ISCIII PI09-01928, PI12-02706, D.S.C is an Airbus Military-Força aerea brasileira fellow
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [638] P 465 - THE ROLE OF NITRIC OXIDE SYNTHASE AND ARGINASE CROSSREGULATION IN L. AMAZONENSIS INFECTIVITY SALA, T.N.; DA SILVA, M.F.L.; MUXEL, S.M.; FLOETER-WINTER, L.M. ISTITUTO DE BIOCIÊNCIAS USP, SÃO PAULO, SP, BRAZIL.
Keyword:arginine metabolism; oxidative burst; arginine metabolism
Abstract: L-arginine is the substrate for two important pathways in macrophages: polyamines metabolism by arginase II (ARG II) and defense mechanisms by inducible nitric oxide synthase (iNOS). These two enzymes are cross-regulated and the induction of ARG II down regulates iNOS decreasing nitric oxide (NO) production and the burst oxidative response to pathogens. On the other hand, Leishmania also express arginase (ARG), which is essential for parasite infectivity and proliferation. By constructing an ARG knockout mutant (arg-) and two genetically complemented mutants with the native ARG ORF (arg-/ARG) or the ORF lacking the SKL glycosomal addressing signal (arg-/arg SKL), we previously showed that ARG glycosomal compartmentalization is crucial for parasite infectivity. Besides, recent studies showed that Leishmania produces NO, by an unknown mechanism, shown to correlate with metacyclic differentiation and infectivity. Altogether NO synthesis and ARG participate in a complex equilibrium between parasite/host enzymes that define L-arginine fate. To evaluate parasite ARG role on NO synthesis in L. amazonensis, we quantified the NO positive (NO+) cells in arg- and wild type (WT) strains after incubation with DAF-FM diacetate (4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate), a reagent that fluoresces in contact with NO. The quantification was performed in promastigotes along the growth curve and the fluorescence was quantified by flow cytometry. The arg- mutant population presented 68% of NO+ cells on the third day of culture, against 25% of WT. This difference is diminished in the subsequent days of culture reaching the stationary phase with almost the same amount of NO+ cells. So, the arg- mutant cells presented an earlier production of NO than the WT. Considering that the arg- is less infective, we hypothesized that ARG expression regulates NO production allowing the metacyclic differentiation and parasite infectivity. Furthermore we will use the arg-/ARG and arg-/arg SKL strains to evaluate the role of ARG glycosomal compartmentalization on NO synthesis modulation. Besides we will analyze NO production in amastigotes, metacyclics and infected macrophages. The better understanding of the mechanisms by which Leishmania synthesizes NO and controls arginine availability/fate are important not only to study the parasite physiology and infectivity, but may also open doors for the development of new chemotherapies. Financial Support: FAPESP and CNPq
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [640] P 466 - POTENTIAL OF SECONDARY METABOLITES FROM TRADITIONAL MEDICINAL PLANTS AS LEADS AGAINST VISCERAL LEISHMANIASIS AFRIN, F.1; ISLAMUDDIN, M.1; CHOUHAN, G.1; SAHAL, D.2 1.JAMIA HAMDARD (HAMDARD UNIVERSITY), NEW DELHI, INDIA; 2.INTERNATIONAL CENTRE FOR GENETIC ENGINEERING AND BIOTECHNOLOGY, NEW DELHI, INDIA.
Keyword:leishmania donovani; apoptosis; secondary metabolites
Abstract: Leishmaniasis is a profound global health problem, ranging in severity from self-healing cutaneous lesions to mucocutaneous and fatal visceral manifestations. The limited and antiquated chemotherapeutics available against visceral leishmaniasis (VL) are deplorable due to systemic toxicity and emerging drug resistance. Absence of vaccines, progressive emergence of HIV-VL co-infection and relapse after treatment in the form of post kala-azar dermal leishmaniasis delineate the gravity of visceral affliction. Thus, there is a pressing necessity for more efficacious antileishmanial agents which is constrained by limited diversity of chemically synthesized compound libraries. Since phytotherapy for the treatment of parasitic diseases is well documented, therefore in the quest for new antileishmanial drugs, we have investigated the potential of some traditional medicinal plants such as Artemisia annua, Curcuma longa and Piper nigrum. The antileishmanial efficacy of A. annua (leaves and seeds), C. longa (rhizome) and P. nigrum (fruits) was ascertained against Leishmania donovani. All the extracts exhibited significant antileishmanial activity with IC50 (concentration that inhibits 50% viability) ranging from 1438 g/ml and 5-15 g/ml against L. donovani promastigotes and amastigotes, respectively. The extracts induced morphological alterations including cell shrinkage, aflagellation and ovoid promastigotes that could not revert in growth reversibility analysis, indicating the cytocidal effect. The bioactive extracts promoted leishmanicidal activity by triggering programmed cell death mediated by blebbing of nuclei, dyskinetoplastidy, loss of plasma membrane integrity as evidenced by exposure of phosphatidylserine, mitochondrial membrane depolarization and reactive oxygen species generation culminating in cell-cycle arrest and DNA nicking reflected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. No adverse toxicity of bioactive extracts on mammalian cells was observed up to a concentration of 200µg/ml. Gas Chromatography-Mass Spectrometry characterized the presence of secondary metabolites in the bioactive extracts. The antileishmanial potential of the extracts in vivo in BALB/c mice is under progress. Taken together, our data conclusively suggest that the secondary metabolites from plants exhibit promising antileishmanial activity that is mediated by apoptosis and may provide new leads in the therapeutic control of leishmaniasis.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [642] P 467 - GAMMA-PEPTIDES AS A NEW VERSATILE NANOTECHNOLOGICAL SCAFFOLD. TIPPING THE BALANCE BETWEEN CELL PENETRATING AND ANTIMICROBIAL ACTIVITIES FOR LEISHMANIA LÓPEZ-SÁNCHEZ, A.1; CARBAJO, D.2; PULIDO, X.2; ALBERICIO, F.3; ROYO, M.2; RIVAS, L.1 1.CENTRO DE INVESTIGACIONES BIOLÓGICAS (CSIC), MADRID, SPAIN; 2.COMBINATORIAL CHEMISTRY UNIT, BARCELONA SCIENCE PARK & CIBER-BBN, BARCELONA, SPAIN; 3.INSTITUTE FOR RESEARCH IN BIOMEDICINE, BARCELONA SCIENCE PARK, BARCELONA, SPAIN.
Keyword:cell-penetrating peptide; gamma-peptide; drug conjugate
Abstract: Cell penetrating peptides act as intracellular delivery vehicle for a wide variety of membraneimpermeable payload molecules, bypassing the requirement for a dedicated transporter/translocator. The two major CPP pathways to gain access into the intracellular milieu of the parasite, endocytosis and direct translocation across the plasma membrane, are challenging in Leishmania, due to their special characteristics with respect to the mammalian cells. The feasibility of CPP as nanotechnological tool for Leishmania was previously tested using Tat as a standard mammalian CPP; nevertheless, development of new CPPs capable to
face protease degradation, both by the host or by the parasite, is needed to envisage a future clinical application. As a first step, we tested a library of 52 -peptides with a 5-cis-amino-proline scaffold. Diversity among their components were achieved by variation of the length of the peptide, nature of the side chain and class of linkage into the 5-amino group of the scaffold. Three of them superseded Tat accumulation by more than 6 fold, assessed by cytofluorometry and confocal microscopy using N-terminal fluoresceinated surrogates; all of them are homo hexamers and share a recurring presence of N cationic substitution, due to an N -quaternary amine or of an -guanidinium group at the side chain. These -peptides enter the parasites by endocytosis, with co-localization with rhodaminated dextran and uptake inhibited at low temperature. Based on these results, a second generation of -peptides selected was tested for activity modulation with peptide length; peptides shorter than hexamer showed nil entrance in the parasite, whereas those longer than octamer evidenced steeply growing Leishmania toxicity with the number of residues. Accordingly, we can finely modulate cell penetrating and leishmanicidal activities with this descriptor. Altogether, cis- -amino-proline peptides are a versatile and proteinase-resistant scaffold, both as new antileishmanial leads or as transport vehicles, with enhanced uptake over CPPs previously tested on Leishmania, thus as a useful vehicle to test the effects of payload molecules on the physiology of the parasite. Supported by projects: EU (HEALTH-2007-223414), ISCIII-FEDER RD 06/0021/0006, RD12/0018/0007 and ISCIII PI09-01928, PI12-02706 (LR); MICINN CTQ2008-00177, MINECO SAF2011-30508-C02-01 and Generalitat de Catalunya (2009SGR 1024).
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [650] P 468 - AGREEMENT BETWEEN INDIRECT IMMUNOFLUORESCENCE TEST (IFAT) AND ENZYME LINKED IMMUNOSORBENT ASSAYS (ELISA) FOR THE DIAGNOSTIC OF CANINE VISCERAL LEISHMANIASIS. NEGREIROS, A.S.1; MACEDO, E.C.1; WERNECK, G.L.2; SILVA, K.M.1; AZEVEDO JUNIOR, J.W.C.1; SILVA, K.R.3; CRUZ, M.D.S.P.1
1.UNIVERSIDADE FEDERAL DO PIAUÍ, TERESINA, PI, BRAZIL; 2.UNIVERSIDADE DO ESTADO DO RIO DE JANEIRO, RIO DE JANEIRO, RJ, BRAZIL; 3.CENTRO DE PESQUISA GONÇALO MONIZ – FIOCRUZ, SALVADOR, BA, BRAZIL.
Keyword:leishmaniasis; canine; diagnosis
Abstract: Canine visceral leishmaniasis (CVL) is caused by protozoan parasites of the Leishmania genus and domestic dogs are considered the main reservoir in urban settings. The aim of this work was to evaluate the agreement between serological methods for CVL diagnosis. Peripheral blood samples of 810 domestic dogs from 10 districts of Teresina were collected and serological tests for CVL performed by means of the indirect immunofluorescence test (IFAT) and two different enzyme linked immunosorbent assays (ELISA), one using the EIE-LVC Bio-Manguinhos® kit and the other using an ELISA in house, developed by FIOCRUZ – BA. Agreement between tests was evaluated by means of the kappa coefficient (k), stratified by the following variables:breed, sex and age of the animals, literacy of household head, administrative area of the city and transmission intensity. Higher number of seropositive samples were detected by
IFAT with cutoff 1:40 (IFAT40) (60.12%), followed by ELISA FIOCRUZ-BA (45.48%), IFAT with cutoff 1:80 (IFAT80) (39.01%) and ELISA BIOMANGUINHOS (12.73%). The results demonstrated a weak agreement between IFAT 40 and ELISA BIOMANGUINHOS, independent of the variables analyzed. IFAT 40 and ELISA FIOCRUZ-BA showed a regular to moderate agreement. IFAT80 AND ELISA BIOMANGUINHOS presented a weak to regular agreement and between IFAT80 and ELISA FIOCRUZ-BA showed a moderate agreement. The agreement between two kinds of ELISA used in this study was weak to regular. None of variables analyzed showed any influence on the concordance between tests. The agreement between the tests evaluated was too low and this problem indicates how difficult is to verify the real situation of CVL in Teresina. The tests IFAT80 and ELISA FIOCRUZ, which presented the better agreement, could be used as diagnostic tools in endemic area. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [651] P 469 - THERAPEUTIC RESPONSE OF ARTEMISIA ANNUA TEA IN THE TREATMENT OF CUTANEOUS LEISHMANIASIS: STUDIES IN VIVO ROBLEDO, S.M.1; LUTGEN, P.2; RESTREPO, A.M.1; MUﾃ前Z, D.L.1; UPEGUI, Y.A.1; VELEZ, I.D.1 1.PECET, UNIVERSITY OF ANTIOQUIA, MEDELLIN, COLOMBIA; 2.BELHERB, LUXEMBOURG, LUXEMBOURG.
Keyword:artemisia annua; cutaneous leishmaniasis; therapeutical response
Abstract: Cutaneous leishmaniasis (CL) is endemic in the tropics and neotropics. Most available drugs are costly, require long treatment regimens and are becoming increasingly ineffective. Therefore, discovery of new drugs is needed. Recently, the hamster model for CL was optimized by inoculation in the dorsal skin. In order to evaluate the therapeutic response of Artemisia annua tea in the treatment of CL, the hamster model of experimental infection in the dorsal skin was used. Previous anesthesia, young adult male and female golden hamsters were injected intradermally at the dorsal skin with 1 to 5 x l07 promastigotes of L. amazonensis. Animals were treated after typical skin lesions had developed with different doses of lyophilized aqueous extract of Artemisia annua (grass ground) cultivated in Luxembourg, containing only 0.1% of artemisinin. Compound was administered orally daily during 20 days and the effectiveness of the treatment was assessed comparing the lesion sizes prior to and after treatment. Complete cure began observed after one month of the end of treatment. After 3 months of the end of treatment, the therapeutic response was 100% in the group of hamsters treated with the highest dose. These results suggest that tea from A. annua is highly effective against Leishmania parasites and could be a promising candidate for oral treatment for CL. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [652] P 470 - NUCLEOSIDE HYDROLASE FROM L.DONOVANI: SUBSTRATE AND IMMUCILLIN INHIBITOR SPECIFICITY AND THE INNHIBITION OF IN VITRO GROWTH OF L. CHAGASI DE FREITAS, E.O.1; SCHRAMM, V.L.2; GUAN, R.2; PALATINK DE SOUSA, C.B.1 1.UNIVERSIDADE FEDERAL DO RIO DE JANEIRO, RIO DE JANEIRO, RJ, BRAZIL; 2.ALBERT EINSTEIN COLLEGE OF MEDICINE, YESHIVA UNIVERSITY, BRONX- NEW YORK, UNITED STATES.
Keyword:nucleoside hydrolases; leishmaniasis; l. chagasi
Abstract: Leishmaniasis, a neglected tropical parasitic disease caused by a protozoan from the genus Leishmania is prevalent in 98 countries spread across three continents and considered one of the major neglected diseases. These parasites are purine auxotrophs and use only host-derived nucleosides and nucleotides as precursors for DNA and RNA synthesis. Chemotherapy is the only current treatment option for leishmaniasis, but the drugs are expensive, not orally active, exhibit severe side effects and cause resistance in the parasite population. The first-line treatment in most affected areas is pentavalent antimonial, while amphotericin B and pentamidine are used as alternative drugs. Research for new drugs is a priority for this disease. Nucleoside hydrolase is involved in the purine salvage pathway and represents an important target for chemotherapy since it is not found in mammals. The L. donovani nucleoside hydrolase accepts a wide range of substrates, with a slight preference for adenosine and inosine. Guanosine is not a good substrate. The catalytic specificities differ substantially from those reported for L. major nucleoside hydrolase. We have analyzed potential nucleoside hydrolase inhibitors, as an approach to block purine salvage. Immucillin-H and Immucillin-A gave Ki values of 19 and 80 nanomolar respectively. Immucillin-H (Forodesine) is currently in human clinical trials against lymphatic cancers. Our results may be useful as leads for anti-leishmania agents. In our in vitro assays an inhibitor of adenosine kinase gave an IC50 value on L. chagasi growth of 5 nM for 72 hr growth, but did not completely inhibit growth at 50 nM. Three others gave IC50 values of 5 µM, 9.6 µM, at 72 hr but did not completely inhibit growth at 250 µM. Another gave an IC50 of 27 µM, and also showed incomplete growth inhibition at 250 µM. The others did not reach an IC50 even at a 250 µM. Animal studies will be required to test in vivo effects. The specific inhibitory effects of the immucilins on the NH of Leishmania donovani are in progress using the recombinant enzyme (NH36). Interestingly, NH36 is also an important antigen for vaccination against leishmaniasis.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [657] P 471 - POTENTIATING ANTIPARASITARY EFFECTS OF PAROMOMYCIN THROUGH ENCAPSULATION IN LIPOSOMES GASPAR, M.M.1; PEREIRA, J.1; GONÇALVES, L.M.1; CASTRO, H.2; TOMÁS, A.M.2; CRUZ, M.E.M.1 1.FACULDADE DE FARMÁCIA, LISBOA, PORTUGAL; 2.INSTITUTO DE BIOLOGIA MOLECULAR E CELULAR, PORTO, PORTUGAL.
Keyword:liposomes; paromomycin ; visceral leishmaniasis
Abstract: Leishmaniasis is a parasitic disease affecting tropical and subtropical areas of the world. In particular its visceral form, the most severe clinical manifestation, is responsible for more than 50,000 deaths each year. Problems associated with currently available drugs are: inadequate delivery, reduced therapeutic efficacy, excessive toxicity and steady loss of effectiveness due to increased resistances. To overcome some of these limitations synthesis of new molecules and/or formulation in delivery systems, namely liposomes, constitute modern strategies of this Millenium. In this line, an old molecule, paromomycin (PRM) has been formulated in liposomes with the purpose to potentiate its antiparasitic performance. Despite the proved antileishmanial activity of PRM its therapeutic efficacy is far from ideal as controversial and irregular results have been observed in several clinical trials. PRM is poorly absorbed into systemic circulation after oral administration, rapidly absorbed from intramuscular sites of injection and highly eliminated by glomerular filtration. The developed PRM liposomal formulations presented here were able to dramatically change the biodistribution profile of this antibiotic. PRM liposomes promoted drug accumulations 3 to 12 fold higher in the liver and 60 to 140 fold higher in the spleen in comparison with the respective free form. The biological activity in a murine model of visceral leishmaniasis was in accordance with the in vivo profile of PRM formulations. For mice treated with PRM liposomes more than 99% reduction of parasites in liver and spleen was observed versus around 80% for mice treated with free PRM. The host immune response to parasite burden was assessed by analyzing the response of spleen cells stimulated with Leishmania crude antigen and by the quantification of cytokines markers of Th1/Th2. The cytokine expression showed a variation during the course of the experiment associated with parasite burden. Gamma interferon showed high mean values in all infected animal groups and remained above those for Interleukin 4 suggesting a predominance of Th1 subpopulation over that of Th2 what is consistent with a polarized Th1-like response. The excellent therapeutic performance of PRM liposomes suggests this formulation to be an attractive candidate for treatment of leishmaniasis in mono or combined therapies. Work was financially supported by PEst-OE/SAU/UI4013/2011; PTDC/CVT/098290/2008.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [661] P 472 - EVALUATION OF PCR TECHNIQUE INVOLVING DIFFERENT TISSUES OF SEROPOSITIVE DOGS MARTINS, T.F.C.1; ROCHA, M.1; YAMASHIRO-KANASHIRO, E.1; GALLO, L.2; EVANGELISTA, M.G.3; LINDOSO, J.A.4; COTRIM, P.1 1.INSTITUTO DE MEDICINA TROPICAL, SAO PAULO, SP, BRAZIL; 2.CENTRO DE CONTROLE DE ZOONOSES, EMBU DAS ARTES, SP, BRAZIL; 3.SERVIÇO DE VIGILANCIA EPIDEMIOLOGICA, EMBU DAS ARTES, SP, BRAZIL; 4.INSTITUTO DE INFECTOLOGIA EMILIO RIBAS, SAO PAULO, SP, BRAZIL.
Keyword:canine visceral leishmaniasis; kdna-pcr; different tissues
Abstract: PCR technique are being described as a rapid and reliable diagnosis technique for leishmaniasis, capable to suggest the specie implicated in the infection. We evaluated the efficacy of kDNAPCR reaction, analyzing different tissues samples from dogs with Canine Visceral Leishmaniasis (CVL) identified and sacrificed in an seroepidemiological survey conducted in Embu das Artes, SP. Data associated with this survey also demonstrated the absence of both, human infection, as well the presence of the classic vector of human disease (Lutzomyia longipalpis), suggesting that a different pattern of transmission may be occuring for CVL in this city. In this work, we investigate some aspects involved in the transmission and epidemiology of CVL in Embu das Artes, SP, and comparing them with the results obtained from PCR, clinical evaluation, and classical diagnostic tests, as: direct parasitology tests and in vitro culture of the isolated parasites. A specific protocol for collection and storage of the samples was carried out with the dogs euthanized. Thus, tissues samples from: spleen, lymphnode, skin with and without lesion, and blood of 26 dogs euthanized after positive serology for leishmaniasis were individually evaluated by the two classical tests, clinical evaluation, and by kDNA-PCR, the latter done in triplicate. From the 26 euthanized dogs, 22 (84.6%) were positive by direct parasitological test, at least in one of the four differents tissues examined. Similar results were observed for in vitro culture, where 21 samples (80.77%) were positive. Among these positive dogs, 2 were asymptomatic by cinical evaluation. As expected, PCR reaction presented higher levels of positivity in most of the tissues analysed when compared with other methodologies. We verified PCR amplification products in 24/26 (92.30%) of samples from skin with lesion and in 23/26 (88.46%) of samples from spleen, indicating that both invasive and non-invasive samples can be used by the PCR technique. PCRRFLP restriction analysis with HaeIII were also performed with amplified products of each infected dog and none of the samples tested were digested, indicating a pattern not suggestive of Leishmania (V.) braziliensis, which is in agreement with a visceral infection which is usually caused in our country by L. (L.) chagasi. Supported by FAPESP, CNPq, FMUSP-LIM48.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [663] P 473 - TAMOXIFEN INHIBITS THE TRANSPORT OF MYO-INOSITOL IN LEISHMANIA (L.) AMAZONENSIS TRONCO, C.T.; SILBER, A.M.; ULIANA, S.R.B.
DEPARTAMENTO DE PARASITOLOGIA, INSTITUTO DE CIÊNCIAS BIOMÉDICAS, UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL.
Keyword:myo-inositol transporter; leishmania amazonensis; tamoxifen
Abstract: Current treatments for cutaneous and visceral leishmaniasis often present unpredictable and unsatisfactory responses. Recently, our group demonstrated that tamoxifen (Tam), an antiestrogen used in the treatment of breast cancer, is effective against several species of Leishmania in vitro and in vivo. Previous studies developed in our laboratory indicated that the mechanism of action of Tam may involve changes in the availability of myo-inositol in L. amazonensis. In Leishmania, inositol is used for the synthesis of important lipid molecules, such as the glycosylphosphatidylinositol (GPI) anchors responsible for the attachment of several surface molecules that have been implicated in the virulence of the parasite (for example, gp63,
gp46/PSA2, lipophosphoglicans and glycoinositolphospholipids). In Leishmania spp. myoinositol can be synthesized de novo or acquired via specific transport. A myo-inositol transporter was previously described in L. donovani as an eletrogenic symporter that utilizes a proton gradient to concentrate myo-inositol within the cell (Drew et al., Mol Cell Biol. 1995;15:5508). In the present work, myo-inositol uptake was characterized in L. amazonensis promastigotes. Activity curves as a function of substrate concentration obtained with radiolabeled myo-inositol exhibited Michaelis-Menten type kinetics, as expected for a saturable transport mediated by a protein carrier. The transport system has shown a Km of 195.1 ± 22.40 µM and a Vmax of 18.38 ± 0.61 pmol/min/107 cells. These values are in accordance with the characteristics previously described for the L. donovani myo-inositol transporter. To evaluate the effect of Tam on the characterized transporter, cells were pre-incubated with 10, 30 and 50 µM of Tam and transport assays were performed at concentrations corresponding to the Km of the substrate (200 µM myoinositol) and at the concentration needed to achieve Vmax (2 mM myo-inositol). Under the Km conditions, 10, 30 and 50 µM of Tam inhibited 43, 42 and 68% of the myo-inositol transport and under the Vmax conditions the transport was inhibited by 37, 46 and 48%, respectively. Preliminary results, achieved through several substrate saturation curves in the presence of Tam, indicate that the L. amazonensis myo-inositol carrier is inhibited by this drug through a combination of competitive and non-competitive mechanisms. Supported by FAPESP and CNPq. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [666] P 474 - DRUG RESISTANCE IN LEISHMANIA SPP.: PROTEOMIC ANALYSIS OF THE SENSITIVE AND RESISTANT PHENOTYPE TO GLIBENCLAMIDE, A BLOCKER OF K+ATP CHANNELS MENDOZA-LEÓN, A.1; ORUÉ, A.1; NARVAEZ, M.L.A.1; SOUZA NOGUEIRA, F.2; PERALES, J.3; DOMONT, G.2; HERMOSO, T.1 1.UNIVERSIDAD CENTRAL DE VENEZUELA, CARACAS, VENEZUELA; 2.UNIVERSIDAD FEDERAL RJ, RIO DE JANEIRO, BA, BRAZIL; 3.INSTITUTO OSWALDO CRUZ, RIO DE JANEIRO, BA, BRAZIL.
Keyword:leishmania; glibenclamide; proteomic
Abstract: Chemotherapy currently available for leishmaniasis relies on the administration of antimonial compounds but their toxicity and the emergence and spread of drug resistance emphasize the urgent need for affordable alternative drugs. It has reported that glibenclamide (Gb), an inhibitor of K+ATP channels and P-glycoprotein, affected the viability of Leishmania both in vitro and in vivo (Gbs), but the mechanism involved is unclear; the effect was dose-dependent and the highest dose proved more effective than glucantime. Cross-resistance to glucantime was found in animals infected with a Gb-resistant (Gbr) line, but combined therapy reduced lesion progression even in the Gbr strain. The 50% effective concentration against promastigotes of Leishmania (L.) mexicana (MHOM/VE/90/9012) growing in Schneider’s Drosophila medium was 50 µM, and a Gbr line was selected at 50 µM Gb (Gbr50). Here we carried out a comparative proteomic study to identify differential protein expression between Gbs/Gbr L. (L.) mexicana M9012 Venezuelan strains. Comparison of the whole proteome profiles between the two phenotypes showed qualitative and quantitative differences. Interesting, we observed an apparently increase of protein phosphorylation in the Gbr50 protein profile. We selected 59 spots to identification and analysis by MALDI-TOF-TOF, 32 and 27 from the Gbs and Gb50r respectively. For each phenotype, 19 proteins were identified, many of them involved in different biological functions, such as proteolysis process, DNA replication, glycolisis, transporter, and drug resistance. Identification of transcripts of selected proteins was carried out by RT-PCR assays, suggesting that there is a not difference between both Gbs and Gbr50 phenotypes, on the transcription level of the transcripts for these proteins. The Gb has been described as a classical inhibitor of the K+ATP channels in pancreatic cells whose target is the
SUR receptor, a protein belonging to the ABC transporter family, which has not been identified in Leishmania. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [671] P 475 - MINING THE MINIMAL LEISHMANICIDAL STRUCTURES OF HUMAN RNASES BY AMPA ALGORITHM. COMPARISON WITH THE NATIVE PROTEIN. ABENGÓZAR, M.A.1; FERNANDEZ-REYES, M.1; TORRENT, M.2; SALAZAR, V.A.2; BOIX, E.2; ANDREU, D.3; RIVAS, L.1 1.CENTRO DE INVESTIGACIONES BIOLÓGICAS (CSIC), MADRID, SPAIN; 2.2DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY, UNIVERSITAT AUTÒNOMA DE BARCELONA, BARCELONA, SPAIN; 3.3DEPARTMENT OF EXPERIMENTAL AND HEALTH SCIENCES, POMPEU FABRA UNIVERSITY, BBRP, BARCELONA, SPAIN.
Keyword:rnase; innate immunity; eosinophils
Abstract: The use of antimicrobial peptides (AMPs) as leishmanicidal agents is well established. Some AMPs exist within larger proteins as cryptopeptides whose activity is directly revealed by proteolysis of the protein or indirectly shown by chemical synthesis of the putative AMP region. The AMPA algorithm (Torrent et al., Bioinformatics 2012 28:130) identifies domains with potential AMP activity in proteins, a paradigmatic example are many strongly cationic regions interacting with nucleic acids. Using AMPA, we have searched the eight human RNAsas for potential AMP regions, synthesized the corresponding peptides and tested their leishmanicidal activity. The higher effect was observed for the (8-45) segment of RNAse3, with an EC50=3.4 µM on L. donovani promastigotes. The parent protein, RNAse 3 (also known as eosinophil cationic protein, ECP) is present in the secondary granules of eosinophils and active on a variety of pathogens, including a recently reported leishmanicidal activity (Singh et al., Biol Chem. 2011 392:337). The activities of RNAse 3(8-45) and native RNAse 3 (EC50= 3.2 µM) are indeed similar; however their mechanisms of action differ widely. Thus, for the (8-45) peptide it is mostly based on plasma membrane permeabilization with rapid depolarization, extensive loss of intracellular material, and low accumulation of peptide inside the parasite. In contrast RNAse 3 is much less harmful to the plasma membrane and translocates into the intracellular space, killing Leishmania by its enzymatic activity; the RNAse3 H15A variant, devoid of enzymatic activity, accumulates inside the parasite but is innocuous. Also, RNAse 3 is much less toxic to mammalian cells and can be successfully used to kill intracellular amastigotes, with preferential accumulation into the parasitophorus vacuole. In contrast the peptide is fairly toxic to mammalian cells. These results demonstrate a potential role for eosinophils in Leishmania lesions and identify one eosinophil component as an effector molecule; they also illustrate how the innate immune response carried out by these cells can be fine-tuned through modulation by protein fragmentation. Projects: EU (HEALTH-2007-223414), ISCIII-FEDER RD 06/0021/0006, RD12/0018/0007 and ISCIII PI09-01928, PI12-02706, MICINN BFU2009-09371; MINECO SAF2011-24899
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [672] P 476 - DETECTION OF LEISHMANIA IN CANINE AND EQUINE SKIN BIOPSIES BY IMMUNOHISTOCHEMISTRY USING A HYPERIMMUNE CANINE SERUM AS THE PRIMARY ANTIBODY
GAMA, A.1; MACHADO, N.2; CORDEIRO-DA-SILVA, A.3; RIBEIRO, A.J.4; COTOVIO, M.1; TILLEY, P.5; MONTEIRO, M.6; PIRES, M.A.1; ALVES, A.1; CARDOSO, L.1
1.DEPARTMENT OF VETERINARY SCIENCES AND CECAV, UNIVERSITY OF TRÁS-OS-MONTES E ALTO DOURO (UTAD), VILA REAL, PORTUGAL; 2.ANIMAL AND VETERINARY RESEARCH CENTRE (CECAV), UTAD, VILA REAL, PORTUGAL; 3.PARASITE DISEASE GROUP, IBMC, UNIVERSIDADE DO PORTO, OPORTO, PORTUGAL; 4.VETERINARY TEACHING HOSPITAL, UTAD, VILA REAL, PORTUGAL; 5.FACULTY OF VETERINARY MEDICINE, TECHNICAL UNIVERSITY OF LISBON, LISBON, PORTUGAL; 6.NATIONAL LABORATORY OF VETERINARY RESEARCH, INIAV, LISBON, PORTUGAL.
Keyword:skin biopsies; canine, equine; immunohistochemistry, leishmania infantum
Abstract: The leishmanioses are diseases caused by protozoa of the genus Leishmania, parasites that infect several mammal species, namely dogs, cats, horses and also human beings. Canine leishmaniosis (CanL) is a chronic systemic disease caused by Leishmania infantum (syn. Leishmania chagasi), a zoonotic species both in the Old and New Worlds, which is endemic in several countries around the Mediterranean basin and Portugal. Infections in cats and horses have also been reported in areas where CanL is diagnosed. Skin lesions are among the most common manifestations of leishmaniosis, but detection of Leishmania in conventional histopathological sections with hematoxylin and eosin or Giemsa staining is not always straightforward and non-visualization of the parasite does not exclude its presence. The aim of the present study was to apply an immunohistochemical method alternative to conventional ones for the detection of Leishmania amastigotes in skin biopsies obtained from dogs and horses with suspected/confirmed leishmaniosis. We used a streptavidin–biotin immunohistochemical method with canine hyperimmune serum as the primary antibody, in 47 canine and two equine skin biopsy samples. Histopathological (HE) and immunohistochemical (IHC) methods for detection of Leishmania were assessed. A diffuse or nodular granulomatous dermatitis was a consistent finding in all canine cases examined on routine histopathology, with visible amastigotes forms in 21 (44.7%) cases. Both equine samples were characterized by a nodular granulomatous dermatitis, associated with the presence of numerous amastigotes forms. Following IHC, amastigote forms of Leishmania were easily observed within macrophages in 24 (51.1%) canine skin biopsies and in both equine samples. Parasite load was considered as discrete in five (21.7%), moderate in six (26,1%) and intense in 12 (52,2%) positive canine cases. In equine samples, parasite load was considered as moderate (n = 1) or intense (n = 1). Our results strongly suggest this technique as a useful tool to diagnose CanL as well as to confirm Leishmania infection in equine skin samples, especially in those cases where the parasites are not clearly detectable on the slide and the histopathologic pattern clearly points out for infections with Leishmania. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [674] P 477 - EVALUATION OF AMPHOTERICIN B AND TAMOXIFEN IN COMBINED THERAPEUTIC SCHEMES FOR CUTANEOUS LEISHMANIASIS TRONCO, C.T.; REIMÃO, J.Q.; YOKOYAMA-YASUNAKA, J.K.; MIGUEL, D.C.; ULIANA, S.R.B. DEPARTAMENTO DE PARASITOLOGIA, INSTITUTO DE CIÊNCIAS BIOMÉDICAS, UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL.
Keyword:combination therapy; tamoxifen; leishmania amazonensis
Abstract: Tamoxifen (Tam) is an antiestrogen used in the treatment of breast cancer. Recently, our group demonstrated that this drug is effective against Leishmania in vitro, reduces the parasite burden in Leishmania (L.) amazonensis and Leishmania (V.) braziliensis-infected BALB/c mice and in hamsters infected with Leishmania (L.) infantum. The aim of this study was to assess the effect of the association of Tam and amphotericin B (AmB), a drug regularly used in the clinical practice for cutaneous leishmaniasis. Our assays were performed after infecting the basis of the
tail of BALB/c female mice with 1 million luciferase-expressing L. (L.) amazonensis stationaryphase parasites. The in vivo association assay was performed by combining the 25%, 50% and 100% effective dose (ED25, ED50 and ED100, respectively) of each drug determined from the dose response curves obtained previously. The obtained ED25, ED50 and ED100 of Tam and AmB were 6.5, 13 and 26 mg/kg/day and 1.2, 2 and 4 mg/kg/day, respectively. The combination schemes were chosen to represent lower, intermediate and higher doses of Tam and AmB as follows: 6.5 mg/kg/day Tam associated with 1.2 mg/kg/day AmB; 13 mg/kg/day Tam associated with 2 mg/kg/day AmB, and 26 mg/kg/day Tam associated with 4 mg/kg/day AmB. Treatment efficacy was evaluated through clinical (lesion size and ulcer area) and parasitological parameters (In Vivo Imaging System). The association of Tam and AmB was more effective than the administration of each drug alone. Moreover, the combined scheme at lower doses (ED25 of each drug) was clinically and parasitologically as effective as the treatment with Tam or AmB alone at doses four times higher. The pattern of response was maintained up to 5 weeks after the end of treatment. These results indicate a positive interaction profile between Tam and AmB. These results indicate that combined therapeutic schemes including Tam may be useful new alternatives for the therapy of leishmaniasis. This work was supported by FAPESP and CNPq. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [676] P 478 - HIGH PARASITE LOAD IN ASYMPTOMATIC INDIVIDUALS CONTRIBUTE IN PROGRESSION OF VISCERAL LEISHMANIASIS SINGH, A.K.; SUDARSHAN, M.; CHOURASIA, A.; CHAKRAVARTY, J.; RAI, M.; SUNDAR, S. DEPARTMENT OF MEDICINE, INSTITUTE OF MEDICAL SCIENCES, BANARAS HINDU UNIVERSITY, VARANASI, INDIA.
Keyword:leishmania; asymptomatic; seropositive
Abstract: Background : In an area endemic for visceral leishmaniasis, subclinical or asymptomatic infections play a crucial role in progression of disease. We determined the parasite load by quantitative PCR (qPCR) in healthy infected population living in an area endemic for Visceral leishmaniasis. Methodology: We enrolled 13366 persons from 11 villages of highly endemic region of Bihar. We conducted two sero- survey with 1 year time interval for identification of incident infected healthy individuals. Parasite load using TaqMan based qPCR were done on these sero-converted individuals and its matched control populations. Individuals having parasite load greater than 1 genome/ml of blood was considered as positive by qPCR. Follow-up visit to the homes of each individuals were made to monitor the disease conversion in this cohort. Agreements between seroconversion and qPCR were accessed by kappa value. Results and conclusion: Total 235 persons were converted their serology within 12 month intervals. Of these 235 sero-converter 105 (44.6%) individuals were also positive by qPCR. However, similar number of controls groups (87/ 237, 37%) also showed positivity by qPCR. The agreement between sero-converter and qPCR was moderate. Among all individuals only one were converted into disease that has parasite load 146 parasite genome/ ml of blood. These findings suggest the usefulness of parasite load in heathy individuals living in an endemic area of Bihar and contribute as a good tools for VL elimination programme.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [680]
P 479 - INFECTION WITH LEISHMANIA SP. IN BLOOD DONORS FROM AN ENDEMIC AREA IN MATO GROSSO DO SUL, BRAZIL FRANÇA, A.O.1; JUNIOR, M.S.C.L.2; MATOS, M.F.C.1; LANDGRAF, V.O.3; PEREIRA, L.O.R.4; OLIVEIRA, M.P.4; LIMA, R.B.4; TORRES, M.S.5; DORVAL, M.E.C.1
1.UNIVERSIDADE FEDERAL DE MATO GROSSO DO SUL, CAMPO GRANDE, MS, BRAZIL; 2.INSTITUTO FEDERAL DE MATO GROSSO DO SUL, NOVA ANDRADINA, MS, BRAZIL; 3.LABORATÓRIO CENTRAL DE SAÚDE PÚBLICA, CAMPO GRANDE, MS, BRAZIL; 4.FUNDAÇÃO OSWALDO CRUZ, RIO DE JANEIRO, RJ, BRAZIL; 5.HEMOSUL, CAMPO GRANDE, MS, BRAZIL.
Keyword:leishmania; donors; blood transfusion
Abstract: The risk of Leishmania transmission by blood transfusion has been described for a number of countries, including Brazil, but no recommendations have been made for routine testing to detect this parasite in blood banks. In endemic areas, this mode of transmission can pose considerable challenges, as does, to an even higher degree, the diagnosis of asymptomatic carriers, since no gold standard method is available for detecting this clinical form of parasitosis. The purpose of the present study was to detect cases of asymptomatic infection with Leishmania sp. in blood donors from Campo Grande, Mato Grosso do Sul state, Brazil. After interviews to gather demographic and socioeconomic data, blood samples were collected from 430 donors of the city’s blood bank. The serological survey was performed by indirect immunofluorescence (Instituto Biomanguinhos, Fiocruz, Rio de Janeiro). Serum titers were considered positive at 1:80 or greater. Molecular detection was performed by conventional polymerase chain reaction (PCR) for Leishmania sp. kinetoplast DNA (kDNA). Respondents’ age ranged from 18 to 68 years, with a median of 32. Males predominated (70.2%). Campo Grande was the current place of residence of 96.3%, with most (79.0%) residing in the urban area. The longest donation period was 1 to 5 years (168 donors, or 39.1%). Only 27 donors (6.3%) had undergone blood transfusions. Of 430 samples, 67 (15.6%) were serologically positive. Of these, Leishmania sp. kDNA was detected in 37 (55.2%). Detection of anti-Leishmania antibodies and Leishmania DNA in the samples demonstrates the relevance of this study for mapping and selecting blood donors, given that recipients are often debilitated or immunocompromised. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [688] P 480 - ANALYSIS OF HISTOPATHOLOGICAL CRITERIA RELEVANTS TO UNDERSTAND THE HUMAN CUTANEOUS LEISHMANIASIS PERES, A.C.1; MACHADO, P.2; GUIMARÃENS, L.H.2; CARVALHO, E.M.2; ARRUDA, S.1 1.FIOCRUZ, SALVADOR, BA, BRAZIL; 2.HUPES, SALVADOR, BA, BRAZIL.
Keyword:leishmaniasis; histopathology; correlation
Abstract: The analysis of histopathology of biopsies from ulcers and papules of patients in endemic areas is still important to diagnose and for further analysis to understand the tissue inflammation and its importance as marker to predict the clinical evolution as well as the leishmaniasis pathogenesis. In order to clarify the possible relationships between tissue observations and the different forms of the disease, we correlated the clinical and histopathological data of 113 biopsies by blind selection manner. We used the Pearson’s Chi-squared contingency test to find correlation between clinical and histopathological parameters. The results of the study suggests that there may be positive correlation of degree of necrosis with presence of vasculitis, expansion of inflammation area and presence of amastigotes in tissue section, as well as positive culture suggest that necrosis could promote the survival of the Leishmania in skin dermis. Surprisingly we also found found that high expanded inflammation area combined with presence of fibrosis in the tissue seemed to prevent the apparition of disseminated forms. Although this study indicate possible strategy of investigation, further studies must be necessary
to confirm that these elements are effectively associated to progress in the comprehension of the disease. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [699] P 481 - ANTILEISHMANIAL ACTIVITY OF ESSENCIAL OILS FROM LEAVES OF LANTANA SALZMANNII SCHAM. SANTANA, A.M.1; SANTANA, A.L.1; BLANK, A.F.2; BACCI, L.2; ALMEIDA, R.P.3; SENA FILHO, J.G.4; ALVES, P.B.5; FERNANDES, R.M.6; SCHER, R.1 1.DEPARTAMENTO DE MORFOLOGIA, UNIVERSIDADE FEDERAL DE SERGIPE, SÃO CRISTÓVÃO, SE, BRAZIL; 2.DEPARTAMENTO DE AGRONOMIA, UNIVERSIDADE FEDERAL DE SERGIPE, SÃO CRISTÓVÃO, SE, BRAZIL; 3.DEPARTAMENTO DE MEDICINA,UNIVERSIDADE FEDERAL DE SERGIPE, ARACAJU, SE, BRAZIL; 4.EMBRAPA TABULEIROS COSTEIROS, ARACAJU, SE, BRAZIL; 5.DEPARTAMENTO DE QUÍMICA, UNIVERSIDADE FEDERAL DE SERGIPE, SÃO CRISTÓVÃO, SE, BRAZIL; 6.DEPARTAMENTO DE FISIOLOGIA, UNIVERSIDADE FEDERAL DE SERGIPE, SÃO CRISTÓVÃO, SE, BRAZIL.
Keyword:leishmania chagasi; medicinal plants; leishmaniasis
Abstract: The current chemotherapeutical treatment of leishmaniasis is far from satisfactory. This occurs due to high costs and to potential toxicity shown by drugs in use nowadays and also to the associated drug resistance acquired by parasites. This scenario makes urgent the development of new antileishmanial drugs. Medicinal and aromatic plants are promising sources of natural bioactive compounds. Based on the reported biological activities and medicinal properties of some plants, the present study was aimed at investigating the leishmanicidal potential of essential oils against promastigotes forms of Leishmania chagasi. To do this screening, 1x10 parasites in logarithmic growth phase were exposed to increasing amounts (0.1ng - 100 g) of essential oils from Cymbopogon martini, Cymbopogon nardus, Croton sonderianus, Pogostemon cablin, Azadirachta indica, Pelargonium hybridum, Vetiveria zizanioides, Eucaliptus citriodora, Lantana salzmannii. All evaluated oils have already been characterized chemically by GS/MS. Parasites viability was determined by colorimetric method using resazurin (Sigma). The assay was carried out in 96-well microtiter plates. After 24 hours of exposition to essential oils, rezasurin (0.3 mM final concentration) was added in each well and plates were further incubated at 26°C for 3 hours. Amphotericin B was used as reference drug and cultures of L. chagasi promastigotes growing in Schneider plus DMSO 0.1% (without oils) were included as controls. Among evaluated oils, only that from Lantana salzmannii showed effect on the growth of L. chagasi promastigotes. A mean of 40% of growth inhibition were observed as from the treatment with 100ng of essential oil what persisted until the highest concentration tested (100 g). The main compounds of L. salzmannii oil are (E)-caryophyllene (15.6%) and selin-11-en-4-ol (11.2%). In addition, GC/MS analyses also demonstrated the presence of considered amounts of monoterpenes (12.4%) in its composition. These compounds possess a wide spectrum of pharmacology activities and may be involved in leishmanicidal activity detected. Thus, we can consider the L. salzmannii essential oil as a potential source of novel agents for the treatment of leishmaniasis. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [700] P 482 - STUDY OF THE ANTI-LEISHMANIA (L.) INFANTUM ACTIVITY OF DEFERIPRONE-TYPE IRON CHELATORS CONJUGATED TO 4-AMINOQUINOLINE PINTO, É.G.1; GEHRKE, S.S.2; STEVERDING, D.3; PLEBAN, K.2; TEMPONE, A.G.1; HIDER, R.C.2; WAGNER, G.K.4 1.DEPARTMENT OF PARASITOLOGY, INSTITUTO ADOLFO LUTZ, SÃO PAULO, SP, BRAZIL; 2.INSTITUTE OF PHARMACEUTICAL SCIENCE, SCHOOL OF BIOMEDICAL SCIENCES, KING’S COLLEGE, LONDON, UNITED KINGDOM; 3.BIOMEDICAL RESEARCH CENTRE, NORWICH MEDICAL SCHOOL, UNIVERSITY OF EAST ANGLIA, NORWICH, UNITED KINGDOM; 4.FDEPARTMENT OF CHEMISTRY, SCHOOL OF BIOMEDICAL SCIENCES, KING’S COLLEGE, LONDON, UNITED KINGDOM.
Keyword:leishmania; iron chelation ; drugs
Abstract: Iron has a central role in numerous biochemical processes due to its redox potential and high affinity for oxygen. In protozoa, as well as fungi and bacteria, iron is an essential growth factor, and iron chelation has been suggested as a potential new strategy to combat parasitic infections. Orally active iron chelators of the 3-hydroxypyridin-4-one (HPO) class as deferiprone are effective drugs with a high selectivity for Fe(III). Deferiprone has been considered a potential anti-malarial candidate. In this study, we have developed novel derivatives of deferiprone with increased anti-leishmanial activity (detected by MTT assay) and reduced cytotoxicity against human cell lines (detected by resazurin assay). Of particular interest are several new derivatives in which the HPO scaffold has been conjugated, via a linker, to the 4-aminoquinoline ring system present in chloroquine. In our assays, promastigotes of L. (L.) infantum were susceptible to chloroquine (IC50 3.7 µM) but not to deferiprone (IC50 > 100 µM). The chloroquinedeferiprone conjugate was at least four times (IC50 = 27 µM) more active than its parent deferiprone, which showed any activity in our experiments. This conjugated compound was also tested against the intracellular amastigotes, but showed no activity at the highest concentration of 60 µM. Our data also showed that the octyl (C8H17) and decyl (C10H21) substituted derivatives of deferiprone showed increased activity against promastigotes (IC50 0.7 to 3.2 µM) and the most hydrophobic derivative (C10H21; log P = 4.45), showed the highest selectivity index of 9. The use of the ion chelator deferiprone as scaffold for drug design studies could represent an interesting strategy to discover more selective anti-leishmanial compounds. Supported by FAPESP and King´s College London. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [701] P 483 - ANTILEISHMANIAL ACTIVITY OF AMB CONJUGATED TO AMINE MODIFIED GRAPHENE M, S.L.L.; RAI, M.; SUNDAR, S. DEPARTMENT OF MEDICINE, INSTITUTE OF MEDICAL SCIENCES, BANARAS HINDU UNIVERSITY, VARANASI, INDIA.
Keyword:leishmaniasis; in vivo; in vitro
Abstract: Background: There are no antileishmanial drugs available for leishmaniasis, second after malaria in mortality worldwide. We evaluated the efficacy and toxicity of Amphotericin B (AmB) attached to Amine modified Graphene (G-NH2) novel formulation and compares it with conventional AmB. METHODS: G-NH2 was prepared in a two step chemical carboxylation and amidation process. Further AmB was attached and attachment is confirmed by FTIR and TEM. Cytoxicity of the formulation is assessed in vitro against J774A.1 macrophage cell lines and in vivo in Swiss mice. Antileishmanial activity of the formulation is tested in hamsters in vivo and against J774A.1 macrophage cell lines in vitro. RESULTS: FTIR and TEM showed successful attachment of AmB to G-NH2. In vivo toxicity study in swiss mice revealed no hepatic or renal toxicity. In vitro toxicity against J774A.1 macrophage cell lines showed the order AmB>G-NH2-AmB> G-NH2. In vitro antileishmanial assay showed increased efficacy of G-NH2-AmB over AmB. Percentage Inhibition of amastigote replication In vivo was significantly higher in G-NH2-AmB (87%) compared to AmB (78%) in hamster. CONCLUSION: These results suggest that G-NH2-AmB formulation has significantly greater antileishmanial efficacy that conventional AmB with no significant cytotoxic effects.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [702] P 484 - DIAGNOSIS OF MEDITERRANEAN VISCERAL LEISHMANIASIS BY DETECTION OF ANTI-LEISHMANIA IMMUNOGLOBULIN G ANTIBODIES AND LEISHMANIA ANTIGENS IN SALIVA SAMPLES BOURATBINE, A.1; GALAÏ, Y.1; HABBOUL, Z.2; BEN-ABID, M.1; BEN-SGHAIER, I.1; AOUN, K.1 1.INSTITUT PASTEUR DE TUNIS, TUNIS, TUNISIA; 2.REGIONAL HOPITAL OF KAIROUAN, KAIROUAN, TUNISIA.
Keyword:saliva; visceral leishmaniasis; diagnosis
Abstract: The ability to monitor health status, disease onset and progression, and treatment outcome through noninvasive means is a highly desirable goal in health care promotion and delivery. Whole saliva is a perfect medium to be explored for health and disease surveillance. It offers distinctive advantages as a biological specimen. Samples can be stored at room temperature, are readily collected and shipped, and provide easy alternatives to whole blood collection. Oral fluid sampling facilitates the diagnostic process under field conditions, particularly in specific population groups, such as children. Consequently, it presents an interesting option in the diagnosis of infantile visceral leishmaniasis (VL). Oral fluids from 30 VL cases and 20 healthy controls were collected by ORACOL devices. Oral fluid specimens from both groups were analyzed by rK39 Enzyme Linked Immuno-Sorbent assay (ELISA) for the detection of antiLeishmania antibodies and latex agglutination assay for the detection of Leishmania antigens. The development of a sensitive biotin-streptavidin ELISA allowed the measuring of specific rK39 antibodies. Antibodies detection in the oral fluid had a sensitivity of 94% and a specificity of 100%. KATEX commercial kit was applied on oral-fluid specimens, as recommended for urine. It showed a sensitivity of 82%and a specificity of 95% in VL diagnosis. Oral fluid-based diagnostic tools for VL show much promise. However, more research is needed to choice the best biomarkers in saliva and to develop point-of-care salivary diagnostic technologies. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [716] P 485 - IN VIVO ANTI-LEISHMANIAL ACTIVITY OF GREEN TEA CATECHIN IN AN EXPERIMENTAL MODEL OF CUTANEOUS LEISHMANIASIS BARROSO, P.A.1; MARCO, J.D.1; RAMOS, F.1; MORA, M.C.1; UNCOS, A.1; NOVICOV, M.2; HOYOS, C.1; MASATAKA, K.3; YASHIGUCHI, Y.4; BASOMBRIO, M.Á.5
1.INSTITUTO DE PATOLOGÍA EXPERIMENTAL-CONICET, SALTA, ARGENTINA; 2.CÁTEDRA DE MICROBIOLOGÍA DE LOS ALIMENTOS, SALTA, ARGENTINA; 3.CÁTEDRA DE PARASITOLOGÍA, NANKOKU, JAPAN; 4.CENTRO DE BIOMEDICINA, QUITO, ECUADOR; 5.INSTITUTO DE PATOLOGÍA-CONICET, SALTA, ARGENTINA.
Keyword:leishmania; treatment; experimental
Abstract: Flavonoids are compounds widely distributed among plants with anti-inflammatory, antimicrobial, antithrombotic, anti-allergic, anti-cancer and antioxidant activities. Recently, we have demonstrated the in vitro leishmanicidal activity of epigallocatechin gallate (EGCG) of Camellia sinensis (green tea). The present study addressed the anti-leishmanial activity of EGCG alone or combined with meglumine antimoniate (MA) in a Leishmania (Leishmania) amazonensis-BALB/c model. Mice were infected in the right ear with promastigotes and the treatments were initiated at day 42 post-infection. The animals were given topical and intraperitoneal (I) treatment with EGCG and MA, respectively. Drugs were administered daily during 21 days, either separately or in combination following different protocols; a) control group receiving an hydrophilic cream, b) cream containing 15% of EGCG, c) MA (120 mg Sbv/kg/d) and d) cream with 15% of EGCG combined with MA (I). Treatment efficacy was
assessed by measuring the lesion size weekly and by determining the parasite load with limiting dilution assay. In addition, determinations of serum glutamic-pyruvic transaminase (GPT) and serum glutamic-oxaloacetic transaminase (GOT) as well as the histopathological analysis were carried out. Lesion sizes after infection of BALB/c mice were similar in all the experimental groups till the onset of therapeutic treatments. Significant differences (p < 0.05) were found in the lesion sizes and the parasite burden of mice treated with EGCG, MA as well as in mice treated with EGCG combined with MA compared with the control group at the end of the treatments. Treated mice showed normal levels of GPT and GOT and no histological alterations were observed in the livers. The results of this study demonstrate the in vivo anti-leishmanial activity of EGCG as monotherapy or combined with MA. The efficacy of these therapies against other Leishmania strains should be explored further. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [720] P 486 - HETEROLOGOUS EXPRESSION OF KDDR (KINESIN DEGENERATED DERIVED REPEAT) FROM LEISHMANIA INFANTUM: A NEW RECOMBINANT ANTIGEN FOR THE DIAGNOSIS OF VISCERAL LEISHMANIASIS DHOM-LEMOS, L.; MENDES, T.; REIS-CUNHA, J.; BUENO, L.L.; GAZZINELLI-GUIMARAES, P.; BARTHOLOMEU, D.C.; FUJIWARA, R.T. UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL.
Keyword:visceral leishmaniasis; diagnosis; kinesin
Abstract: A reliable diagnosis of Leishmania infantum infection is crucial for the control of visceral leishmaniasis (VL). High sensitivity and specificy are desirable remarks to avoid unnecessary treatment of false-positive individuals and, more importantly, to avoid the false negative diagnosis. In the current study, we aimed to produce a new recombinant protein for the diagnosis of visceral leishmaniasis, based in the repetitive portion of the kinesine from L. infantum. Cloning of the repetitive sequence of the kinesine was performed using a expression vector (pET-TEV) and primers that allowed amplified a specific portion of the kinesine from L. infantum. Serological diagnosis using sera from infected and control dogs demonstrated that recombinant KDDR displayed higher sensitivity (78%) when compared to standard rK39 (51%) while similar specificity was detected for both proteins (94% and 97%, respectively). In conclusion, the present study showed a good performance of rKDDR for detection of CVL. Further testings are still required to evidence the use of rKDDR in the field for precise detection of CVL determining the specificity to discriminate other parasitic infections and immunized animals with commercial vaccines. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [727] P 487 - GREEK BIODIVERSITY AS AN IMPORTANT SOURCE OF BIOACTIVE MOLECULES TO FIGHT LEISHMANIASES ROCHA, V.1; SMIRLIS, D.2; NONATO, F.R.1; ALIGIANNIS, N.3; ARGYROPOULOU, K.3; KOLOURA, I.3; AMOAH, S.4; HALABALAKI, M.3; SOTERIADOU, K.2; SKALTSOUNIS, A.3; SOARES, M.B.P.1 1.LABORATORY OF TISSUE ENGINEERING AND IMMUNOPHARMACOLOGY, GONÇALO MONIZ RESEARCH CENTER, OSWALDO CRUZ, SALVADOR, BA, BRAZIL; 2.LABORATORY OF MOLECULAR PARASITOLOGY, MICROBIOLOGY DEPARTMENT, HELLENIC PASTEUR INSTITUTE, ATHENS, GREECE; 3.DEPARTMENT OF PHARMACOGNOSY AND NATURAL PRODUCTS CHEMISTRY, FACULTY OF PHARMACY, UNIVERSITY OF ATHE, ATHENS, GREECE; 4.DEPARTMENT OF PHARMACEUTICAL SCIENCES, FEDERAL UNIVERSITY OF SANTA CATARINA, FLORIANOPOLIS, SC, BRAZIL.
Keyword:leishmaniases; treatmente; biodiversity
Abstract: Leishmaniases comprise a broad spectrum of diseases caused by Leishmania parasites. The firstline drugs for the treatment of leishmaniases are the pentavalent antimonials. However this treatment can be complicated by side effects, less sensitivity or resistance of some Leishmania species, variations in pharmacokinetics, drug-host immune response interaction and high cost. Therefore, the development is pivotal of new medicines to treat leishmaniases. The biodiversity is an important source of bioactive molecules which can be exploited for the development of new antileishmanal drugs. The objective of the current work is to evaluate the pharmacological activity of extracts (ethyl acetate and methanol) from Greek plants. Fifty plant extracts derived from 30 plants endemic in Greece were screened at 100 µg/mL for antileishmanial activity, against Leishmania amazonensis and L. chagasi (infantum) promastigotes. The parasite viability was measured by direct counting or with the use of the Alamar blue assay. The IC50 concentration was calculated based on a nonlinear regression. Extracts that displayed potent antiparasitic activity against promastigotes were further evaluated against macrophagescontaining amastigotes at non-toxic concentrations. We found that the constituents of the ethyl acetate extracts possessed antiparasitic activity. Interestingly, plant extracts tested displayed better activity against L. amazonensis rather than L. chagasi promastigotes. The most bioactive extracts clustered into the Lamiacae family. Extracts from Satureja spinosa and Sideritis clandestina presented potent antiparasitic activity against intracellular amastigotes of L. amazonensis (2.9 and 2.3 µg/mL, respectively) compared to promastigotes (40.4 and 31.9 µg/mL, respectively). The cytotoxicity of the abovementioned plants was evaluated using BALB/c mice splenocytes and the viability of cells was determined by the incorporation of 3Hthymidine. These extracts displayed low cytotoxicity (17.7 and 35.7 µg/mL, respectively), suggesting a high selective index. Further studies including the microfractionation of these plant extracts as well as in vitro and in vivo assays will help us to identify new bioactive molecules from natural products useful to the treatment of leishmaniasis. Acknowledgment The authors would like to thank the ChemBioFight project (PIRSES-GA-2010-269301) for the financial support.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [731] P 488 - ANTI-AMASTIGOTE ACTIVITY OF NOVEL ANALOGUES OF LASSBIO-1064 AGAINST LEISHMANIA AMAZONENSIS AND LEISHMANIA BRAZILIENSIS QUEIROZ, A.C.1; ALVES, M.A.2; ARAÚJO, M.V.1; SANTOS, M.S.1; TENÓRIO, A.P.M.1; BARREIRO, E.J.L.2; LIMA, L.M.2; ALEXANDRE-MOREIRA, M.S.1 1.LAFI-UFAL, MACEIÓ, AL, BRAZIL; 2.LASSBIO-UFRJ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:chemotherapy of leishmaniasis; leishmania amazonensis; leishmania braziliensis
Abstract: Leishmaniasis is a public health issue and is among the five parasitic diseases worldwide. The current treatment disease includes limitations of toxicity, variable efficacy, high costs and inconvenient doses and treatment schedules. These observations prompted us to investigate the leishmanicidal activity of novel synthetic derivatives, designed from molecular modification on the prototype LASSBio-1064. Leishmanicidal effect against intramacrophage amastigotes of Leishmania amazonensis (MHOM/BR/77/LTB0016) and Leishmania braziliensis (MHOM/BR/87/BA125) were evaluated at concentration of 100 M – 1 M to calculate LC50 (concentration required to give 50% of death). The cell line J774 viability was determined with MTT assay. Data obtained from experiments were expressed as the mean ± S.E.M. of triplicate
cultures of representative assays. Statistical differences between the treated and the vehicle groups of in vitro experiments were evaluated by ANOVA and Dunnett hoc tests. Differences with a p<0.05 were considered significant. The derivatives LASSBio-1302, 1483, 1485, 1486 and 1488 exhibited high antileishmanial activity against amastigotes of L. amazonensis, with maximum effect greater than 65%. The reference drug pentamidine presented maximum effect against amastigotes of L. amazonensis of 58.1 ± 4.9%. Moreover, LASSBio-1302, 1483 and 1488, beyond of the standards LASSBio-1064 and pentamidine, also significantly diminished the number of intramacrophage amastigotes of L. braziliensis, presenting maximum effect greater than 50%. LASSBio-1302, 1483 and 1488 were as potent as pentamidine to dead amastigotes of L. amazonensis [LC50 of pentamidine was 22.0 µM (14.1 - 29.8)] and L. braziliensis [LC50 of pentamidine was 78.4 µM (58.3 - 98.4)]. However, LASSBio-1486 at concentration of 100 µM showed deleterious activity to the host cell evidenced by MTT assay, with CL50 value of 35.7 (32.5 – 38.8). The derivatives LASSBio-1302, 1483 and 1488 can be considered lead candidates for antileishmanial drugs development because presented pronounced leishmanicidal activity without cytotoxic effect to host cells. Supported by:INCTINOFAR (#573.564/2008-6), CNPq, FAPERJ and FAPEAL. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [738] P 489 - LEISHMANIASIS VISCERAL CANINE IN THE MUNICIPALITY OF ESPÍRITO SANTO DO PINHAL, SÃO PAULO, BRAZIL: AVALIATION OF THE NEW DIAGNOSTIC PROTOCOL RECOMMENDED BY THE MINISTRY OF HEALTH, 2008 – 2012. DE LUCCA, H.R.L.R.1; SILVA, R.M.1; JACOBUCCY, J.F.1; BATISTA, J.A.S.1; BARALDI, S.R.1; NAVARRO, A.L.2; SOUZA, H.3; SILVA, T.O.3; RANGEL, O.4; UCHOA, F.C.5; TANIGUCHI, H.H.6; GUERRA, J.M.6; ELIAS, C.R.6; BARBOSA, J.E.R.6; HIRAMOTO, R.M.6; TOLEZANO, J.E.6 1.INSTITUTO ADOLFO LUTZ, RIO CLARO, SP, BRAZIL; 2.XXVI GVE, SÃO JOÃO DA BOA VISTA, SP, BRAZIL; 3.CENTRO DE CONTROLE DE ZOONOSES, ESPIRITO SANTO DO PINHAL, SP, BRAZIL; 4.SUCEN REGIONAL DE CAMPINAS, CAMPINAS, SP, BRAZIL; 5.SUCEN REGIONAL DE MOGI-GUAÇU, MOGI-GUAÇU, SP, BRAZIL; 6.INSTITUO ADOLFO LUTZ, SÃO PAULO, SP, BRAZIL.
Keyword:leishmaniasis visceral canina; diagnostic; serology
Abstract: Espírito Santo do Pinhal, São Paulo, Brazil, since 2005 is classified epidemiologically as the municipality with canine visceral leishmaniasis transmission, to date no recorded of human cases. Over the past five years three canine serological surveys ( 2008, 2010 and 2012) were carried out according to the recommendations by the Programme Monitoring and Control of American Visceral Leishmaniasis (PVCLVA). In 2008 and 2010 the diagnostic protocol consisted of the use of enzyme linked immunosorbent assay (ELISA) for the screening and identification of seronegative dogs and the indirect immunofluorescence assay (IFA) as a confirmatory test, with a prevalence of 34.78% and 7.06 % respectively. At the end of 2011 from the Technical Note 01/2011 - CGDT-CGLAB/DEVIT/SVS/MS, set up a new scenario for this diagnosis using the immunoassay Biomanguinhos ® DPP (DPP-TR ®) as sorting. For the new algorithm the result “no reagent” in DPP-TR ® correspond to negative diagnosis for LVC and positive diagnosis those with reagent results as in TR-DPP as in ELISA, now defined as the confirmatory test. In a geographic sector identified as the highest prevalence in previous investigations a new canine survey were conduct in 2012, as part of the actions of PVCLVA, using the new diagnostic protocol. From 1908 dogs examined, 10% (183) were reactive in DPPTR ® however, 84.1% (154/183) of these were not reactive by ELISA, diagnoses classified as inconclusive. After one or two months, blood samples were recollected from 71.4% (110/154) of these dogs inconclusive. This second test, 61.8% (68/110) dogs continued positive in DPPTR ® and 12% (13/110) of them were also positive by ELISA. The prevalence in 2012 was 2.25%. Biological samples were collected from 12 symptomatic dogs, household removed to euthanized, 83% (10/12) of them were positive in directed parasitological exam, confirming
infection with Leishmania sp. In 2010, the infection was confirmed by direct examination in 79% (15/19) of dogs euthanized, mostly asymptomatic. Studies are in progress with the aim to identify the meaning of inconclusive results of serological and parasitological exams, as well as to understand the decline in prevalence of canine in that district, without any control measures in the last years. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [739] P 490 - CANINE LEISHMANIOSIS: 16 YEARS OF VETERINARY LABORATORIAL DIAGNOSIS SANTOS, M.F.; MARQUES, C.S.; GOMES, L.; MEIRELES, J.; DE CARVALHO, L.M.; FONSECA, I.P. CENTRO INTERDISCIPLINAR DE INVESTIGAÇÃO EM SANIDADE ANIMAL(CIISA), FACULDADE DE MEDICINA VETERINÁRIA, LISBOA, PORTUGAL.
Keyword:diagnosis; leishmania; dog
Abstract: Canine leishmaniosis (CanL) caused by Leishmania infantum is an endemic zoonotic disease affecting millions of dogs around the world. Dogs are considered the main reservoir of the parasite and play a key role in the life cycle. Epidemiological data is essential for a growing understanding of this disease and its evolution throughout the years. This study aimed to gather all data about dog Leishmania infection diagnosis in the scope of activity of Laboratory of Parasitic Diseases of the Faculdade de Medicina Veterinária of Universidade Técnica de Lisboa, Portugal, to provide information to the scientific community. Field records from a period of 16 years (January 1997 to December 2012) were reviewed and whenever possible, information was collected about dog species, breed, gender and age. Information about biological samples, diagnostic method used, test results and presence of coinfections was also registered. In our laboratory, Indirect Immunofluorescence assay (IFI) with a cut-off of 1:80 has been the gold standard method/assay used since 1998 for the diagnostics of Leishmania infection. A total of 5207 dog samples were tested, including animals of both gender and ages ranging from 2 months to 17 years old. From those, 4945 samples were tested by this serological method (94.97%, N=4945), followed by direct observation of amastigotes in lymph node (10.56%, N= 550) and bone marrow smears (4.51%, N=235). In 10.39% (N=541/5207) of the animals at least two methodologies were done simultaneously. Dog samples were positive in 26.71% (N=1391) of the samples. Antibody titers measured by IFI varied between1:80 (N=227), 1:160 (N=314) and 1:320 (N=704). From the 1391 positive samples, 186 (13.37%) were also tested for other infectious diseases. In Leishmania positive dog samples co-infections detected included: Mycoplasma spp., Dirofilaria immitis, Acanthocheilonema reconditum, Babesia spp., Ehrlichia spp., Demodex canis, Sarcoptes scabiei and Dipylidium caninum. Concerning Leishmania negative samples, 992 were also tested for other parasites and some were positive for: Mycoplasma spp. (15.93%, N=158), Dirofilaria spp. (1.11%, N=11), Babesia spp. (1.01%, N=10) and Rickettsia spp. (0.50%, N=5), among others. These results enhance the importance of a complete screening of canine vector born disease agents, among others parasites, as differential diagnosis in dogs from Leishmania endemic areas and contribute to better knowledge of this zoonotic disease.
Funding: CIISA and Portuguese Foundation for Science and Technology and the European Union (FEDER) through Project PTDC/CVT/118566/2010; PhD Grant SFRH/BD/77886/2011 - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [744] P 491 - IMMUNODIAGNOSIS OF CANINE VISCERAL LEISHMANIASIS USING THE RECOMBINANT PROTEIN HSP90-LIKE SOUZA, D.M.1; GOMES, M.S.2; BUENO, L.L.1; BARTHOLOMEU, D.C.1; FUJIWARA, R.T.1 1.UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL; 2.UNIVERSIDADE FEDERAL DE UBERLANDIA, UBERLANDIA, MG, BRAZIL.
Keyword:diagnosis; visceral leishmaniasis; heat shock protein
Abstract: Establishment of novel serological devices and the validation of alternative methodologies, as well as discovery of new antigens, still represent a broad field for investigation of an accurate differential diagnosis of canine visceral leishmaniasis (CVL) in a population that include animals infected by others canine infection with high prevalence in endemic areas for Leishmaniasis. In this study, we present data on the development of enzyme-linked immunosorbent assay (ELISA)-based techniques for the detection of antibodies against the recombinant HSP90-like protein of L. braziliensis and a comparison of the results with those employing EIE-LVC Kit, the gold standard test for CVL diagnosis in Brazil. Serological assay rHSP90-like showed higher combined sensitivity and total specificity value (Se=94.23% and TSp=96.15%) when compared to EIE-LVC (Se=88.46% and TSp=83.07%). Maximum positive predictive value (PPV) was achieved by rHSP90-like (90.74%) followed by EIE-LVC (67.65%). High values for negative predictive value (NPV) were observed for rPeroxidoxin (97.66%) compared to EIE-LVC (94.74%). The best positive likelihood ratio (LR+) was obtained by rHSP90-like (24.50) and the lowest negative likelihood ratio (LR-) were observed in rHSP90like (0.06). The rHSP90-like presented the high AUC value (0.9861), showing a high accuracy (AC=95.60%). Evaluating the specificity in dogs with infection by other pathogens, we observed lower number of false positives results in ELISA using antigen rHSP90-like for with other canine infections as American Trypanosomiasis, Babesiosis, and Ehrlichiosis, as well as, Leishmania-vaccines, as Leishmune®. In this sense, the rHSP90-like showed the best of the reactions when compared to kit (Biomanguinhos), once showed great ability concomitant to correctly identify infected and uninfected dogs, greater ability to discriminate dogs infected with other relevant species of canine infection also showed greater ability to discriminate between vaccinated and infected animals, an important requirement for a public campaign control of CVL. Finally, the present study demonstrated that rHSP90-like could be among the target molecules which could be used as an immunodiagnostic and potential antigen for development of Leishmania vaccines. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [745] P 492 - LEISHMANICIDAL ACTIVITY OF NAPHTOQUINONES IN ANTIMONYSENSITIVE AND -RESISTANT STRAINS OF LEISHMANIA SSP. LANZA, J.S.1; MAIA, R.G.1; NETO, R.D.M.1; GUIMARÃES, T.T.; PINTO, M.D.C.2; DE MELO, I.M.1; DIOGO, E.B.1; FERREIRA, V.F.3; CAMERA, C.A.4; VALENÇA, W.O.4; DE OLIVEIRA, R.N.4; MELO, M.N.1; FREZARD, F.J.1; JUNIOR, E.S.1
1.UFMG, BELO HORIZONTE, MG, BRAZIL; 2.UFRJ, RIO DE JANEIRO, RJ, BRAZIL; 3.UFF, NITEROI, RJ, BRAZIL; 4.UFRPE, RECIFE, PE, BRAZIL.
Keyword:lapachones; chemotherapy; leishmania
Abstract: Brazil stands out with most cases in the three clinical manifestationsof leishmaniasis. Human treatment is done using antimonial (Sb) compounds, pentamidine, amphotericin B or miltefosine. The actual treatments have several limitations, including high toxicity and emergence of drug resistance by parasites. This context justifies the search for new less toxic molecules, with high effectiveness against both Sb-sensitive and -resistant strains. Efforts are being dispended in order to find in nature, bioactive compounds capable to be used in therapy of parasitic diseases. In medicinal chemistry, some natural naphthoquinones have emerged as promising subject of research due to their structural properties, which can generate reactive oxygen species leading to oxidative stress and subsequently parasite death. We recently reported the synthesis of naphthoquinones coupled 1,2,3-triazoles and nor- -lapachone-based 1,2,3triazoles and their activity against bloodstream trypomastigotes, the infective forms of T. cruzi. Their trypanosomicidal activity with IC50/24h values in the range of 10-359 µM, led us to their further evaluation against Leishmania: Leishmania infantum (syn. L. chagasi) the etiological agent for visceral leishmaniasis and L. amazonensis related to the cutaneous form of the disease in the New World. In the present study, seven 1,4-naphthoquinones coupled 1,2,3-triazoles, five nor- -lapachone-based 1,2,3-triazoles and ten -lapachone-based 1,2,3-triazoles, and other naphthoquinonoid compounds were synthesized and evaluated for their activity against promastigotes forms of Sb-sensitive and -resistant strains of Leishmania infantum (syn. L. chagasi) and L. amazonensis and their toxicity to mammalian cells, continuing our screening program for new anti-parasite compounds. The substances were identified as more potent than the antimonial drug, with IC50 values in the range of 1.0-50.7 µM. Nor- - lapachone derivatives showed the highest antileishmanial activity with selectivity index in the range of 10-15 and emerged as important prototypes for further investigation as antileishmanial agents. Additionally, one of these compounds exhibited cross-resistance in Sb-resistant Leishmania which could provide a molecular tool for investigating the new multidrug-resistance mechanisms in Leishmania parasites. Supported by CNPq, CAPES and FAPEMIG - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [749] P 493 - COMPARISON OF PCR IN URINE AND BONE MARROW WITH BONE MARROW ASPIRATE TO VISCERAL LEISHMANIASIS DIAGNOSIS SOARES, C.R.; SILVA, M.A.L.; OLIVEIRA, A.; ROCHA, A.A.A.; QUARESMA, J.; MEDEIROS, Z.M.; MELO, F.L. ACENTRO DE PESQUISAS AGGEU MAGALHÃES, FUNDAÇÃO OSWALDO CRUZ, RECIFE, PE, BRAZIL.
Keyword:visceral leishmaniais; diagnosis; pcr
Abstract: Visceral leishmaniasis (VL) is a disease caused by parasites of the Leishmania donovani complex. This disease is endemic in several regions of the world and fatal if untreated The methods usually adopted for diagnosing disease have some deficiencies. The gold standard method referring to the direct examination for the diagnosis of disease based on bone marrow aspiration, liver, spleen and lymph nodes. However, this is a very invasive technique, besides requiring trained professionals can present problems for specificity and sensibility of the technique. A promising alternative for the diagnosis of VL is based on molecular test, Polymerase Chain Reaction (PCR). This method has been admitted as sensitive and specific technique and can be used with different biological samples, even with low amount DNA, especially with urine, which has a non-invasive collection. The objective of this study is to compare the PCR in urine and in bone marrow with bone marrow aspirate. The urine samples were collected simultaneously with the bone marrow aspirate and stored at -20 ° C until
posterior process. PCR was performed with primers RV1 and RV2, which amplify a region of the kDNA minicircle (145 bp). It was analyzed by electrophoresis on a 2% agarose gel, stained with ethidium bromide at 0.5 ug / ml and visualized by UV light. At whole, 8 samples were analyzed due to the difficulty of bone marrow collection. Of these, 4 patients were positive by parasitological technique and 4 were negative for parasitological and antibodies techniques. When bone marrow was used, PCR had a sensitivity and specificity of 100%. However, using urine as a biological sample, the PCR has showed low performance, with a sensitivity of 25% and specificity of 50%. This study has evidenced that the PCR in urine was not as sensitive as the PCR in bone marrow for VL diagnosis, whereas the presence of inhibitors such as salts and bilirubin in the urine sample hinders the performance of the PCR. Therefore the extraction method should be further investigated for improvement in the elimination of these inhibitors. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [757] P 494 - VIRTUAL SCREENING TO SELECT THE BEST HIV PROTEASE INHIBITORS AGAINST PROTEASE DOMAIN OF LEISHMANIA DDI1 PROTEIN VALDIVIESO, E.1; MARTIN, V.2; PERTEGUER, M.J.3; DAGGER, F.4; GARATE, T.5; SERRANO, M.L.2 1.LABORATORIO DE BIOLOGÍA CELULAR,INSTITUTO DE BIOLOGÍA EXPERIMENTAL, UNIVERSIDAD CENTRAL DE VENEZUELA, CARACAS, VENEZUELA; 2.UNIDAD DE QUÍMICA MEDICINAL, FACULTAD DE FARMACIA, UNIVERSIDAD CENTRAL DE VENEZUELA, CARACAS, VENEZUELA; 3.SERVICIO DE PARASITOLOGÍA, CENTRO NACIONAL DE MICROBIOLOGÍA, INSTITUTO DE SALUD CARLOS III, MAJADAHONDA, SPAIN; 4.INSTITUTO DE BIOLOGÍA EXPERIMENTAL, UNIVERSIDAD CENTRAL DE VENEZUELA, CARACAS, VENEZUELA; 5.CENTRO NACIONAL DE MICROBIOLOGÍA, INSTITUTO DE SALUD CARLOS III, MAJADAHONDA, SPAIN.
Keyword:protease; hiv; ddi1
Abstract: Experimental evidence suggests that HIV-protease inhibitors (Pls), routinely used in antiretroviral therapy, directly exert antileishmanial effects. Pls may prove to be important chemotherapeutic agents for anti-leishmanial treatment. Recently, the presence in Leishmania of a similar protein to the DNA damaged inducible protein (Ddil), initially described in Sacharomyces cerevisae, was reported. This molecule is a functional protease with a homologous domain to the virus HIV aspartyl-protease (RP). This protein is the only member of the aspartyl-protease family described in this parasite, and might be the target for Pls in Leishmania. Since the global scaling-up of antiretroviral treatments, HIV-1 Pls are becoming increasingly available for visceral leishmaniasis- HIV-endemic regions. The aim of our work was the In Silico studies of the selectivity of some Pls against the Ddil protein of Leishmania major and Leishmania infantum. 3D structures of the parasite Ddil protein RP domain were determined by homology modeling using the S. cerevisiae Ddil RP domain (PDB: 211A) as a template (ID 45%). Molecular dynamics refinement was performed in a 100 Å water sphere using VEGAZZ 2.32 /NAMD 2.8 and CHARM 22. The final model was validated with Procheck (Verify 3D). A Docking evaluation with a library of compounds from the Drug Bank, was performed 20 times for each compound in the active site of Leishmania Ddil RP by the Lamarkian genetic algorithm (GriDock, Auto Dock 4) for the conformational analysis. From 10 compounds evaluated the best antiretroviral, with the greatest parasitic Ddil RP selectivity, were Nelfinavir, Saquinavir and Indanavir. These results are in agreement with the Pls effect observed on the parasites. In contrast the compounds Lopinavir, Darunavir and Atazanavir, currently of common use as first and second line of antiretroviral therapy for positive HIV patients in endemic co- infection zones Leishmania/HIV, were less selective to the parasites protease. Our results indicate that Pls Nelfinavir, Saquinavir and Indinavir are the best option for VIH/VL infection treatments in endemic zones. Furthermore, this study validates the virtual library screening as an economic and fast method for the search of new more selective compounds against the Ddil Leishmania protein.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [758] P 495 - GENE EXPRESSION PROFILING AND MOLECULAR CHARACTERIZATION OF MILTEFOSINE RESISTANCE IN LEISHMANIA DONOVANI TIWARY, P.; KUMAR, D.; RAI, M.; SUNDAR, S. BANARAS HINDU UNIVERSITY, VARANASI, INDIA.
Keyword:leishmani donovani; miltefosine; microarray
Abstract: Drug resistance is a major problem in leishmaniasis chemotherapy. RNA expression profiling using microarrays is a suitable approach to study simultaneous events leading to a drugresistance phenotype. Genomic analysis has been performed primarily with SAG resistant Leishmania species and here we investigate molecular alterations in Miltefosine resistance in L. donovani. We selected clinically Miltefosine resistant population of L. donovani promastigotes. Gene expression of highly resistant mutants was studied using RNA microarrays. RNA expression profiling of Miltefosine -resistant L. donovani revealed the over expression of few genes involved in drug resistance. Genes having more than two fold of over expression was validated by quantitative real-time RT-PCR. Further studies is going on to the identification of resistance molecular markers.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [763] P 496 - PROPOSAL OF SEROLOGICAL ALGORITHM APLICABLE TO ROTINE LABORATORIAL DIAGNOSIS OF HUMAN VISCERAL LEISHMANIASIS NUNES, F.F.C.1; LEMOS, E.M.2; FONSECA, G.S.2; ROCHA, R.R.1; ANDRADE, M.C.1; CARVALHO, S.F.G.3; REIS, A.B.4; DE CARVALHO, A.T.1; FILHO, O.A.M.1
1.CENTRO DE PESQUISAS RENÉ RACHOU, BELO HORIZONTE, MG, BRAZIL; 2.UNIVERSIDADE FEDERAL DO ESPÍRITO SANTO, VITÓRIA, ES, BRAZIL; 3.UNIVERSIDADE ESTADUAL DE MONTES CLAROS, MONTES CLAROS, MG, BRAZIL; 4.UNIVERSIDADE FEDERAL DE OURO PRETO, OURO PRETO, MG, BRAZIL.
Keyword:diagnosis; serological methods; leishmaniose visceral
Abstract: The diagnosis of VL is complex because most clinical features are shared with other commonly occurring febrile hepatosplenic diseases that can be endemic along with VL. There are a number of tests that have been developed and evaluated for diagnosis of VL, including parasitological methods (direct parasite detection- DPD), and conventional serological methods (enzyme linked immunosorbent assay- ELISA and Immuno Fluorescence Assay- IFA). However, a combination of different diagnosis techniques is needed to obtain more precise results and to increase significantly the sensitivity of VL diagnosis. Two categories of unconventional methods have been described as complementary tools in the LV diagnosis, including the recombinant ELISA (rK39) and anti-fixed Leishmania chagasi promastigote IgG1 antibodies (FC-AFPA-IgG1). Considering these findings the aim of this study was created a new analysis algorithm to increase the diagnosis VL sensitivity, especially in the identification of false-negative cases that were not detected by DPD. In this respect two approaches to screening algorithms have been proposed: 1- using IFA, which serological test is available from the Ministério da Saúde, Brazil and 2 - using the rK39, which is a practical and highly specific test. The study population included seventy-seven children with of VL diagnosis confirmed by the clinical improvement
after specific etiologic treatment. Our data demonstrated that all tested methods alone showed a sensitivity higher than that observed on DPD (IFA=74%, ELISA=88%, rK39=79%, FCAFPA=94%, DPD=48%). Furthermore, of the 40 samples (52%) with negative results on direct examination, 30 (75%), 35 (88%), 32 (80%) and 37 (93%) were positive in tests IFA, ELISA, rK39 and FC-AFPA respectively. Of the 20 samples (26%) with negative result at IFA, 17 (85%), 15 (75%) and 16 (80%) were positive in ELISA, rK39 and FC-AFPA respectively. Finally, of the 16 samples (21%) that were negative in rK39, 11 (69%), 13 (81%) and 16 (100%) have conclusive results in tests IFA, ELISA and FC-AFPA respectively. Detailed analysis showed comparable performance of the rK39 and FC-AFPA-IgG1 to include samples with negative DPD and discordant conventional serology in the LV diagnosis, adding complementary sensitivity to the conventional methods. The results presented here demonstrate that use of an algorithm based on serological conventional and unconventional methods contributes to diagnostic inclusion of all samples tested. Supported by: FIOCRUZ/CNPq/FAPEMIG. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [768] P 497 - IN VITRO ANTIPARASITIC ACTIVITIES OF COLOMBIAN PLANT EXTRACTS FROM EUPHORBIACEAE FAMILY AGAINST TRYPANOSOMATIDS ESCOBAR, P.; NEIRA, L.F.; LEAL, S.M.; STASHENKO, E. UNIVERSIDAD INDUSTRIAL DE SANTANDER, BUCARAMANGA, COLOMBIA.
Keyword:essential oil ; euphorbiaceae ; tripanosomatid
Abstract: Euphorbiaceae family is a heterogeneous group of plants distributed in Colombian territory used for medicinal proposes. Studies in essential oils (EO) of the genus Croton have shown activity against Lesihmania. (Leishmania) amazonensis, and from C. macrostachyus against L. donovani and L. aethiopica. Activities against New World L. (Viannia) panamensis and L. (V) braziliensis involved in cutaneous leishmaniasis are scarce. The aim of this work was determine cell activities of plant extracts from Euphorbiaceae family against trypanosomatids. The EO of Croton pedicellatus Kunth (EO1) and C. leptostachyus Kunth (EO2) and the extract of Phyllanthus acuminatus Vahl (Ext1) were assessed against extracellular and intracellular forms of Trypanosoma cruzi, L. (Viannia) panamensis, L. (V.) braziliensis and Vero and THP-1 mammalian cells. The antiparasitic and mammalian cells activities were determined by standard methods (microscopically and MTT colorimetric assay). The results were expressed as the inhibitory (IC50) or cytotoxic (CC50) concentration for 50% of parasites or cells and selective index (SI) calculated divided CC50 by IC50. EO1 and EO2 inhibited T. cruzi, L.(V.) panamensis and L. (V..) brazilienses free life forms (IC50 7.14 to 8.78 µg/mL) and L. (V.) braziliensis intracellular forms (EO1: IC50 36.74, SI: 0.45 and EO2: 19.77µg/mL, SI: 0.47) . The antiparasitic activities of Ext1 were IC50 55.61-91.22 µg/mL against free parasites forms and IC50 57.8 µg/mL, SI: 1.10 against intracellular forms of L. (V.) braziliensis. The EO cell toxicities were CC50 9.29 to 64.12 µg/mL on THP-1 cells and CC50 24.86 to 63.52 µg/mL on Vero cells. Amphothericin B activities on L. (V.)panamensis and L. (V.)braziliensis intracellular amastigotes were IC50 0.03 to 0.05 µg/mL, SI: 314. EO antiparasitic activities were demonstrated in this work; however given the mammalian cell toxicities showed, evaluation of their EO major components are required in order find antiparasite-selectives compounds. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [770] P 498 - USE OF RAPID TEST IN THE DIAGNOSIS OF COINFECTION HIV / VISCERAL LEISHMANIASIS IN PERNAMBUCO
BARBOSA JR, W.L.1; ANDRADE, L.D.1; SANTOS, A.M.A.1; FILHO, D.M.2; SILVA, E.D.1; MEDEIROS, Z.M.1
1.CENTRO DE PESQUISAS AGGEU MAGALHÃES, FUNDAÇÃO OSWALDO CRUZ, RECIFE, PE, BRAZIL; 2.FACULDADE DE CIÊNCIAS MÉDICAS, UNIVERSIDADE DE PERNAMBUCO, RECIFE, PE, BRAZIL.
Keyword:visceral leishmaniasis; lv/hiv-aids coinfection; rapid tests
Abstract: Visceral leishmaniasis associated with HIV/Aids coinfection (LV/HIV-Aids coinfection) can be characterized as an aggressive disease, which complicates accurate diagnosis. This study performs a comparison between the results of the rapid test detection of anti-Leishmania antibodies in the sera of patients (rK39) and the detection of parasite antigens in urine (KAtex) obtained from patients with suspected of coinfection, admitted to referral hospitals for the HIV/Aids in the State of Pernambuco, Brazil. According to the criteria of this study, 24 patients were selected with signing a consent form, sampling the blood and urine, in addition to information regarding Leishmania research in bone marrow aspirate (parasitological test). The coinfection LV/HIV-Aids diagnosis is based on the positive parasitological test. In the present study, this test defined 6 patients as positive, five of which were confirmed for rK39 and two with KAtex. Parasitological tests have not identified coinfection in 18 patients, two of which were positive by rK39 and four positive by KAtex. The use of a single test as a tool for diagnosis is usually not advisable to define cases of coinfection. The continuity of this study, with a larger number of cases, it is necessary for the rapid diagnosis using a small amount of biological material, making the case definition fast and low cost. This has encouraged the adoption of rapid tests in laboratories and field, being needed to be incorporated in the health services, which receive cases of LV / HIV-Aids coinfections. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [778] P 499 - OCULAR AND PERIOCULAR ALTERATIONS IN NATURALLY INFECTED DOGS BY LEISHMANIA INFANTUM FREITAS, J.C.1; BOUTY, L.F.1; LIMA, A.L.1; SANTOS, G.J.1; CAPITANI, C.O.2; NUNESPINHEIRO, D.C.1 1.UNIVERSIDADE ESTADUAL DO CEARÁ, FORTALEZA, CE, BRAZIL; 2.CENTRO DE CONTROLE DE ZOONOSES, FORTALEZA, CE, BRAZIL.
Keyword:naturally infected dogs; ocular and periocular regions; clinical score
Abstract: Leishmanioses are a complex of chronic diseases with high variability of clinical manifestations, thus hindering the diagnosis. Ocular and periocular alterations have been reported in canine visceral leishmaniasis, however, a detailed ophthalmologic evaluation and description of each clinical manifestation in dogs were rarely described. This study described the ocular alterations and their prevalences in naturally infected dogs by Leishmania infantum. The experimental protocol was approved by the Ethics Committee for Animal Use of the State University of Ceará (CEUA/UECE), protocol nº 08622833-1. Ten mongrel dogs, from the Zoonosis Control Center, varying in age and weight, were used. They were selected by immunofluorescence assay (IFA), enzyme linked immunosorbent assay (ELISA), being considered positive with titers 1:40, and by ocular lesions presence. Initially, the animals were subjected to corporal, ocular and periocular region clinical examination. Dogs were then classified into clinical scores, according to the appearance of clinical signs characteristic of visceral leishmaniasis, such as cachexia, onychogryphosis, alopecia, hepatosplenomegaly and skin lesions, ranging from 0 to 5, with 0 being the absence of signals, 1, 2, 3 and 4 symptoms of mild to moderate and 5 as severe symptoms. Ocular lesions were also described in detail and classified into scores, ranging from 0 to 2, being 0 the absence of signs, 1 as light symptoms and the 2 as intense symptoms. After
semiquantitative analysis, the findings were expressed in percentages. 50% of dogs presented body condition score 2, 30% score 3 and 20% score 1.5. In the evaluation of ocular alterations, all animals had clinical manifestations in both eyes, and 20% of dogs had score 2 and 80% score 1. The main changes were observed in the region were keratoconjunctivitis (90% of animals), periocular ulceration with alopecia (80% of animals), uveitis (60% of animals), blepharitis (50% of animals) and edema in 30% of animals. So, from the above, we can conclude that the ocular manifestations may be considered characteristic findings of the canine visceral leishmaniasis, which is very important to assist in differential diagnosis. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [780] P 500 - PREPARATION, CHARACTERIZATION AND IN VITRO CELL TOXICITY OF PLGA NANOPARTICLES LOADED WITH ITRACONAZOLE AS CANDIDATE AGAINST CUTANEOUS LEISHMANIASIS ESCOBAR, P.1; DA SILVA, J.R.2; BISWARO, L.2; DE AZEVEDO, R.B.3
1.UNIVERSIDAD INDUSTRIAL DE SANTANDER, BUCARAMANGA, COLOMBIA; 2.UNIVERSIDAD DE BRASILIA, BRASILIA, DF, BRAZIL; 3..
Keyword:itraconazole ; poly (d,l lactide-co-glycolide) ; nanothechnology
Abstract: Itraconazole (ITZ) is a synthetic triazole drug used to treat various fungal infections and with moderate activities against cutaneous and visceral leishmaniasis. It acts by inhibiting the 14- demethylase through blocking the conversion of lanosterol to ergosterol, which disrupts fungal cell membrane synthesis. The technological advances have developed new systems for carrier drugs as a controlled drug delivery and drug targeting systems, many of those systems are prepared using biodegradable and biocompatible polymers. Poly (D,L Lactide-co-glycolide) (PLGA) nanoparticles constitute a good delivery system for drug distributions because of their biodegradable, biocompatible, bioadhesive properties and low toxicity. The main objective of this study was to develop a polymeric drug delivery system for ITZ intended to be intravenously administered, capable of improving the efficacy of ITZ in cutaneous leishmaniasis treatment. PLGA-ITZ nanoparticles were prepared by an oil-in-water emulsion solvent extraction/evaporation technique. Then characterized by high performance liquid chromatography (HPLC), drug loading, size (dynamic light scattering-DLS), surface charge (Zeta potential–Zeta-sizer) and particle morphology (scanning electron microscopy-SEM). The final concentration of ITZ was 1.25 mg/mL. The mean particle size of ITZ loaded nanoparticles was 236.83 ± 3.97 nm, the size distribution for all samples was unimodal with Polydispersity index ranging between 0.128 to 0.144. All the nanoparticles were spherically shaped with zeta potential of -2.07 ± 0.2 mV. For the in vitro assays, mammalian cell toxicity was determined using the MTT colorimetric assay while antileishmanial activities by direct microscopic analysis. Human THP-1 cells transformed into macrophages were infected with Leishmania (Viannia) braziliensis and L.(V.) panamensis parasites then treated with a serial dilution (1:3) of PLGA-ITZ (0-100µM) . No cell toxicity was induced in THP-1 cells after 72 h of treatment. No antileishmanial activities were observed after 72 h of treatment; moreover, activities after 120 h of treatment are discussed in this work. - GENETICS, EVOLUTION AND TAXONOMY [781] P 501 - VALIDATION OF PCR-RFLP (ITS1) AND NESTED PCR (T2-B4/L1-L4) FOR DETECTION AND IDENTIFICATION OF LEISHMANIA IN ARCHIVED GIEMSASTAINED SLIDES OF CUTANEOUS LEISHMANIASIS PATIENTS IN TUNISIA SAADI, Y.1; YACOUB, A.2; KRAIEM, M.1; FATHALLAH-MILI, A.2; BENSAID, M.2; GUIZANI, I.1
1.LEEP/LMEEP-INSTITUT PASTEUR DE TUNIS, TUNIS, TUNISIA; 2.SERVICE DE PARASITOLOGIE – FACULTÉ DE MÉDECINE/HOPITAL FARHAT HACHED, SOUSSE, TUNISIA.
Keyword:leishmania; diagnosis; pcr
Abstract: Cutaneous leishmaniasis (CL) is highly endemic in many countries over four continents, particularly in Africa and Mediterranean region. In Tunisia, three clinico-epidemiological forms of cutaneous leishmaniasis exist (zoonotic, sporadic and chronic) and are caused by three Leishmania species (L. major, L. infantum and L. tropica). During the last years, changes in the geographical distribution, increased observation of atypical clinical presentations and refractory response of some patients to treatment were observed. Therefore, detection and identification of Leishmania species from patients is central to diagnosis and treatment decision, continuous appraisal of the epidemiological situation, and to control strategies of the disease. Investigation of past CL cases and outbreaks will bring base line information to surveillance and control programs in endemic countries like Tunisia where CL is epidemic. The aim of this research was the validation of two molecular tools: the internal transcribed spacer (ITS1) PCR- RFLP, and nested PCR (T2-B4/L1-L4) followed by sequencing for the detection and identification of Leishmania DNA extracted from archived Giemsa stained slides of CL patients diagnosed in the Hospital Farhat Hached, Sousse, Central Tunisia. The assays were first validated on a selection of previously identified parasites having different geographical and host origins. They were then applied to a selection of positive Giemsa stained slides to identify the parasites. Results obtained with both techniques on isolated promastigotes are congruent with previous parasite identification (100%). Of the 33 DNA extracted from slides and amplified by either technique, 27 were identified as L. major; 4 need further analysis and 2 were negative by both assays. However, nested PCR was more sensitive for detection and identification than PCR-RFLP (ITS1) (66% versus 44%). These results showed that these PCR based assays could detect and identify Leishmania DNA in archived slides and carry potential for laboratory diagnosis of leishmaniasis and for epidemiological investigation of transmission areas across their recent history. The study received financial support from MESRS-Tunisia (LR00SP04 /LR11IPT04) and from AUF-PCSI 2010 program. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [786] P 502 - HISTOPATHOLOGICAL CHANGES IN EYE OF NATURALLY INFECTED DOGS BY LEISHMANIA INFANTUM LIMA, A.L.; BOUTY, L.F.M.; SANTOS, G.J.L.; FREITAS, J.C.C.; MORAIS, G.B.; EVANGELISTA, J.S.A.M.; NUNES-PINHEIRO, D.C.S. UNIVERSIDADE ESTADUAL DO CEARÁ, FORTALEZA, CE, BRAZIL.
Keyword:eye; leishmania infantum; naturaly infected dogs
Abstract: Canine visceral leishmaniasis (CVL) is a disease caused by infection with one of different protozoa of the genus Leishmania that have a cycle with a stage in the insect vector and one in the mammalian host. The transmission of the parasite occurs through the bite of insects of the genus Phlebotomus and Lutzomyia. The CVL can affect many organs and systems causing different clinical signs and the dogs can develop an asymptomatic and symptomatic disease. The immune response can be associated with the clinical signs onset. In the eye, immunocomplexes can be deposited on different structures causing tissue damage. Recent studies have reported the presence of amastigotes in eye structures. This makes us wonder if the cause of the lesions is due to the presence of Leishmania. Because there are few reports in the literature, the objective
of this study was to describe the histological aspects of the eye, eyelids and eye muscles in dogs naturally infected by L. infantum. The experimental protocol was approved by the Ethics Committee for Animal Use of the State University of Ceará (CEUA/UECE), protocol nº 08622833-1. Mongrel dogs (n=10), weight, age and sex varied, provided by the Center for Zoonosis Control of Fortaleza, Ceará, were used. Clinical signs, presence of ocular lesions and serological titer of antibodies measured by ELISA and IFA 1:40 were used for animal inclusion. After euthanasia was performed orbital exenteration. Eyes, lower eyelids and eye superior rectus orbital muscle were separated for histological processing. Slides were stained with hematoxylin and eosin and analyzed by light microscopy. The results were described according to macro and microscopic aspects. Uveitis, corneal opacity and purulent mucus secretion was observed in the eyes. The sclera presented edema, inflammatory infiltrate and infected macrophages in the microscopic analysis. The superior rectus orbital muscle were slightly swollen, it was possible to see a mild inflammatory infiltrate and rare infected macrophages in the perimysium with an optical microscope. The eyelids were swollen up, with hemorrhagic spots. In microscopic evaluation was observed moderate signs of edema, presence of red blood cells, intense inflammatory infiltrate and rare infected macrophages. We conclude that the pathology of ocular signs can be associated with the presence of parasite and the deposit of circulating immune complexes in the structures of the eye causing an inflammatory process. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [787] P 503 - EVALUATION OF DIFFERENT PCR PROTOCOLS FOR THE DETECTION OF LEISHMANIA DNA IN THE DIAGNOSIS OF CANINE LEISHMANIASIS ALBUQUERQUE, A.1; CORTES, S.2; CARDOSO, L.3; CAMPINO, L.4
1.U. PARASITOLOGIA MÉDICA/INSTITUTO HIGIENE MEDICINA TROPICAL(IHMT)-UNIVERSIDADE NOVA LISBOA(UNL), LISBOA, PORTUGAL; 2.U. PARASITOL. MÉDICA/CMDT/IHMT-UNL, LISBOA, PORTUGAL; 3.DCV/ECAV/UNIVERSIDADE TRAS-OS-MONTES, VILA REAL, PORTUGAL; 4.U. PARASITOL. MÉDICA/IHMT-UNL. DEP.CIÊNCIAS BIOMÉD. E MED.UNIVERSIDADE DO ALGARVE, LISBOA, PORTUGAL.
Keyword:pcr protocols; canl; leishmaniasis
Abstract: Canine leishmaniasis (CanL), caused by Leishmania infantum, is a veterinary health problem, in southwestern European countries, namely in Portugal. In addition, dog is considered the main reservoir host of leishmaniasis. Presently, laboratory diagnosis of CanL is mainly performed by direct parasitological and serological methods, which are not effective enough. Therefore, sensitive and specific molecular diagnostic tools are essential to detect Leishmania infection, both in symptomatic and asymptomatic dogs, since the latter group can also be a source of infection to the vectors. The polymerase chain reaction (PCR) is now a simple and powerful molecular tool that has proven to be more specific and sensitive than conventional diagnostic methods and can be performed on any biological sample. In this study we compared two PCR protocols with kinetoplastid primers (MC-PCR) and internal transcribed spacer 1 primers (ITS1-PCR) and a Leishmania nested PCR (LN-PCR) with ribosomal primers . Additionally, NNN (Nicole-Novy-MacNeal) cultures were performed. A total of 200 bone marrow canine samples from symptomatic and asymptomatic dogs from the Alto Douro region, Portugal, were tested. The results showed that the highest rate of positives was detected by LN-PCR with 36%, following MC-PCR with 13% and ITS1-PCR with 9,5%, against the 1,5% detected by NNN culture of the biological samples. The LN-PCR revealed to be the most sensitive protocol and therefore an important tool to detect parasites’ DNA, in endemic and non-endemic areas, even in asymptomatic dogs, allowing the intervention in the early stages of this disease. This work was supported by funding from the research project PTDC/CVT/112371/2009 from Fundação para a Ciência e a Tecnologia (FCT), Ministério da Ciência, Tecnologia e Ensino
Superior, Portugal. S. Cortes holds a fellowship from FCT (SFRH/BPD/44450/2008). LN-PCR primers, were gently provided by Dr. Marina Gramiccia, Istituto Superiore di Sanitá, Rome, Italy. scortes@ihmt.unl.pt - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [789] P 504 - FATTY ACID PROFILES IN LEISHMANIA NATURALLY RESISTANT TO NITRIC OXIDE AND TRIVALENT ANTIMONY DE AZEVEDO, A.F.1; DUTRA, J.L.L.1; LINO, M.T.B.2; SANTOS, D.A.3; DE MOURA, T.R.2; DE ALMEIDA, R.P.4; ALVES, P.B.3; FERNANDES, M.F.5; SCHER, R.2; FERNANDES, R.P.M.1
1.DEPARTAMENTO DE FISIOLOGIA, UNIVERSIDADE FEDERAL DE SERGIPE, SÃO CRISTÓVÃO, SE, BRAZIL; 2.DEPARTAMENTO DE MORFOLOGIA, UNIVERSIDADE FEDERAL DE SERGIPE, SÃO CRISTÓVÃO, SE, BRAZIL; 3.DEPARTAMENTO DE QUÍMICA, UNIVERSIDADE FEDERAL DE SERGIPE, SÃO CRISTÓVÃO, SE, BRAZIL; 4.DEPARTAMENTO DE MEDICINA,UNIVERSIDADE FEDERAL DE SERGIPE, ARACAJU, SE, BRAZIL; 5.EMPRAPA TABULEIROS COSTEIROS, ARACAJU, SE, BRAZIL.
Keyword:lipids; biomarker; leishmaniasis
Abstract: Fatty acids, especially those from phospholipids (PLFA), are essential membrane components, make up a relatively constant proportion of the biological membranes under natural conditions. However, under harmful conditions such as pathophysiology, changes in the environment and chemical exposure, the lipids proportion could undergo specific changes. Once these changes could be identified and revel to be specific for such adverse situation, it could be used as a biomarker. Thereby, clinically useful biomarkers can help, for example, to identify pathogenic organism carrying resistance to particular chemotherapeutic agent. Therefore, a specific biomarker can lead to a better therapeutic decision, regarding candidate drugs and their indications which could improve the effectiveness of treatment. The objective of this study was to compare the fatty acid profiles of L. chagasi and L. amazonesis isolates resistant and sensitive to trivalent antimony and to nitric oxide (NO). Parasites were obtained from the leishmania bank of the Medicine Department, Universidade Federal de Sergipe. Total lipids were extracted from lyophilized promastigotes forms in late logarithmic growth phase (50 mL; 5x108/mL cells). Fatty acid methyl esters (FAMEs) were obtained from total lipids, ester-linked lipids and esterlinked phospholipids by MIDI, ELFA and PLFA techniques respectively. FAMEs were analyzed by chromatography and mass spectrometry. Differences in FAME profiles associated with species or NO and antimony resistance were assessed by non-metric multidimensional scaling, multiresponse permutation procedures and indicator species analyses using PC-ORD 6.0. Previously to these analyses, molar masses of individual FAMEs were relativized by FAMEs totals within each sample. Isolates resistant and sensitive to NO and antimony had different MIDI-FAME profiles, regardless the species. However, neither ELFA nor PLFA profiles differed between sensitive and resistant isolates. The fatty acid 18:1 9c was increased in sensitive isolates (p<0.001), whereas an as-yet-unidentified peak showed the opposite trend (p<0.01). We can conclude that these two fatty acids have potential as biomarkers for NO and antimony resistance in L. chagasi and L. amazonesis, which can help to introduce the right therapy to patients faster than today. Support: Fapitec/SE; Capes DS; CNPq - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [790] P 505 - EVALUATION OF IN VITRO AND IN VIVO ANTILEISHMANIAL ACTIVITY OF CALCIUM CHANNEL BLOCKERS AND LIPOSOMAL NIMODIPINE COLOMBO, F.A.1; REIMAO, J.Q.2; PEREIRA-CHIOCCOLA, V.L.1; TEMPONE, A.G.2 1.LABORATORY OF MOLECULAR BIOLOGY, DEPARTMENT OF PARASITOLOGY, INSTITUTO ADOLFO LUTZ, SAO PAULO, SP, BRAZIL; 2.LABORATORY OF APPLIED TOXINOLOGY ON ANTI-PARASITIC DRUGS, INSTITUTO ADOLFO LUTZ, SAO PAULO, SP, BRAZIL.
Keyword:calcium channel blockers; liposome; qpcr
Abstract: Calcium channel blockers (CCB) are a class of drugs widely used in the therapy of hypertension, cerebrovascular spasms, and other pathogeneses. CCB have been considered promising anti-parasitic candidates, and can partially reverse multidrug resistance, presumably by increasing cellular drug accumulation. Additionally some CCB have been shown to successfully inhibit Leishmania (L.) donovani in vitro infection and in BALB/c mice. Furthermore, nimodipine was in vitro effective against promastigotes and intracellular amastigotes of Leishmania (L.) infantum, resulting in strong ultrastructural damage to the mitochondria and plasma membrane. Considering the need for alternative treatments for leishmaniasis, the present work evaluated the in vitro activity of the CCB amrinone, fendiline, lidoflazine and mibefradil against Leishmania (L.) amazonensis, Leishmania (L.) braziliensis and L. (L.) infantum. The in vitro cytotoxicity in mammalian cells was determined to provide the selectivity index. Finally, a liposomal formulation of nimodipine containing phosphatidylserine (PS) was developed for evaluation of CCB in vivo efficacy, golden hamsters were previously infected with L. (L.) infantum amastigotes (1.8×108/animal). Forty-five days after infection, the hamsters were i.p. treated for ten consecutive days with amlodipine (10 mg/kg/day), fendiline (3 mg/kg/day), nimodipine (40 mg/kg/day), liposomal nimodipine (0.86 mg/kg/day) or Glucantime (50 mg/kg/day) (n = 5/group). The animals were euthanized 57 days post-infection and the parasite burden was evaluated by real time PCR. The in vitro results demonstrated that fendiline and lidoflazine were active against promastigotes, with 50% effective concentrations (EC50) ranging from 2 to 21 µM, and against L. (L.) infantum intracellular amastigotes, with EC50 values of 11 and 15 µM, respectively. The in vivo results revealed lack of activity for amlodipine, fendiline and nimodipine. Liposomal nimodipine caused a significant decrease (97 %) in the parasite burden in the spleen, but failed to treat the liver. These results suggest preferential spleen targeting to the spleen rather than the liver, but the use of higher doses of drugs entrapped in the liposomes might overcome this problem. These results corroborate the efficiency of PS-liposomes in the targeted delivery of drugs to Leishmania, which is primarily due to the interaction with macrophage scavenger receptors. This work was supported by FAPESP. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [792] P 506 - NEUTROPHIL PROPORTION AND OXIDATIVE METABOLISM IN CANINE VISCERAL LEISHMANIASIS SANTOS, G.J.L.; LIMA, A.L.; FREITAS, J.C.C.; NUNES-PINHEIRO, D.C.S. UNIVERSIDADE ESTADUAL DO CEARÁ, FORTALEZA, CE, BRAZIL.
Keyword:naturally infected dogs; oxidative stress; neutrophil
Abstract: Visceral leishmaniasis is an anthropozoonosis chronic, with high public health importance. Shortly after the bite of the insect and the parasite inoculation, neutrophils are recruited to the site of infection, as a protective immune response. This defense mechanism occurs by increased production of reactive oxygen substances, also known as oxidative stress. Therefore, the objective of this study was to evaluate the correlation and the influence of neutrophil proportion on oxidative stress in naturally infected dogs by Leishmania infantum. This study was approved by the ethics committee for animal use of the state university of Ceará (CEUA/UECE), nº 11223318-0/50. Twenty mongrel dogs, varying in age and weight, were used. They were selected by immunofluorescence assay (IFA) and by enzyme linked immunosorbent assay (ELISA), being considered positive with titres 1:40 and the parasitological examination of Leishmania infantum amastigote forms in bone marrow smears. The dogs were grouped in negative dogs (ND = 10) and symptomatic dogs (SD = 10). Blood samples (10mL) were obtained, by jugular puncture, and distributed into tubes containing EDTA, for differential
counting of neutrophils, and activator gel for obtaining serum. Serum samples were stored at 20°C until use. Oxidative stress was evaluated by the thiobarbituric acid reactive substances method (TBARS), which measures the levels of lipid peroxidation. Data were expressed according to a standard curve of malondialdehyde (MDA) (µM x 102 of TBARS) in relation to the neutrophil count (K/ml) of blood samples. To identify the association and the influence of the neutrophil proportion on oxidative stress were used the Spearman correlation test and linear regression, respectively. In all cases p 0.05 was considered significant. Statistical analysis was performed using the software GraphPad Prism 5.0. A positive correlation was observed between the neutrophil proportion and TBARS levels, in both groups. Furthermore, TBARS presented more elevated and suffered greater influence of neutrophil proportion, in SD group. We can conclude that there is a direct dependency between the neutrophil proportion and oxidative stress. Moreover, we observed the importance of neutrophils in the protective response to infection with Leishmania infantum. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [796] P 507 - IN VITRO ACTIVITY OF CRUDE EXTRACT OF MORINDA CITRIFOLIA FRUIT (NONI) AGAINST LEISHMANIA CHAGASI ALMEIDA-SOUZA, F.1; OLIVEIRA, R.M.2; AMARAL, A.C.F.1; TANIWAKI, N.N.3; SOUZA, C.S.F.1; SOUZA, B.V.C.1; CALABRESE, K.S.1; ABREU-SILVA, A.L.2 1.FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 2.UNIVERSIDADE ESTADUAL DO MARANHÃO, SÃO LUIS, MA, BRAZIL; 3.INSTITUTO ADOLF LUTZ, SÃO PAULO, SP, BRAZIL.
Keyword:leishmania chagasi; morinda citrifolia; electron microscopy
Abstract: In the search for new alternatives of leishmaniasis treatment, the discovery of therapeutic properties of active substances present in plant extracts has attracted the interest. Morinda citrifolia, commonly known as Noni, stands out since it has several active substances that have antimicrobial, antiviral and antifungal and potential activity against protozoa such as the members of Leishmania genus. We carried out the phytochemical study to evaluated, in vitro, the cytotoxicity of crude extract of M. citrifolia fruit, using macrophage cells and antileishmanial activity against Leishmania chagasi. The crude extract was obtained from the release of the liquid by the fruits and was carried out the phytochemical screening and the high performance liquid chromatography with diode array detector and light scattering. Antileishmanial activity was tested against promastigotes and intracellular amastigotes. The crude extract showed a yield of 6.31% and the phytochemical screening showed the presence of anthraquinones, flavonoids, alkaloids, terpenoids, saponins, coumarins, phenolic compounds, tannins, anthocyanidins and chalcones. The high performance of liquid chromatography with diode array detector and light scattering identified phenolics and aromatics compounds as the major constituents. Antileishmanial activity was tested against promastigotes and intracellular amastigotes. The extract showed a dose-dependent activity and the IC50 was of 196,9µg/mL for promastigotes and 57,8µg/mL for intracellular amastigotes. The cytotoxicity assay showed that the extract is toxic for the protozoan and not to the cell. Transmission electron microscopy was performed to evaluate the ultrastructural alterations in promastigotes treated, for 24 hours, with 15 to 120µg/mL of crude extract and results showed cytoplasmatic vacuolization, lipid inclusion and increased activity of exocytosis. The crude extract of M. citrifolia fruit is active against L. chagasi in the in vitro model and promising for further researches of new treatments for leishmaniasis. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [797] P 508 - GENOTYPIC ANALYSIS OF LEISHMANIA INFANTUM RESISTANCE TO CONVENTIONAL DRUGS AND NEW CHEMICALLY SYNTHESIZED COMPOUNDS
SANTOS-MATEUS, D.1; ESTEVES, A.2; RODRIGUES, M.A.1; CARVALHEIRO, M.3; LOPES, R.3; CRUZ, E.3; SANTOS-GOMES, G.M.1 1.CMDT, INSTITUTO DE HIGIENE E MEDICINA TROPICAL, UNIVERSIDADE NOVA DE LISBOA, LISBON, PORTUGAL; 2.NATIONAL LABORATORY FOR ENERGY AND GEOLOGY, FUEL CELLS AND HYDROGEN UNIT, LISBON, PORTUGAL; 3.NANOMEDICINE & DRUG DELIVERY SYSTEMS GROUP, IMED.UL, FACULDADE DE FARMÁCIA, UNIVERSIDADE DE LISBOA, LISBON, PORTUGAL.
Keyword:drug resistance; gene amplification; membrane pumps
Abstract: Previous studies demonstrated that drug resistance in Leishmania spp. is caused mainly by changes in membrane permeability allowing a reduction of intracellular drug accumulation or a decreased of drug in-take. This can be not only a consequence of a higher expression of multidrug resistance genes but also the ability of selectively increasing the number of gene copies associated to pumps. The augment of gene copies leads to higher expression and, consequently to accumulation of pumps in the cellular membrane of the parasite. Three proteins seem to be relevant in this multidrug resistance scenario: ABC-thiol transporter (MRPA), pteridine reductase 1 (PTR1) and p-glycoprotein (MDR1). Two other proteins, belonging to the glutathione metabolism and folate biosynthesis pathways, respectively, seem to be important as well: gamma-glutamylcysteine synthetase (GSH1) and dihydrofolate reductase-thymidylate synthase (DHFR-TS).Therefore, this study analyses multidrug resistance gene copies of Leishmania infantum promastigotes that had previous contact with common antileishmanial drugs and new chemically synthesized compounds, assessing the importance of pump associated genes. L. infantum isolates obtained from previously infected BALB/c mice treated with sodium stibogluconate or two Trifluralin derivatives (TLF-A3 or TLF-A7), both free and incorporated in liposomes, were used for DNA extraction. Cultured L. infantum promastigotes resistant to these compounds, as to meglumine antimoniate and miltefosine were used as positive controls. The number of MRPA, PTR1, MDR1, GSH1 and DHFR-TS gene copies were quantified by realtime PCR.Comparing the results of the five analyzed genes it is possible to predict the sensitivity/resistance level of each isolate. This new approach showed that studies focused only in mRNA expression, though relevant, should be completed by the analysis of target genes copy number which plays an important role in Leishmania multidrug resistance. This study was funded by the Portuguese Foundation for Science and Technology and the European Union (FEDER) through projects PTDC/CVT/098290/2008 and PTDC/CVT/113121/2010. Rodrigues A. holds a PhD scholarship (SFRH/BD/73386/2010). - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [798] P 509 - TRIVALENT ANTIMONY ENCAPSULATED IN CONVENTIONAL LIPOSOMES AS AN EFFICIENT DELIVERY SYSTEM FOR TREATMENT OF MURINE VISCERAL LEISHMANIASIS CASTRO, R.A.O.1; LICIO, C.S.A.2; SOUZA, J.B.2; FERREIRA, F.M.3; BATISTA, M.A.4; FRÉZARD, F.J.G.5; BARCELLOS, N.M.S.2; REZENDE, S.A.1
1.LABORATÓRIO DE PESQUISAS CLÍNICAS, PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS FARMACÊUTICAS , UFOP, OURO PRETO, MG, BRAZIL; 2.LABORATÓRIO DE FARMACOLOGIA, PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS FARMACÊUTICAS – CIPHARMA/UFOP, OURO PRETO, MG, BRAZIL; 3.LABORATÓRIO DE PESQUISAS CLÍNICAS, PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS FARMACÊUTICAS, UFOP, OURO PRETO, MG, BRAZIL; 4.LABORATÓRIO DE IMUNOPARASITOLOGIA, PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS – NUPEB/UFOP, OURO PRETO, MG, BRAZIL; 5.DEPARTAMENTO DE FISIOLOGIA E BIOFÍSICA, UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL.
Keyword:visceral leishmaniasis; liposomes; trivalent antimony
Abstract: Visceral leishmaniasis is a chronic debilitating disease endemic in tropical and subtropical areas worldwide. The first therapeutic approach consisted in the parenteral administration of trivalent
antimony (SbIII), which was later abolished due to the high toxicity and side effects of such compound. The employment of a drug delivery system could result in reduced dosage and consequently, diminished toxicity associated to SbIII, enabling its reinclusion as an efficient therapeutic strategy against leishmaniasis. The present investigation evaluated the utilization of conventional liposomes as carriers of SbIII in a murine model of leishmaniasis. For this purpose, liposomes composed of diestearoylphosphatidylcholine and cholesterol (5:3) were obtained according to the freeze / thaw technique and hydrated in a solution of tartar emetic. Empty liposomes, hydrated in phosphate buffer, served as control vehicles. Isogenic BALB/c mice, aged 6 to 8 weeks, were intravenously inoculated with 1 x 107 Leishmania (Leishmania) chagasi promastigotes (C43 strain). Six weeks post infection, the animals were divided into four groups (n=4) and treated intraperitoneally with either (1) phosphate buffer, (2) tartar emetic at 9 mg/kg, (3) empty liposomes or (4) SbIII at 9 mg/kg encapsulated in liposomes. Ten days after treatment, animals were euthanized and the parasite load associated to the liver, spleen and bone marrow evaluated through limiting serial dilution. In order to evaluate any potential toxicity associated to the formulations, creatine phosphokinase, transaminases and creatinine levels were assayed from animal sera. Liposome characterization revealed size, polydispersity index and encapsulation efficiency of 222.5 nm, 0.214 and 15%, respectively, for a formulation containing 4 mg SbIII/mL. After treatment it was observed 58%, 21% and 33% reductions in parasite load in the liver, spleen and bone marrow respectively. Serum biomarkers provided no evidence of cardiac, renal or hepatic impairments. No difference in parasite load was observed after treatment with free SbIII for any of the evaluated organs. The results of the present study attest for the ability of such liposomal system to reach organs containing cells of the mononuclear phagocytic system and innovate with data regarding efficient drug delivery to the bone marrow. Financial support: Fapemig, LGqA/DEGEO, Rede Mineira de Bioterismo. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [802] P 510 - PREPARATION AND CHARACTERIZATION OF A CHLOROALUMINUM PHTHALOCYANINE (CLALPC) NANOEMULSION AND ITS IN VITRO PHOTODYNAMIC ACTIVITY AGAINST NEW WORD LEISHMANIA PARASITES ESCOBAR, P.1; LEAL, S.M.1; JUNIOR, E.R.2; DE AZEVEDO, R.B.3 1.UNIVERSIDAD INDUSTRIAL DE SANTANDER, BUCARAMANGA, COLOMBIA; 2.UNIVERSIDAD FEDERAL DE RIO DE JANEIRO, RIO DE JANEIRO, RJ, BRAZIL; 3.UNIVERSIDAD DE BRASILIA, BRASILIA, DF, BRAZIL.
Keyword:chloroaluminum phthalocyanine ; nanotechnology ; nanoemulsion
Abstract: Chloroaluminum phthalocyanine (ClAlPc) has been used as photosensitizer in photodynamic therapy (PDT) constitutes an important alternative for cutaneous leishmaniasis (CL) treatment. Nanotechnology constitute a useful tool for develop drug delivery systems for transdermic applications to achieve greater drug penetration, biological activity and avoid adverse effects. The aim of this study was to develop a nanoemulsion (NE) for ClAlPc and evaluate its in vitro activity against Leishmania (Viannia) braziliensis and L.(V) panamensis after PDT. NEs (oil/water) with and without-ClAlPc were prepared by ultrasonication process. The aqueous phase containing type I water, glycerol and Tween20 and the oil organic Phospholipid PL90, oleic acid and ClAlPc. The oil phase was added slowly to the aqueous phase, sonicated, rotoevaporated and stored at 4ºC. The formulations were evaluated for the particle size, polydispersity index (dynamic light scattering-DLS), surface charge (Zeta potential-Zeta-sizer), phospholipids (Bartlett acid-digestion procedure) and the final ClAlPc concentration (spectrofluorometry). For the in vitro activities, human THP-1 macrophages (0.0001-10 µM) and Leishmania-intracellular amastigotes (0.004-0.5µM) were treated with serial dilution of compounds for 24 h. The cells were irradiated at a fluency of 5J/cm2 using a photoreactor, lamps (50 W, 120 V), 0.33 mV power (spectral range 597-752 nm). Mammalian cell toxicities were determined by colorimetric MTT colorimetric assay and antileishmanial activities
microscopically by direct cell counting. ClAlPc-NEs were prepared more than 10 times showing average of size values between 55.25 to 212.5 nm, polydispersity index 0.08 to 0.4, zeta potential -2.26 to -22.4mV, ClAlPc concentration 92.52 to 200.13 µM and phospholipids 4700 to 8000 nmol/mL. Physicochemical parameters varied with each preparation, but homogeneity and stability when stored at 4ºC were excellent. For the in vitro test, 0.5 and 0.1 µM CLAlPcNE-PDT inhibited 98 and 82% of L.(V.) braziliensis and 67 and 25% of L.(V.) panamensis intracellular amastigotes. No parasite-inhibition was observed in dark conditions neither after NE-without ClAlPc and PDT. ClAlPc-NE was toxic for THP-1 cells before and after PDT. A ClAlPc-NE was physicochemical stable with in vitro antileishmanial activity, but toxic for THP1 cells. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [803] P 511 - LEISHMANICIDAL ACTIVITY OF PLANT EXTRACTS ON THE INTRACELLULAR MULTIPLICATION OF LEISHMANIA AMAZONENSIS TEIXEIRA, S.C.; DOS SANTOS, P.C.F.; TEIXEIRA, T.L.; DE SOUZA, M.A.; DA SILVA, C.V. UNIVERSIDADE FEDERAL DE UBERLÂNDIA, UBERLÂNDIA, MG, BRAZIL.
Keyword:leishmania; treatment; herbal extracts
Abstract: Leishmaniasis is caused by many species of protozoa the genus Leishmania. Leishmaniasis threatens about 359 million people in 88 countries around the world. There are about 12 million people infected worldwide. The chemotherapy used in leishmaniasis is considered ineffective, there are various strains resistant and can cause serious side-effects, making treatment difficult and expensive. In previous studies we observed effect of extracts derived from plants belonging to the genus Ruta, Ageratum and Tabebuia on promastigotes (infective forms) of Leishmania amanzonensis in axenic parasite culture. However, we had not observed the effect of these extracts during amastigotes forms multiplication inside mammalian cells. Thus, the aim of this study was determine the effect of plant extract Ruta graveolens, Tabebuia impetiginosa and Ageratum conyzoides on the multiplication of amastigotes. Methods: peritoneal macrophages (BALB/c) were plated in 24 well plates, after 24 hours parasites were added to invade cells during five hours. Then cells were treated with the extracts in predetermined concentrations. After 72 hours, coverslips were fixed and observed under fluorescence microscope after staining the cell with polyclonal antibody against the parasite. Results: Two hundred infected cells were counted and the number of amastigotes internalized in each cell. A significant decrease (P<0.05) in the number of amastigotes in cells treated was observed compared to control cell. Treatment with extracts from T. impetiginosa, A. conyzoides and R. graveolens generated a reduction in the number of amastigotes of about 30%, 40% and 45% respectively. Conclusion: The plants extracts have activity against amastigotes and promastigotes of L. amazonensis. Financial Support: Fapemig/ UFU/ CNPq.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [805] P 512 - MOLECULAR DIAGNOSTICS FOR DETECTION AND DIFFERENTIATION OF COMPLEX LEISHMANIA SILVA, R.T.; NEVES, P.C.B.C.; QUINTAL, A.P.N.; MACHADO, F.C.; DA SILVA, C.V. UNIVERSIDADE FEDERAL DE UBERLÂNDIA, UBERLÂNDIA, MG, BRAZIL.
Keyword:multiplex pcr; species differentiation; diagnosis
Abstract:
Leishmaniasis are vector-borne parasitic diseases, caused by protozoa of the genus Leishmania. Three complexes have great importance: Braziliensis, Mexican and Donovani which have different clinical presentations as mucocutaneous, cutaneous and visceral form. The aim of this study was to develop a molecular diagnostic kit for leishmaniasis which is possible to detect and differentiate three major complexes of the genus Leishmania, especially in areas of co-existence with Chagas disease. We performed DNA extraction with phenol / chloroform (SAMBROOK et al., 1989) from 28 human blood samples from the University Hospital of Uberlândia, whose patients were suspected for leishmaniasis. Multiplex PCR was performed with primers descriptor by Harris et al. (1998) and visualized in 1.5% of agarose gel. Multiplex PCR could differentiate the three complexes that produce distinct molecular weights (146-149, 218-240 and 351-394pb to complex L. braziliensis, L. donovani and L. mexinaca, respectively). In some cases it was possible to verify the mixed infections of Leishmania species and there was no cross-reactivity with Trypanosoma cruzi strains tested in the experiments, suggesting a good specificity for the technique. The lyophilization process did not impair PCR reaction and appears to allow storage of the compounds for long periods of time without loss of quality. The multiplex PCR was able to make the differential diagnosis of Leishmania complexes and may be applicable in the near future. Financial support: FAPEMIG/CAPES. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [807] P 513 - EVALUATION OF RK39 RAPID DIAGNOSTIC TESTS FOR CANINE VISCERAL LEISHMANIASIS: LONGITUDINAL STUDY AND META-ANALYSIS QUINNELL, R.1; CARSON, C.2; REITHINGER, R.3; GARCEZ, L.4; COURTENAY, O.2
1.UNIVERSITY OF LEEDS, LEEDS, UNITED KINGDOM; 2.UNIVERSITY OF WARWICK, COVENTRY, UNITED KINGDOM; 3.GEORGE WASHINGTON UNIVERSITY, WASHINGTON DC, UNITED STATES; 4.INSTITUTO EVANDRO CHAGAS, BELEM, PA, BRAZIL.
Keyword:rk39; dipstick; diagnosis
Abstract: Sensitive and specific rapid diagnostic tests (RDT) for canine visceral leishmaniasis are needed for confirmation of clinically suspected cases, and for identification of infected dogs in epidemiological studies and control programmes. Immunochromatographic dipstick RDTs using rK39 antigen have been widely used, but their reported diagnostic performance is variable. Here, we investigate the sensitivity of rK39 dipstick tests to detect infection, disease and infectiousness in a longitudinal cohort study of natural infection in Brazil, and use meta-analysis to estimate the sensitivity and specificity of rK39 dipstick tests from published studies. The sensitivity of the rK39 RDT in Brazil to detect infection, disease and infectiousness was 46%, 77% and 78% respectively. Sensitivity increased with time since infection and disease severity (measured by antibody titre, parasite load and clinical score). Meta-analysis of data from 16 published studies showed that the combined sensitivity of rK39 RDTs was 87% to detect clinical disease and but only 59% to detect infection. Combined specificity was 99%. Sensitivity and specificity varied considerably between studies. We discuss the implications of the low sensitivity to detect infected dogs for epidemiological studies and control programmes. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [809] P 514 - RED FLUORESCENT PROTEIN (DSRED) TRANSFECTED LEISHMANIA DONOVANI AND ITS APPLICATION IN DRUG SCREENING DUBE, A.; JAISWAL, A.K.; RAI, A.; KHARE, P. CENTRAL DRUG RESEARCH INSTITUTE, LUCKNOW, INDIA.
Keyword:red fluorescent protein (dsred); multiparametric assay for drug screening; flowcytometry and bioimaging
Abstract: Emergence of flowcytometry and cell imaging has put tremendous expectation from Leishmania biology. A major challenge in this area has been to develop a fluorescent Leishmania parasite, which could be efficiently traced within the harboring macrophages and experimental host. This will immensely help the biologist to pinpoint on only infected macrophages and not the uninfected fellow macrophages. Using this technology, a stable cellular system of DsRed, a red fluorescent protein (RFP) expressing Leishmania donovani which has a broad excitation (400550 nm) and emission (550-700 nm) spectrum, was developed for the screening of agents that modulate activation of the caspase cascade and thus induce apoptosis. It was interesting to note that the DsRed expressing L. donovani infects macrophages at a much faster rate and achieves 80-90% infections within a short span of 12 hrs. Upon treatment with amphotericin B and miltefosine, apoptosis of parasites were accompanied with the decline in fluorescence of DsRed, suggesting its suitability for screening purpose. On the other hand, the proliferation of DsRed expressing parasite was also demonstrated using CFSE. In addition to this, redox status of the macrophages infected with the DsRed expressing parasite were also measured using DCFDA dye, re-affirming suppression of the induction of reactive oxygen species upon infection. Furthermore, imaging of the hamster infected with DsRed expressing L. donovani shows the localization of fluorescent parasite within lymph nodes and their subsequent dissemination within next 15 days. Though, some improvements are yet to be done, DsRed expressing L. donovani flexibly accommodates commonly used dyes (emission ~540nm; FITC, DCFDA, CFSE, vibrant green etc.) as it occupies the mainly FL-2 detector channel (excitation/emission maximum of DsRed ~ 558nm/583 nm) in flowcytometer. As a result, it leaves us with more choice to perform multiple combinations, because FL-1 channel (emission ~540nm) is free and so many dyes having same excitation spectra can be used. We have accomplished to perform the multiparametric flowcytometry on macrophages infected with DsRed expressing L. donovani, demonstrating apoptosis, redox status etc. on single cell level. Along with Green Fluorescent Protein (GFP) tagged L. donovani, DsRed expressing L. donovani offers a promising advancement in this area. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [812] P 515 - PROFILE OF DRUG TRANSPORTERS AND OXIDATIVE STRESS GENE EXPRESSION IN HUMAN MACROPHAGES TREATED WITH GLUCANTIME® TÉLLEZ, J.1; ROMERO, I.1; STEINDEL, M.1; GRISARD, E.C.1; ROMANHA, A.J.2
1.UFSC, FLORIANÒPOLIS, SC, BRAZIL; 2.UFSC, CENTRO DE PESQUISAS RENÉ RACHOU – FIOCRUZ, FLORIANÒPOLIS, BELO HORIZONTE, SC, BRAZIL.
Keyword:transporters; oxidative-stress; macrophages
Abstract: Leishmaniasis affects millions of people worldwide and represents one of the major public health problem. Leishmania spp. control relies primarily on chemotherapy, with the mainstay being pentavalent antimony (SbV) complexed to carbohydrates in the form of sodium stibogluconate (Pentostam®) or meglumine antimoniate (Glucantime®). Due to the emergence of drug resistance, the absence of effective vaccines, and the spread of insecticide-resistant vectors, Nevertheless, treatment is becoming extremely difficult. Although antimonials derivatives have been used for chemotherapy for over 100 years. The mode of action of SbV is still poorly understood. Thus, identification of affordable and readily available drugs against resistant parasites is of global demand. To explore the effect of SbV on human macrophages gene expression, a PCR array analysis was performed using drug transporters and oxidative stress pathways to compare gene expression profiles of Leishmania braziliensis-infected human monocytes derived macrophages (MDM) and non-infected cells treated or not treated with Glucantime®. The SbV treatment induced changes on the expression of a few host genes,
regardless of the L. braziliensis infection. On the other hand, L. braziliensis infection had a greater effect on modulation of the host gene expression, especially in the glutathione biosynthesis pathway. Analysis of the gene expression indicated that the expression of twentytwo genes was modified by at least threefold upon SbV treatment, with ten genes up-regulated and twelve down-regulated. The expression of ABCB6 and SCARA3 genes was up-regulated in infected primary human monocyte-derived macrophages by SbV treatment. Our analysis suggests that the expression of a few genes is altered by SbV treatment, and some of these encoded proteins may be implicated in the molecular and biochemical mechanisms resistance to antileishmanial drugs. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [814] P 516 - IN VITRO LEISHMANICIDAL ACTIVITY AND ACUTE ORAL TOXICITY ASSAYS OF THE CRUDE ETHANOL EXTRACT AND ENRICHED FRACTION OF CAESALPINIA ECHINATA STEMS PARREIRAS, P.M.1; FILHO, E.P.S.1; ARAUJO, M.S.S.1; ALVES, B.G.V.1; ALVES, T.M.A.1; JANUARIO, C.I.1; TEIXEIRA, E.M.1; MELO, M.M.2; SERAKIDES, R.S.2; MAGALHAES, V.P.1; FILHO, O.A.M.1; COTA, B.B.1 1.CPQRR FIOCRUZ MG, BELO HORIZONT, MG, BRAZIL; 2.ESCOLA VETERINÁRIA - UFMG, BELO HORIZONTE, MG, BRAZIL.
Keyword:caesalpinia echinata; leishmanicidal activity; acute toxicity
Abstract: Crude ethanolic extract from stems of Caesalpinia echinata Lamarck showed in vitro leishmanicidal activity (90%) against amastigote-like of Leishmania (Leishmania) amazonensis and it when avalued for mononuclear cells from human peripheral blood at 20 g/ml did not show toxicity. Crude ethanolic extract (EE) from stems of C. echinata was submitted to gel permeation chromatographic fractionation and furnished an enriched fraction (EF) that inhibited the growth of L. (L.) amazonensis at 80% at 20 g/ml. Next it was evaluate the safety of EE and EF of C. echinata in Swiss-Webster mice orally treated by a single oral dose of 5000 mg/kg. The LD50 was estimated be greater than 5000 mg/kg by oral route. Based on others in vivo studies of genus species and the lack of mortality, clinically significant adverse effects and morphological parameters changes, Swiss-Webster mice received a single low oral dose EE and EF of 300 mg/kg to evaluate daily weight gain, clinical toxicity test, histopathologic evaluation and the hematological and biochemical profiles of the blood. Our present study showed that crude ethanolic extract (EE) and an enriched fraction (EF) of C. echinata had in vitro leishmanicidal activity at 20 g/ml. Although Brazilwood or “Pau-brasil” has not been used by traditional medicine, EE and EF from stems showed a pharmacological potential. The results indicated that C. echinata, administered acutely, was not toxic orally in mice, since mortality or adverse symptoms after the dose limit of 5000 mg/kg was not observed neither at 300 mg/kg as expected. Indeed, animals behaviour such as appetite, water ingestion and morphological analyses suggested that EE and EF of C. echinata were not toxic. Although in the biochemical analysis the acute administration of EE and EF of C. equinata afected creatinine results, those results were not clinically significants for the animal health as they are in the acceptable rate expected in this analysis for this animal line. Histopathological examinations showed no evidence of necrosis in the liver or kidney of mice corroborating with these biochemical parameters. Other studies in vivo will be conducted to confirm the leishmanicidal activity of these extracts. Apoio Financeiro-Fapemig.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [816] P 517 - IN SILICO COMPARATIVE 3D STRUCTURE ANALYSIS OF LEISHMANIA INFANTUM AND HUMAN METHYLTHIOADENOSINE PHOSPHORYLASE AS CANDIDATE DRUG TARGET. ABID, H.1; MEJRI, T.2; BARHOUMI, M.1; BORCHANI, L.3; KAROUI, H.2; GUIZANI, I.1
1.LMEEP/ LEEP, INSTITUT PASTEUR, TUNIS, TUNISIA; 2.LMEEP, INSTITUT PASTEUR, TUNIS, TUNISIA; 3.LVBT, INSTITUT PASTEUR, TUNIS, TUNISIA.
Keyword:mtap, leishmania; three-dimensional (3d) structure; active site, drug target
Abstract: Development of new drugs constitutes a Leishmaniasis research priority. For many years, the purine salvage pathway of parasitic protozoa is regarded as an attractive chemotherapeutic target. The Methylthioadenosine phosphorylase (MTAP) enzyme plays an essential role in metabolic pathways. In presence of phosphate, the MTAP recycles the 5’-deoxy-5’-methylthioadenosine (MTA), a toxic by-product of the polyamine pathway, into adenine and 5’methylthioribose-1-phosphate then incorporated respectively into the salvage pathways of purines and methionine. Thus, MTAP constitutes the initial enzyme in these pathways. In this paper, the homology modeling of the Leishmania infantum (Li) MTAP three-dimensional (3D) structure, by the use of the I-tasser server and analysis of the alignment of the obtained 3D model with the human one, via the Pymol software, were accomplished. The two MTAP 3D structures were globally compared, especially around the active site looking at the amino acids characteristics, the electrostatic potential and distances of some residues to the substrate (MTA). The in silico comparison of the two MTAP 3D models showed differences at different levels. Indeed, 5 of the 17 amino acids, involved in the active site, are different. The electrostatic profile of the Leishmania MTAP model, around the active site, is anionic with a negative electrostatic potential reaching - 38 volts. While the one, around the human protein active site, is cationic with a positive electrostatic potential which reaches 58 volts, in the bottom of the active site pocket. Distances of the majority of involved active-site-residues, in the human MTAP 3D model are nearest to the substrate than the corresponding ones in the Leishmania protein model. Shifts were also observed, on the aligned 3D structures, on the N and C terminal extremities and on a central helix. These sequence divergences are concordant with those observed in the primary sequence alignment of the proteins. Because of the extent of 3D structure differences between Leishmania MTAP and the human protein, these could be exploited to design specific inhibitors of the parasitic enzyme. The study constitutes a step towards validation of MTAP as a candidate drug target. The study received financial support from Ministry of Higher Education and Research in Tunisia (LR00SP04 & LR11IPT04) and from the Institut Pasteur Calmette & Yersin Programme.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [817] P 518 - ANTILEISHMANIAL ACTIVITY OF THE NITRIC OXIDE AND NITROXYL DONOR DEA/NO, NITROXYL DONOR WR014614 (R1-HNOH) AND WR167996 (R2HNOH) IN VITRO PERVITSKY, D.; MENDEZ, J.; BRASOV, I.E.; DIAO, L.Y.; HANNOLD, C.; MELENDEZ, V.; GROGL, M.; WEINA, P.J. WRAIR, SILVER SPRING, UNITED STATES.
Keyword:leishmaniasis; drug development; nitroxyl
Abstract: Leishmaniasis is endemic in 92 countries, with 350 million people at risk of becoming infected. More than 10 million people are suffering from cutaneous leishmaniasis and over 1.5 million new cases are diagnosed annually. The life cycle of Leishmania protozoa involves two stages: the promastigote that exists in the vector sand fly, and the amastigote that reproduces in macrophages of the host. Curative effect of Nitric Oxide (NO) in therapy for cutaneous leishmaniasis has been proven in vitro and in vivo. The aim of this study was to investigate in vitro the antileishmanial activity of the NO donor Diethylammonium (Z)-1-(N,Ndiethylamino)diazen-1-ium-1,2-diolate (DEA/NO, C4H11N3O2•C4H11N), WR014614, and WR167996. DEA/NO and WR014614 (R1-HNOH) in pH 7.4 solution form NO and/or Nitroxyl (HNO). HNO is the reduced form of NO and similarly to NO it has potential applications in medicine. WR167996 (R2-HNOH) is a 3-electron reduced form of NO. We investigated the effect of these compounds against two species of the Leishmania parasites: L. major (Old World) and L. panamensis (New World). The promastigotes were cultured in Schneider’s Insect Medium supplemented with fetal bovine serum (FBS) at 220C, and the amastigotes in macrophages were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with FBS at 370C under 5% CO2 atmosphere. The tested compounds killed promastigotes and amastigotes in a time- and dose-depended manner. DEA/NO (400µg/ml) completely suppressed parasitemia in freshly prepared solution of macrophages and promastigotes (1:10 ratio of macrophages to promastigotes). The IC50 for WR014614 and WR167996 against promastigotes was 200 and 40µg/ml, respectively, while the IC50 for WR014614 against amastigotes in macrophages was less than 1µg/ml. This study identified two classes of chemicals with the potential to be used as treatment of cutaneous leishmaniasis. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [840] P 519 - THE USE OF BISNAPHTHALIMIDOPROPYL DERIVATIVES TO CONTROL VISCERAL LEISHMANIASIS INFECTIONS TAVARES, J.1; LIMA, S.C.1; KONG, P.2; OUAISSI, A.3; CORDEIRO-DA-SILVA, A.1 1.INSTITUTO DE BIOLOGIA MOLECULAR E CELULAR (IBMC) DA UNIVERSIDADE DO PORTO, PORTO, PORTUGAL; 2.3SCHOOL OF PHARMACY AND LIFE SCIENCES, ROBERT GORDON UNIVERSITY, ST. ANDREW STREET, ABERDEEN, UNITED KINGDOM; 3.4INSERM, CNRS UMR 5236, UNIVERSITÉ MONTPELLIER II, MONTPELLIER, FRANCE.
Keyword:bisnaphthalimidopropyl polyamines; leishmania silent information regulator 2; drug delivery systems
Abstract: Current therapy against visceral leishmaniasis is far from satisfactory, due to serious limitations such as drug unresponsiveness, difficult route of administration, emergence of resistance, high cost and toxicity. Targeting trypanosome protozoan polyamines and their associated enzymes has become interesting as a novel anti-parasitic therapy (Oliveira 2007, Tavares 2010, Tavares 2012), since in trypanosomatid protozoa, polyamines participate actively in the endogenous redox equilibrium. Based on this, the bisnaphthalimidopropyl (BNIP) polyamines derivative compounds were designed and identified as inhibitors of the Leishmania Silent Information Regulator 2 (SIR2) NAD+-dependent deacetylase (Tavares 2008). A small library of BNIP compounds was synthetized and screened on the proliferative stages of Leishmania infantum (L. infantum) parasite, the causative agent of visceral leismaniasis. The potential of these compounds was demonstrated in vitro and in vivo using BALB/c mice chronically infected with L. infantum as a model (Tavares 2012). These experiments led to the identification of BNIPdiaminooctane (BNIPDaoct) as an effective compound able to induce significant reduction of the parasite load in the spleen and liver of infected mice. Moreover, no
indications of haematological toxicity were detected, hence making BNIPDaoct a potential therapeutic agent against leishmaniasis. However, these compounds have some drawbacks, including their low aqueous solubility and some toxic effects in higher doses. To overcome these limitations we have designed drug delivery systems (DDS) to enhance delivery of BNIP derivative compounds which reduce adverse toxic effects, and hence increasing their biopharmaceutical efficacy. One of the DDS was based on BNIPs encapsulated on polymeric nanoparticles of pegylated poly(lactic acid) which exhibitied in vitro antileishmanial activity with significant reduction of mammalian cellular cytotoxicity (Lima 2012a). Furthermore, the poly(D,L-lactide-co-glycolide) nanoparticles containing BNIPDaoct were able to reduced drug cellular toxicity, retaining its in vitro anti-leishmanial activity and leading to a significant in vivo reduction on the parasite burden in the infected mice spleen when compared with the free drug or amphotericin B (Lima 2012b). These BNIPs drug delivery systems seem to be a promising therapeutically approach to control visceral leishmaniasis in the future. References Tavares, J et al., Biochem J. 2008;415: 377-86; Oliveira, J et al., Bioorg. Med. Chem.2007, 15: 541-545; Tavares, J et al., ChemMedChem. 2010, 5:140-7; Tavares, J et al., Parasitol Int. 2012, 61:360-3; Lima, SAC et al., Int J Antimicrob Agents. 2012a, 39:424-30; Lima, SAC et al., Nanomedicine (Lond). 2012b, 7:1839-49. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [843] P 520 - EVALUATION OF THE IN VITRO ANTILEISHMANIAL ACTIVITY USING DIFFERENT COMPOSITIONS DERIVED FROM CHITOSAN RIBEIRO, T.G.1; CHÁVEZ-FUMAGALLI, M.A.; FRANÇA, J.R.1; MARTINS, L.E.2; MARTINS, V.T.3; LAGE, P.S.2; LAGE, D.P.4; ANDRADE, P.H.4; DUARTE, M.C.2; RÉGIS, W.C.B.5; TAVARES, C.A.P.3; CASTILHO, R.O.1; FARACO, A.A.G.1; COELHO, E.A.F.4
1.PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS FARMACÊUTICAS, FACULDADE DE FARMÁCIA, UFMG, BELO HORIZONTE, MG, BRAZIL; 2.PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE:INFECTOLOGIA E MEDICINA TROPICAL, UFMG, BELO HORIZONTE, MG, BRAZIL; 3.DEPARTAMENTO DE BIOQUÍMICA E IMUNOLOGIA, ICB, UFMG, BELO HORIZONTE, MG, BRAZIL; 4.DEPARTAMENTO DE PATOLOGIA CLÍNICA, COLTEC, UFMG, BELO HORIZONTE, MG, BRAZIL; 5.PUC, BELO HORIZONTE, MG, BRAZIL.
Keyword:chitosan; antileishmanial activity; molecular weight
Abstract: Leishmaniasis is a severe public-health problem. Efforts to search new, effective and safe agents for the treatment of disease have occurred for several decades. Chitosan is a naturally polysaccharide obtained by deacetylation of chitin. Chitosan can be found in different molecular weights (MW) and degree of deacetylation, and its diversity can be exponentially increased by including chemical modifications. The present study aims to investigate the in vitro antileishmanial activity of low and medium MW chitosans against different Leishmania species. In the results, these products were effective against stationary-phase promastigotes of L. amazonensis, presenting IC50 values of 61.2, 82.1 and 77.1 g/ml, respectively for low MW chitosan, and of 72.5, 67.1 and 77.7 g/ml for medium MW chitosan, respectively. The treatment of murine macrophages infected with L. amazonensis promastigotes using the low and medium MW chitosans showed reductions in the parasite load in the order of 77% and 92%, respectively. The applied low and medium MW chitosans presented a low cytotoxicity in murine macrophages and a null hemolytic activity in type O+ human red blood cells. No nitric oxide (NO) production was observed in macrophages after stimulation with the low and medium MW chitosans, indicating a possible direct effect of the products in the amastigotes intramacrophages. In conclusion, the results demonstrate that low and medium MW chitosans
present a possible direct effect in the infected host cells and can be used to provide a safe and less expensive treatment for leishmaniasis on its own, or also in association with other drugs. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [844] P 521 - EFFECT OF METHANOLIC EXTRACT OF SELAGINELLA SELOWII (SELAGINELLACEAE) IN CUTANEOUS LESIONS INDUCED BY LEISHMANIA AMAZONENSIS IN HAMSTERS QUEIROZ, D.S.; ARAÚJO, V.P.; CAROLLO, C.A.; RIZK, Y.S.; BOSQUIROLI, L.S.; COSTA, E.C.; ARRUDA, C.P. UNIVERSIDADE FEDERAL DO MATO GROSSO DO SUL, CAMPO GRANDE, MS, BRAZIL.
Keyword:leishmaniasis cutaneous; selaginella selowii; plant extracts
Abstract: American cutaneous leishmaniasis (ACL) is an infectious, non-contagious disease caused by different species of protozoa of the genus Leishmania, which affects the skin, cartilage and mucous membranes of the upper respiratory tract. It is widely distributed in Brazil, with cases registered in all states. The pentavalent antimonials are the first choice drugs in their treatment, but they have important limitations regarding security, presenting hepato, cardio and nephrotoxicity. The use of plant extracts is promising for the treatment of ACL, since many secondary metabolites with structural variety have proved to be active against the parasite, with low toxicity, and allowing other forms of administration. The aim of this study was to evaluate the anti-Leishmania activity of Methanolic Extract of Selaginella selowii (MCE) in vivo in hamsters infected with Leishmania amazonensis. The animals were infected with 106 L. amazonensis promastigotes (0.05 mL) in the left footpad. Four weeks after infection, they were treated orally with MCE (50mg/kg) or PBS (0.05 ml) once a day for 20 days. Other groups were treated with five intralesional injections of MCE (50mg/kg) or PBS with intervals of four days. Seven days after the end of treatment, thickness of the infected paws was determined weekly by measuring with a caliper and parasite load was evaluated in the site of infection, lymph nodes and spleen. Histopathological study was carried out in various organs at the same point of time. The oral treatment with MCE reduced paw thickness and parasite load compared to the group of untreated animals. The treatment by intralesional route resulted in a significant increase in paw thickness, but with significant reduction of the parasitic load. These data strongly suggest the in vivo activity of methanolic extract of S. sellowii in hamsters infected with L. amazonensis, demonstrating the potential of this natural product for future use in the treatment of cutaneous leishmaniasis. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [853] P 522 - DEVELOPMENT OF AN ANTIGEN BASED RAPID TEST FOR SERODIAGNOSIS OF CANINE VISCERAL LEISHMANIASIS FRAGA, D.B.M.1; DA SILVA, E.D.2; OLIVEIRA, I.Q.3; SILVA, R.A.3; PACHECO, L.V.3; SUCUPIRA, M.V.2; CONRADO DOS SANTOS, W.L.3; DE CARVALHO, L.C.P.3; MONTEIRO, G.4; VITAL, W.C.5; REIS, A.B.5; JERÔNIMO, S.M.B.6; OLIVEIRA, G.G.S.3; VERAS, P.S.T.7 1.ESCOLA DE MEDICINA VETERINÁRIA, UNIVERSIDADE FEDERAL DA BAHIA; INCT-DT, SALVADOR, BA, BRAZIL; 2.BIOMANGUINHOS – FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 3.CENTRO DE PESQUISAS GONÇALO MONIZ - FIOCRUZ, SALVADOR, BA, BRAZIL; 4.CENTRO DE BIOCIÊNCIAS, UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE; INCT-DT, NATAL, RN, BRAZIL; 5.NÚCLEO DE PESQUISAS EM CIÊNCIAS BIOLÓGICAS, UNIVERSIDADE FEDERAL DE OURO PRETO; INCT-DT, OURO PRETO, MG, BRAZIL; 6.CENTRO DE BIOCIÊNCIAS, UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE; INCT-DT, NATAL, RN, BRAZIL; 7.CENTRO DE PESQUISAS GONÇALO MONIZ - FIOCRUZ; INCT-DT, SALVADOR, BA, BRAZIL.
Keyword:multi-antigen; mapia; recombinant antigens
Abstract: In Brazil, visceral leishmaniasis control is based on diagnosis and euthanasia of seropositive dogs. The recommended diagnostic protocol is Dual Path Platform named DPP速 LVC confirmed by ELISA EIE-LVC速. This protocol shows good sensitivity to identify symptomatic dogs, but low detection of asymptomatic cases. Tests using multiple antigens are more sensitive since different dogs, when infected hardly produce antibodies against the same set of antigens. We aim to develop a multiple antigen rapid test for serodiagnosis of canine visceral leishmaniasis (CVL). Twelve recombinant antigens (Lci1, Lci2, Lci3, Lci4, Lci5, Lci6, Lci7, Lci8, Lci10, Lci11, Lci12 and Lci13) were previously selected using Leishmania infantum genomic and cDNA libraries. Selected fragments were produced using Escherichia coli, transformed with pRSET plasmid constructions, and purified by affinity chromatography. Reactivity of sera from L. infantum naturally infected dogs was evaluated against recombinant antigens (rAgs) using multi-antigen print imunoassay (MAPIA). rAgs that showed more promising results using MAPIA were individually impregnated in nitrocellulose membranes and tested against sera from parasite-positive dogs in the multi-antigen DPP. Lci1, Lci2, Lci4, Lci5, Lci8 and Lci12 were the more recognized rAgs by infected dog sera, using MAPIA. Reactivity of the six selected rAgs was evaluated using DPP against 55 sera from parasite-positive dogs. Considering the individual positive reaction of each rAgs, the serum reactivity varied from 0% to 69%, while the reactivity increased to 82% when the sera reacted with at least one of the rAgs. Two (Lci1 and Lci2) out of these six antigens showed highest sensitivities, and were further selected to be independently tested in two different concentrations in DPP, by 3 INCTDT laboratories: BA, MG and RN. Lci1 in the lowest concentration were identified by 34% of sera tested at BA, 16% at MG and 30% at RN, sensitivity increased to 68%, 71% and 91% respectively, when used in higher concentration. Lci2 in the lowest concentration were identified by 62% of sera tested at BA, 51% at MG and 63% at RN, sensitivity increased to 82%, 78% and 91% respectively, when used in higher concentration. Test sensitivity using higher concentration achieved the best results when the sera reacted with at least one of the rAgs: BA 82%, MG 86% and RN 94%. These results confirm that select rAgs are promising candidates for compose a multiple antigen for CVL diagnostic test. Supported by FAPESB, CNPq, INCT-DT, PDTIS-FIOCRUZ, Bio-Manguinhos. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [854] P 523 - SYNTHESIS AND ANTILEISHMANIAL ACTIVITY OF NOVEL 5-[3-METHYL -1(PHENYL)-1H-PYRAZOL-4-YL]-1H-TETRAZOLE DERIVATIVES BECKER, K.M.1; FARIA, J.V.2; DOS SANTOS, M.S.2; BERNARDINO, A.M.R.2; LEON, L.L.1; CANTO-CAVALHEIRO, M.M.1 1.IOC-FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 2.UFF, NITEROI, RJ, BRAZIL.
Keyword:leishmaniasis; leishmania braziliensis; tetrazole compounds
Abstract: Leishmaniasis is a protozoan disease that affects about 12 million people in the world, particularly in subtropical and tropical regions, causing a serious public health problem. Current chemotherapy of leishmaniasis is unsatisfactory. In this way, efficacious and safe new drugs are needed. In the present work, the antileishmanial efficacy of novel pyrazol- tetrazole derivatives was determined against Leishmania braziliensis. The pyrazol rings structure is already established as a template for antiparasitic drugs and tetrazoles compounds are mentioned in connection with the creation of new pharmaceutical products. The 5-[3-methyl -1-(phenyl)-1Hpyrazol-4-yl]-1H-tetrazole derivatives were obtained, in good yields and all the substances were fully characterized by usual methods (IR, 1H, 13C NMR). The antileishmanial efficacy of fifteen substituted and nonsubstituted pyrazol-4-yl]-1H-tetrazole was determined in vitro against
L. braziliensis promastigotes (MCAN/BR/98/R619 strain). Parasites were cultured with and without the drugs in Schneider´s medium at 25ºC, using Pentamidine as the standard drug. After 24 hours incubation, parasite viability was determined using the MTT (tetrazolium blue) assay. The results showed that among compounds assayed the substituted 3- chlorophenyl (IC50 =15±0.14 µM) and 3,4 di-chlorophenyl (IC50 =26±0.09 µM) were the most potent in inhibiting promastigotes forms of L. braziliensis. These results point out the 5-[3-methyl -1(chlorophenyl)-1H-pyrazol-4-yl]-1H-tetrazole derivatives as potential lead compounds for designing new candidates for leishmaniasis treatment. - OTHER SPECIAL TOPICS [868] P 524 - ASSESSMENT OF LIVER HISTOLOGICAL ALTERATIONS IN DOGS NATURELLY INFECTED WITH L. INFANTUM. LIMA, I.S.1; SILVA, J.S.1; LARANJEIRA, D.F.2; SOUZA, P.A.N.3; VERAS, P.S.T.1; CONRADO DOS SANTOS, W.L.1; FREITAS, L.A.R.1 1.CENTRO DE PESQUISAS GONÇALO MONIZ, FUNDAÇÃO OSWALDO CRUZ, SALVADOR, BA, BRAZIL; 2.FACULDADE DE MEDICINA VETERINÁRIA DA UFBA, SALVADOR, BA, BRAZIL; 3.CENTRO DE REFERÊNCIA EM DOENÇAS ENDÊMICAS PIRAJÁ DA SILVA (PIEJ), JEQUIÉ, BA, BRAZIL.
Keyword:canine visceral leishmaniasis ; dogs; liver
Abstract: Canine visceral leishmaniasis (CVL) is a protozoan disease caused by Leishmania chagasi/ infantum and is endemic for both humans and dogs in many regions in Brazil. Dogs are widely considered to be the main domestic reservoir, and because of the close association between dogs and men, dogs play an important role for the transmission of disease to man. Furthermore, dogs constitute an excellent model to study leishmaniasis, because the symptoms in dogs are similar to those developed in humans, and both also share a number of pathological features. The aim of the present study was to assess the liver histological alterations in dogs naturally infected with Leishmania chagasi. We examined 31 animals from an endemic area, the city of Jequié, Bahia. The animals were grouped into three categories: a) infected dogs (positive splenic culture and ELISA); b) intermediate dogs (only positive ELISA); c) non-infected dogs (negative splenic culture and ELISA). The clinical status was defined by the number of characteristic signs of CVL, with 0 signs being considered asymptomatic and 4 or more signs being considered symptomatic. Histological evaluation was performed in a blind manner. The results shows that granulomas in portal tracts (p<0.02) and parasitism (p<0.02) were more frequent in infected dogs than in intermediate and non-infected dogs. Infected dogs did not differ significantly from the others with respect to the frequency of portal inflammation, Kupffer cells granulomas, clinical status and disruption of the splenic structure. It is interesting to note that granulomas in infected animals were found to be permissive to parasites, while in the intermediate group, no dog had parasites. Findings herein suggest that the evaluation of liver biopsies may provide important information on the evolution of CVL and indicate that the functional analysis of granulomas is required to provide relevant information regarding the factors related to the mechanisms involved in parasite survival. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [876] P 525 - LEISHMANIA INFANTUM ENDONUCLEASE G PRESENTS A SELF-INHIBITOR DOMAIN THAT REGULATES ITS ACTIVITY OLIVA-CORREIA, C.1; RICO, E.1; SÁNCHEZ-MURCIA, P.2; BUEREN, J.A.2; GAGO, F.2; JIMÉNEZ-RUIZ, A.1 1.DPTO BIOQUÍMICA Y BIOLOGÍA MOLECULAR, UNIVERSIDAD DE ALCALÁ, MADRID, SPAIN; 2.DPTO FARMACOLOGÍA, UNIVERSIDAD DE ALCALÁ, MADRID, SPAIN.
Keyword:endonuclease g; self-inhibitory domain; leishmania infantum
Abstract: Leishmania infantum Endonuclease G (LiEndoG) has been shown to participate in the degradation of the DNA during parasite cell death. In order to increase our knowledge of this enzyme, we have developed a new molecular model using the servers of protein structure prediction Phyre2, Dali and Robetta. The main structure of the protein was constructed by comparison with the structures of the human mitochondrial Endo/Exonuclease, the D. melanogaster EndoG forming a complex with its inhibitor (DmEndoG/DmEndoGI) and the CPS-6 of C. elegans (PDB codes 4A1N, 3ISM and 3S5B, respectively) LiEndoG presents an insertion rich in acidic residues that, according to our model, is located in the same spatial region occupied by DmEndoGI in the DmEndoG/DmEndoGI crystal, in close proximity to the active site. Previous results from our laboratory demonstrate that the enzyme is inhibited at pH 8.0. The presence of various aspartic residues in this insertion led us to postulate that it could be acting as a self-inhibitory domain of the enzyme responsible for the pHdependent activity. In order to test this hypothesis we constructed a mutated version of LiEndoG that lacks this domain and compared its activity with that of the wild type protein. The results of our study confirm that deletion of this region gives place to a recombinant protein (LiEndoG i) far more active than its wild type counterpart over a wide pH range. In addition, overexpression of LiEndoG i under an inducible system (L. tarentolae) decreases parasite growth rate. This effect could be attributed to the increased activity of the enzyme by the removal of its regulatory region.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [877] P 526 - IDENTIFICATION OF CLINICAL PREDICTORS SIGNS FOR CANINE VISCERAL LEISHMANIASIS MACEDO, B.M.1; BORDONI, M.2; MERELLES, S.L.2; SOLCÀ, M.S.2; BORJA, L.S.2; SANTOS, L.C.S.1; GUEDES JÚNIOR, J.C.O.1; PEREIRA, G.C.3; CONRADO DOS SANTOS, W.L.2; DE ALMEIDA, M.C.C.2; VERAS, P.S.T.4; FRAGA, D.B.M.5 1.ESCOLA DE MEDICINA VETERINÁRIA – UNIVERSIDADE FEDERAL DA BAHIA, SALVADOR, BA, BRAZIL; 2.CENTRO DE PESQUISAS GONÇALO MONIZ FIOCRUZ, SALVADOR, BA, BRAZIL; 3.CENTRO DE CONTROLE DE ZOONOSES, CAMAÇARI, BA, BRAZIL; 4.CENTRO DE PESQUISAS GONÇALO MONIZ FIOCRUZ; INCT-DT, SALVADOR, BA, BRAZIL; 5.ESCOLA DE MEDICINA VETERINÁRIA – UNIVERSIDADE FEDERAL DA BAHIA; INCT-DT, SALVADOR, BA, BRAZIL.
Keyword:predictive model; diagnosis; dogs
Abstract: In urban areas, dog is the main reservoir of visceral leishmaniasis (VL). These animals present a varied number and intensity of clinical signs, including: weight loss, lymphadenopathy, hepatosplenomegaly, conjunctivitis, onychogryphosis, queratitis, and cutaneous lesions. Diagnosis of canine visceral leishmaniasis (CVL) is difficult because clinical signs are similar to those presented in other diseases. Studies evaluating the contribution of clinical signs in accurate CVL diagnosis are scarce and have shown conflicting results. This study was conducted to determine clinical predictors of CVL. A cross sectional study was performed in an endemic area for VL in Bahia, Brazil. This study was designed to determine which are the signs that predict the CVL diagnosis in symptomatic dogs. Eight hundred animals were clinically evaluated according to the presence and intensity of the signs. CVL diagnosis was made by serology (ELISA) or splenic aspirate culture. Multivariate poisson regression models were performed to identify the clinical signs associated with CVL diagnosis. CVL tested positive in 21% of the dogs and all of these animals presented at least one clinical sign. The most significant nonadjusted variables (p<0.05) associated with CVL diagnosis includes: ulcerated and crusty
lesions, dermatitis, onychogryphosis, fur alterations, ocular congestion, conjunctivitis, periocular dermatitis, alopecia, ulcerated and crusty lesions on ears, hyperkeratosis or depigmentation of the muzzle, submandibular and popliteal lymphadenopathy. Among these, five were considered as the most significant using stepwise forward poisson regression: onychogryphosis, crusts and ulcers in the pinna of the ear, depigmented muzzle and periocular dermatitis. The performance measures of the predictive model were 75% of area under ROC curve, 58% of positive predictive value, and 83% of negative predictive value. The model had 82% of probability of ascertain the CVL diagnosis. These findings suggest that the use of this model may help to obtain a more accurate diagnosis of CVL. Supported by FAPESB, CNPq, UFBA. - OTHER SPECIAL TOPICS [878] P 527 - ANALYSIS OF ULTRASTRUCTURAL ASPECTS OF PATIENTS WITH NONULCERATED LESIONS OF CUTANEOUS LEISHMANIASIS BY ELECTRON MICROSCOPY GARCIA, M.1; PERES, A.C.1; GUIMARÃES, L.H.2; MACHADO, P.2; CARVALHO, E.M.2; ARRUDA, S.1 1.FIOCRUZ, SALVADOR, BA, BRAZIL; 2.HUPES, SALVADOR, BA, BRAZIL.
Keyword:leishmaniose; ultrastructural; cells
Abstract: During the sandfly bite the skin lesions are present in the microvasculature and blood lake formation, favoring the insect meal. Besides the parasite components of saliva are inoculated and both will induce local immune response. The primary lesion arises from the appearance of a papule, which tends to develop insidiously and may ulcerate since its initial phase. These phases of infection by Leishmania sp. are well described in experimental models, including research demonstrated the development of skin lesions similar to human disease. In our study with human material are collected biopsies from the center of skin lesions using a punch of 4 mm in diameter, after local anesthesia, patients of the Health Center Corte de Pedra, Bahia, considered endemic area of American cutaneous leishmaniasis. The skin fragments are fixed in Karnovsky and processed according to the protocol of transmission electron microscopy. Already using the technique of electron microscopy can visualize cellular ultrastructures, defined from 3,200× to 200,000×, making the correct identification of intra-and extracellular components. The inflammatory response in skin lesion arising from the spread of L. brasiliensis is a chronic inflammatory immune response in the presence of the parasite. Inflammatory response cells migrate from the circulation into the interstitial tissue, are the first cells to arrive at the site of injury, neutrophils, however, the persistence of tissue damage favors the emergence of mononuclear cells, macrophages and lymphocytes. Cellular in resolution or resistance to Leishmania infection confers an important aspect to be discussed, with regard to the activation of certain cells and their contribution to the inflammatory response, in view of contribution under immunoprophylaxis and understanding the pathogenesis infection. The cellular infiltrate and amastigotes have been identified and counted in attempting to find types of cell infiltrate to distinguish the amastigotes inside and outside of phagocytic cells. The junction dermis-epidermis has been evaluated in attempting to characterize types of cells that lyses the epidermis contribuiting for ulcerating.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [881] P 528 - IN VITRO LEISHMANICIDAL ACTIVITY OF ENZYME INHIBITORS RIBOSE-5PHOSPHATE ISOMERASE DA MATTA, C.B.B.1; ARAÚJO, M.V.1; DA SILVA, A.E.1; SANTOS, M.S.1; TENÓRIO, A.P.M.1; LEITE, A.B.1; QUEIROZ, A.C.1; GONÇALVES, V.T.2; DA COSTA, J.B.N.2; SANT'ANNA, C.M.R.2; ALEXANDRE-MOREIRA, M.S.1 1.FEDERAL UNIVERSITY OF ALAGOAS, MACEIÓ, AL, BRAZIL; 2.FEDERAL UNIVERSITY OF RIO DE JANEIRO, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:leishmaniasis; inhibitors ; ribose-5-phosphate isomerase
Abstract: Leishmaniasis is a group tropical diseases caused by protozoan parasites of the genus Leishmania. It is known that 12 million people world-wide are infected with this parasitosis. Due to the emergence resistance to antileishmanial drugs, like pentavalent antimony and pentamidine, development of new effective drugs against this parasite is an immediate need. In this study we evaluated the leishmanicidal activity of enzyme inhibitors ribose-5-phosphate isomerase that catalyzes an important step in the pentose phosphate pathway. Cytotoxicity of 18 inhibitors towards J774 cells was estimated by MTT assay and the in vitro effect on parasite viability was evaluated under several Leishmania species (L. amazonensis, L. chagasi and L. braziliensis) at 100, 10, 1, 0.1, 0.01 and 0.001 M concentrations. Inhibitory concentration (IC50) growth of promastigotes was determinated and pentamidine was used as standard drug. Except V67 inhibitor and pentamidine, none of the inhibitors ribose-5-phosphate isomerase evaluated were significantly toxic to the J774 cells until maximal concentration. All inhibitors evaluated, except V21, were active against promastigotes of L. amazonensis (p < 0.01). Furthermore, the V65, V66 and V67 inhibitors shown to be as effective as pentamidine, however, V67 presented power exceeding the standard drug. When tested against promastigotes of L. brasiliensis showed activity. Except V18, V19, V20 and V37, other inhibitors interrupted parasite growth significantly (p < 0.01) and exhibited efficacy exceeding 50%. Furthermore, V5, V12, V16, V28, V38, V39 and V66 proved as potent as pentamidine. However, the viability assay under promastigotes of L. chagasi, only substances V65 and V66 inhibited parasite growth significantly (p < 0.01) with greater than 50% efficacy and potency similar to standard drugs. Nonetheless, inhibitors V5, V26, V38 and V39 showed IC50 < 0.001 and significant efficacy. Thus, these results are very promising and encourage the continued study of leishmanicidal activity with inhibitors of the enzyme ribose-5-phosphate isomerase. Supported by:INCTINOFAR (#573.564/2008-6), CNPq, FAPERJ and FAPEAL. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [884] P 529 - NOVEL ASSAY TO ASSESS DIMER DISRUPTION OF L. INFANTUM TRYPANOTHIONE REDUCTASE BY PEPTIDES AND SMALL MOLECULES. TORO-LONDOÑO, M.1; MORENO-MATEOS, D.1; SÁNCHEZ-MURCIA, P.2; CAMARASA, M.J.2; GAGO BADENAS, F.3; VELÁZQUEZ, S.2; JIMÉNEZ-RUIZ, A.1 1.DPTO. BIOQUÍMICA Y BIOLOGÍA MOLECULAR, UNIVERSIDAD DE ALCALÁ, MADRID, SPAIN; 2.INSTITUTO DE QUÍMICA MÉDICA, CSIC, MADRID, SPAIN; 3.DPTO. FARMACOLOGÍA, UNIVERSIDAD DE ALCALÁ, MADRID, SPAIN.
Keyword:trypanothione reductase; peptide inhibitors; dimer disruption
Abstract: Trypanothione Reductase (TryR) is a homodimeric enzyme essential for the survival of the diverse species of the Leishmania genus. Because of the lack of catalase and glutathione peroxidase activity, these parasites rely solely on the trypanothione system to detoxify the intracellular milieu from the free radicals produced by the oxidative stress generated either by
the parasite metabolism or by the host immune response. For this reason, this enzyme is considered an excellent target for drug design. TryR is a homo-dimeric enzyme that requires both monomers to efficiently reduce trypanothione. Based on this fact, we have developed a novel strategy aiming to detect peptides and small molecules that inhibit enzyme activity by disruption of the TryR dimers. Based on the work described by Sluis-Cremer et al. (2006) we designed an ELISA assay in order to assess the percentage of dimer lost. In this assay both of the TryR subunits have a T7-tag, and each either has a HIS-tag or a FLAG-tag. The proteins are co-expressed in the same bacteria (BL21(DE3)) and purified using a Ni-NTA matrix. Then, the purified dimers are bound to an ELISA plate coated with anti-FLAG antibody. The dimers that lack a FLAG-tag are washed away and only “Heterodimers” with both HIS and FLAG-tags are detected using anti-HIS HRP-conjugated antibodies. If the peptide or molecule is able to get into the dimerization interface and disrupt the dimer, the ELISA signal is diminished, as the HIS subunit separates from the FLAG subunit. Using this assay we demonstrated that series of rationally designed peptides targeted to the dimerization interface of TryR (alpha-helix spanning residues P435 to M447) were able to disrupt enzyme dimers and inhibit the oxidoreductase activity. - VACCINE DEVELOPMENT AND TRIALS [891] P 530 - COMPARISSON OF PENTAMIDINE ISETHIONATE AND NMETHYLGLUCAMINE IN THE TREATMENT OF AMERICAN CUTANEOUS LEISHMANIASIS. A CLINICAL TRIAL. SOUSA, M.C.S.; SILVA, J.S.F.; DE PAULA, C.D.R.; GOMES, C.M.; RIBEIRO SAMPAIO, R.N. UNIVERSIDADE DE BRASÍLIA, BRASÍLIA, DF, BRAZIL.
Keyword:pentavalent antimony; pentamidine isethionate; treatment
Abstract: Pentavalent antimony (PA) is considered the first choice treatment for american cutaneous leishmaniasis (ACL). However, some limiting factors such as toxicity and resistance are matters of concern in many countries. Fourty-one patients with ACL were randomised into two groups of 19 cases and 22 controls treated with 3 intramuscular doses of 4mg/kg/day pentamidine isethionate (PI) and 20 intravenous doses of 15mgSb/Kg/day N-methylglucamine respectively. Clinical cure was found in 52.63% of the pentamidine group and 72.72% of the Nmethylglucamine group. Patientes who used PI presented a cure ratio of 0.7237 compared to those treated with PA (IC95% = 0.4401, 1.19). Despite the advantages of shorter treatment duration and lower cardiological toxicity compared to PA, the use of PI showed less eficacity without statistic significance. Based on these results, we conclude that PA remains the principal therapy for treating ACL. Although other drugs have shown promising results in some studies, more scientific evidence is needed to ascertain their true equivalence to PA. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [893] P 531 - MASSIVE SEARCH OF ANTI-LEISHMANIAL COMPOUNDS BY VIRTUAL SCREENING MUSKUS, C.E.1; FLOREZ, A.2; WATOWICH, S.3; OCHOA, R.1 1.UNIVERSIDAD DE ANTIOQUIA, MEDELLIN, COLOMBIA; 2.2GERMAN CANCER RESEARCH CENTER (DKFZ), HEIDELRBERG, GERMANY; 3.UTMB, GALVESTON, UNITED STATES.
Keyword:leishmania ; drug serach; docking
Abstract: The classical treatments for all forms of leishmaniasis can cause severe side effects, including death. Furthermore, drug resistant parasites are causing major problems in many endemic countries. For those reasons there is an urgent need for new, safe and inexpensive antiLeishmania compounds. In this work we are using the computing power of the World Community Grid from IBM to search for chemical compounds through molecular docking, which may lead to new drugs for treating this disease. Docking is perhaps one of the most promising bioinformatics tools in the search for drugs against diseases and in fact, several drugs against some diseases have been discovered by docking simulations. At the beginning of this work, 53 non-redundant proteins were available at PDB and mainly derived from L. major (66) and L. mexicana (38) and in a smaller proportion of L. donovani (14) L. infantum (6) and L. tarentolae (5). Each protein structure was modeled into 100 different structures using the NAMD software. The 53 original proteins from PDB were docked against 600.000 chemical compounds selected from ZINC database, using the program AutodockVina. Promising drug candidates have been selected based on the docking score results and converted to Ki. The promising drug candidates will be tested in in vitro and in vivo assays. - OTHER SPECIAL TOPICS [897] P 532 - AMERICAN TEGUMENTARY LEISHMANIASIS: IMAGES, IDEAS, CONCEPTS AND HEALING STRATEGIES. SANTOS, J.L.C.; MELO, M.B.; FERREIRA, R.A.; FONSECA, A.F.Q.; VARGAS, M.L.F. ESCOLA DE SAÚDE PÚBLICA DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL.
Keyword:american tegumentary leishmaniasis; ethnicity xacriabá; action research
Abstract: This research sought to identify the images, ideas, conceptions, attitudes and conditions related to preventive practices and care of American Tegumentary Leishmaniasis (ATL) in Xacriabá Indian Reserve; municipality of São João das Missões; state of Minas Gerais, Brazil. This is an action-based research of qualitative approach. It were performed interviews with indigenous leaders, an indigenous healthcare agent, healthcare and educational managers. Besides the interviews, it were accomplished focus groups with healthcare and educational professionals and users of the Health System (Sistema Único de Saúde - SUS). The following categories were selected from the content analysis technique: images, ideas and conceptions concerning the desease; understanding about elements of the desease cycle and the socioeconomic and cultural aspects hindering it's prevention. It was evident that the prevention and control of ATL in Indian Xacriabá villages isn't a simple matter to solve. Environmental interventions and insect transmitter control are difficult to implement. Educational actions constitute important tools for disease's understanding and formulation and implementation of preventive strategies. Therefore, before any intervention, it is of prime importance comprehending the images, ideas and conceptions that locals have about the disease, in order to conceive control and prevention strategies alongside them.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [909] P 533 - PREVALENCE AND INCIDENCE OF CANINE VISCERAL LEISHMANIASIS DETECTED BY SEROLOGICAL AND MOLECULAR METHODS IN AN URBAN AREA OF BRAZIL COURA-VITAL, W.1; MACHADO, E.M.M.1; ROATT, B.M.2; MORAIS, M.H.F.3; MARQUES, M.J.4; VELOSO, V.M.2; REIS, L.E.S.2; BRAGA, S.L.2; AGUIAR-SOARES, R.D.2; GIUNCHETTI, R.C.1; CORRÊA-OLIVEIRA, R.5; CARNEIRO, M.1; REIS, A.B.2 1.UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL; 2.UNIVERSIDADE FEDERAL DE OURO PRETO, OURO PRETO, MG, BRAZIL; 3.SECRETARIA MUNICIPAL DE SAÚDE, PREFEITURA DE BELO HORIZONTE, BELO HORIZONTE, MG, BRAZIL; 4.UNIVERSIDADE FEDERAL DE ALFENAS, ALFENAS, MG, BRAZIL; 5.CENTRO DE PESQUISA RENÉ RACHOU, BELO HORIZONTE, MG, BRAZIL.
Keyword:canine visceral leishmaniasis; incidence; prevalence
Abstract: The Visceral Leishmaniais Control and Surveillance Program (VLCSP) in Brazil is based on early diagnosis and treatment of human cases, vector control and dog culling. This study aimed to evaluate the prevalence and incidence of canine L. infantum infection using PCR followed by RFLP and to compare its results with serological diagnostic (ELISA/LZOON [Biomanguinhos/RJ/ FIOCRUZ] and ELISA/LIMP ‘‘in house’’). The study was conducted in Belo Horizonte, Minas Gerais, Brazil. To determine the prevalence and incidence for CVL, a canine survey (n=1443) was conducted, after a cohort study was established (n=455) which 3 evaluations were carried out during an approximately 1.5-year follow-up period. Blood sample was collected in dogs and serologic (ELISA) and molecular test (PCR-RFLP) were performed. Among the examined dogs, 230 (15.9%) were seropositive according to at least one of the ELISA. The estimated positivity by Laboratory of Immunopatology (LIMP) was 12.0% (95% CI 10.4-13.7) and at Laboratory of Zoonosis (LZOON) was 9.4% (95% CI 7.9-10.9). The analysis of the molecular diagnosis revealed that 24.7% (95% CI 22.5-26.0) of the investigated dogs were PCR positive by L. infantum. The overall incidence seroconversion between the 2 laboratories was 12.7/1000 dogs-months (95% CI 10.7–15.2). According to LZOON, 65 failure events (seroconversion) were observed within the cohort, with overall incidence of 6.5/1000 dogs-months (95% CI 5.1–8.2). In LIMP 109 failure events were observed, with an incidence of 11.2/1000 dogs-months (95% CI 9.3–13.5). Already in the PCR 234 failure events were observed, with an incidence of 57.5/1000 dogs-months (95% CI 50.5–65.3). The results showed that the prevalence and incidence of canine infection detected by serology has been considerably underestimated in under control areas. However, to carry out studies using PCR with a large number of samples under field conditions needs to be better appraised. We not recommend the use of PCR to incriminate dogs as reservoirs when used isolating. It is necessary to investigate the meaning of a PCR-positive in seronegative dogs in the transmission of visceral leishmaniasis and their capacity to transmit L. infantum to the vectors. Supported by: CNPq, CAPES, FAPEMIG, UFMG & UFOP
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [924] P 534 - COMPARISON AMONGST AN IN HOUSE ELISA TEST AND TWO OTHERS COMMERCIALLY AVAILABLE FOR AMERICAN TEGUMENTARY LEISHMANIASIS DIAGNOSIS SANTOS, P.V.W.G.; NASCIMENTO, M.P.; MACHADO, A.R.S.R.; KIM, O.M.; ROSELINO, A.M. UNIVERSITY OF SÃO PAULO, FACULTY OF MEDICINE OF RIBEIRÃO PRETO, RIBEIRÃO PRETO, SP, BRAZIL.
Keyword:elisa; leishmaniasis; serology
Abstract: The diagnosis of American tegumentary leishmaniasis (ATL) is frequently based on epidemiological and clinical data associated with the visualization on biopsy sample, or isolation of the parasite, which is a poorly sensitive and time-consuming method. Studies have shown that ELISA (Enzyme-linked immunosorbent assay) is a rapid test with good sensitivity that may improve the diagnosis of ATL. Brazilian cases of ATL are mainly caused by L. (Viannia) braziliensis. However, there are no ELISAs commercially available for ATL diagnosis which ones are prepared with other species. Thus, this study intends to compare an in house ELISA prepared with L. (V.) braziliensis (ELISAb), with 2 others tests commercially available, a L. infantum (ELISAi) and L. mexicana (ELISAm) ELISA tests. Antigens from promastigotes of L. (V.) braziliensis were prepared with Trizol and with a protein extraction commercial kit and, as the protein extraction with the commercial kit was much better, this was the one chosen for ELISAb. Immunoblotting using L. (V.) braziliensis protein extracted as antigens revealed specific peptides for ATL diagnosis and detect all patients in the sample tested. Serum samples from 36 cutaneous form and 17 from mucosal leishmaniasis patients, and 62 controls were used for all 3 ELISA tests. The ODs from ATL samples showed Gaussian distribution only on ELISAb. The values of ODs were statically higher for ELISAi (median 1.316) when compared to ELISAb (0.3080) and ELISAm (0.2370). When ATL samples were compared to controls on ELISAm (0.2410; 0.1680) and ELISAb (0.3310; 0.0310) there was statistical significance, while the ODs in control group were lower in ELISAb. There was no difference when comparing the ODs between the forms of ATL in any of the tests. In conclusion, these results support the recommendation of ELISAb as a diagnostic method in L. braziliensis-endemic areas. - CLINICAL LEISHMANIASIS [927] P 535 - COMPARISON OF EFFICACY AND SIDE EFFECTS OF SODIUM STIBOGLUCONATE AND MEGLUMINE ANTIMONIATE FOR TREATMENT OF NEW WORLD CUTANEOUS LEISHMANIASIS IN PERU VALENCIA, B.M.1; RAMOS, A.P.2; MARTINEZ, D.Y.2; MIRANDA-VERASTEGUI, C.3; CALDERON, W.3; LAZO, M.3; LLANOS-CUENTAS, A.2 1.INSTITUTE OF TROPICAL MEDICINE ALEXANDER VON HUMBOLDT, UNIVERSIDAD PERUANA CAYETANO HEREDIA, LIMA, PERU; 2.INSTITUTE OF TROPICAL MEDICINA ALEXANDER VON HUMBOLDT, UNIVERSIDAD PERUANA CAYETANO HEREDIA, LIMA, PERU; 3.UNIVERSIDAD PERUANA CAYETANO HEREDIA, LIMA, PERU.
Keyword:sodium stibogluconate; meglumine antimoniate; new world cutaneous leishmaniasis
Abstract: Pentavalent antimonials (SB5+) have been the first line therapy for American Tegumentary Leishmaniasis (ATL) in Peru for almost 30 years. Meglumine Antimoniate (MA) and sodium stibogluconate (SSG) were used during this period but SSG was largely preferred mainly for cost rather than efficacy/tolerability. Since studies comparing adverse events from both Sb5+ drugs were not evaluated in Peru, it's important to identify potential differences of tolerability and efficacy between MA and SSG in Peruvian individuals. A retrospective cohort study was performed at Leishmania Clinic, Hospital Nacional Cayetano Heredia. Medical records from 130 subjects treated with SSG (SSG group) and 70 subjects treated with MA (MA group) with confirmed diagnosis of Cutaneous Leishmaniasis were evaluated. Median age by group was 30 and 29 years-old. Demographic and clinical characteristics were similar except size of lesion and number of lesions with greater number and size of lesions in the MA group. There was a trend towards higher cure rates in MA group but differences were not statistically significant. Risk of treatment failure was higher in individuals who do not live in/come from endemic areas [OR: 2,44, p=0,037 (CI:1,05-5,64)] and those with disease duration less than 6 weeks [OR: 4,18, p=0,013 (CI:1,34-12,96)]. Type of Sb5+ drug was not associated with treatment outcome. There was a non-clinically significant drop in total leukocyte, lymphocyte and platelet count as well as
prolongation of QTc. Three older patients treated with SSG required interruption of treatment due to QTc>0,48 milliseconds and refractory emesis associated with mild Amylase elevation. Only increase ALT/AST levels were frequent in the SSG group (p<0,001). Symptoms reported in both groups were dysgeusia and dizziness during infusion (37 and 32%), headache (36%), articular pain (31%), lymphangitis (21%), muscle pain and abdominal pain (12%). Dysgeusia, dizziness and headache were more frequent during SSG treatment (p>0,001) as well as a prolonged period with symptoms in those from the SSG group (p=0,012). Subjects older than 25 years had higher chances to present those symptoms [OR: 2,45; p=0,023 (CI:1.13-5.33)].Our findings show that efficacy of MA and SSG was similar and only ALT/AST elevations, dysgeusia, dizziness and headache were more frequent during SSG treatment. Age was an important determinant of laboratorial and clinical side effects. Partially funded (statistical analysis) by Sanofi. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [928] P 536 - DIAGNOSIS OF URBAN VISCERAL LEISHMANIASIS IN NEIVA AYALA, M.S. INSTITUTO NACIONAL DE SALUD, BOGOTA, COLOMBIA.
Keyword:visceral leishmaniasis; visceral leishmaniasis; visceral leishmaniasis
Abstract: INTRODUCTION: Visceral leishmaniasis (VL) is one of the world's neglected diseases. In Colombia, mainly affects children under 15 years and can be potentially fatal without prompt treatment. Diagnostic confirmation of cases is done by direct observation of the parasite in samples of tissue smears obtained by splenic aspiration with a sensitivity of 96 to 98%, 70% in bone marrow and 58% in lymph nodes and by quantifying IgG antibodies by indirect immunofluorescence. According to the guidelines established in the event monitoring protocol, serological tests are considered useful in the diagnosis and monitoring of disease. MATERIALS AND METHODS: To 7 children under 3 years who met the probable case definition of LV, prolonged fever, and one or more of the following symptoms: hepatosplenomegaly, anemia, thrombocytopenia, and repeated infections, underwent direct and serological diagnosis by indirect immunofluorescence (IIF), technique developed in the Parasitology Reference Laboratory of the National Institute of Health (NIH) and made from strains of L. infantum circulating in the country that were isolated, cultured and identified. RESULTS: One of the children was positive for antileishmanial IgG antibodies (14.3%) versus 85.7% (6 children) positive by direct examination obtained by : splenic aspirate (12.5%) and 87.5 % by bone marrow aspiration, which is the most used test for medical staff due to security reasons its execution and lower risks to the patient. DISCUSSION: Given the high lethality of LV is essential to diagnose and provide timely care to prevent the occurrence of severe or fatal infections. According to the results, the low reactivity in the immune response of patients in critical clinical condition and the high sensitivity of direct examination suggests implementing the practice of direct diagnostic test in endemic areas of the country and strengthen the skills of medical personnel to direct sampling of spleen due to the preferential localization of parasites in this organ.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL
[930] P 537 - PRODUCTION OF RECOMBINANT CHIMERIC PROTEINS FOR VACCINE AND DIAGNOSIS OF CANINE VISCERAL LEISHMANIASIS FARIA, A.R.; DAMASCENO, L.M.; GAZZINELLI, R.T.; ANDRADE, H.M. UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL.
Keyword:diagnosis; recombinant; protein
Abstract: Visceral Leishmaniasis (VL) is a neglected parasitic disease, widely distributed and with increasing incidence in urban and peri-urban areas. The dogs act as disease reservoirs, when it is caused by Leishmania infantum chagasi. Since the control of canine disease is managed by the diagnosis and subsequent euthanasia of infected dogs, there is a need for reliable diagnostic tests. Moreover, the development of a vaccine that allows protection against the infection it is also necessary. To contribute to disease control, the goal of this work is the production and the characterization of two chimeric recombinant proteins, which will be tested for LVC diagnostic and vaccine. To be used in the diagnosis, a chimeric protein was constructed with ten peptides previously identified and tested in ELISA with sensitivity and specificity around 80% and accuracy (AUC) of 0.9 (Faria et al, 2011). These 10 peptides were included in a synthetic gene, which had been cloned and expressed in Escherichia coli. After purified, it has been used in ELISA and in the development of an immunochromatographic assay. Sensitivity and specificity of 91.8% and 88.8%, respectively, and AUC=0.947, were achieved using this chimeric protein in ELISA, when testing canine sera (n=61 infected and n=10 uninfected ones). In parallel, another chimeric protein was produced, which will be used in dog immunization. For this, we used 20 peptides previously described as good T cell epitopes (Costa et al, 2011). In summary, our studies demonstrate that the use of chimeric proteins has great potential in diagnosis and immunization, what may benefit Canine Visceral Leishmaniasis control programs. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [932] P 538 - ENZYME INHIBITION VERSUS NITRIC OXIDE PRODUCTION BY BENZOPHENONE DERIVATIVES DE ALMEIDA, L.1; ALVES, K.F.1; MACIEL-REZENDE, C.M.1; PIRES, F.R.1; IZIDORO, M.A.2; DOS SANTOS, M.H.1; MARQUES, M.J.1 1.UNIVERSIDADE FEDERAL DE ALFENAS, ALFENAS, MG, BRAZIL; 2.UNIVERSIDADE FEDERAL DE Sﾃグ PAULO, Sﾃグ PAULO, SP, BRAZIL.
Keyword:leishmania; nitric oxide; cysteine protease
Abstract: Considered as neglected tropical diseases, leishmaniasis are caused by protozoa of the genus Leishmania. The drugs available for the treatment of this disease exhibit various side effects. New compounds classes, benzophenones, have been described for your activity on several human pathogens, including T. cruzi and Leishmania. The objective of this study was to try to find possible action mechanisms of benzophenone derivatives that have proven to be effective on both forms of Leishmania (L.) amazonensis, since this class of compounds have been showing effectiveness on several human pathogens. Therefore with the derivatives (LFQM-116, LFQM-117, LFQM-119, LFQM-120 and LFQM-121) was performed dosage of nitric oxide (NO) and evaluation of enzyme inhibition potential of cysteine proteases representatives (papain, cruzain). To assess the nitric oxide (NO) production in cell culture was used Griess reaction, which evaluates the production of nitrite, a degradation product of NO. Culture supernatants from murine peritoneal macrophages infected with L. (L.) amazonensis and in contact with the substances tested or LPS were collected after 48 hours. For testing enzyme inhibition, papain and cruzain were pre-activated with DTT. The benzophenone derivatives were
added and the dosage of enzyme activity was initiated by adding of substrate Z-FR-MCA in the reaction. Moreover, molecular docking was also performed using AutoDock Vina. It can be seen that not all derivatives induce macrophages to produce the equivalent amount of NO that is produced by cells in contact with LPS. The compounds LFQM-119, LFQM-120 and LFQM-121 (NO = 13.61, 13.01 and 11.44 µM, respectively) induced more the NO production than the other ones. To the papain’s inhibitory activity the best results were exhibited by derivatives LFQM116, LFQM-117 and LFQM -119 (IC50 values = 27.91, 42.78 and 48.20 µM, respectively). But LFQM-119 showed no Hydrogen bonds (H-bonds) interactions with the enzyme’s active site residues. The best results for cruzain’s inhibitory activity were showed by LFQM-119, followed by LFQM-116 and LFQM-117 (IC50 values = 28.13, 31.24 and 33.69 µM, respectively), additionally these two last ones exhibited H-bonds interactions with enzyme’s active site residues. At the end of these tests can be observed that the derivatives LFQM-117 and LFQM116 were the main inhibitors to the proteases evaluated. While derivatives LFQM-119, LFQM120 and LFQM-121 stimulated more the NO production. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [943] P 539 - CONJUNCTIVAL SWAB PCR AS A SPECIFIC AND SENSITIVE TOOL IN THE DIAGNOSIS OF CANINE LEISHMANIASIS USING GENERIC AND SPECIFIC PRIMERS OLIVEIRA, T.M.F.S.1; PEREIRA, V.F.2; BENASSI, J.C.3; DA SILVA, D.T.4; STARKE-BUZETTI, W.A.4 1.FACULDADE DE ZOOTECNIA E ENGENHARIA DE ALIMENTOS DA UNIVERSIDADE DE SÃO PAULO, PIRASSUNUNGA, SP, BRAZIL; 2.FACULDADE DE MEDICINA VETERINÁRIA E ZOOTECNIA DA UNIVERSIDADE DE SÃO PAULO, PIRASSUNUNGA, SP, BRAZIL; 3.FACULDADE DE ZOOTECNIA E ENGENHARIA DA UNIVERSIDADE DE SÃO PAULO, PIRASSUNUNGA, SP, BRAZIL; 4.FACULDADE DE ENGENHARIA DA UNIVERSIDADE ESTADUAL PAULISTA, ILHA SOLTEIRA, SP, BRAZIL.
Keyword:conjuntival swab; dogs; pcr
Abstract: Vector-borne diseases, leishmaniasis are endemic in many countries, and an increasing problem in veterinary medicine and public health, due to their zoonotic importance. Parasites of the genus Leishmania are etiological agents of both, cutaneous and visceral forms of human and viscero-cutaneous canine leishmaniasis (CL). Leishmaniasis diagnose can be made by direct methods, such as smear prepared with different lymphoid organs and polymerase chain reaction (PCR), or by indirect methods such as enzyme immunoassay (ELISA) and Indirect Immunofluorescence (IFAT). Currently, molecular techniques, such as PCR, are being widely used for its sensitivity and specificity and it is known that PCR efficiency is directly associated to used primer and biological sample. Thus, the aim of the present study was diagnose CL using generic and specific primers with DNA samples from conjunctival swab (CS). CS is easy to collect and a noninvasive technique that has been shown to be a good biological sample to screen positive animals by PCR, even without clinical signs. To test and compare the use of CS samples by PCR analysis, CS and blood were collected from 213 dogs in an epidemiological survey in an endemic area from Brazil. The present study used three different pair of primers; two of them generic (L1/ L2 and 13A/13B) and one specific to L. infantum (MC). The CS PCR results showed 29 (61.8%) positive dogs using 13A/13B primers and 27 dogs (57.5%) using L1/L2. Specific primers MC1/MC2 showed 15 (32%) CS PCR positive dogs, demonstrating the possible presence of more than one Leishmania specie. Results of 13A and 13B primers were compared to blood PCR and IFAT from the same dogs. Using IFAT as gold standard, sensitivity and specificity of CS PCR were respectively 58.6% and 94.0%, positive predictive value 61.0%, negative predictive value 94.0%, and kappa index 0.53, which demonstrates moderate agreement between tests. PCR blood demonstrated a sensitivity of 24.1%, specificity 88.5%, positive predictive value 25% and negative predictive value of 88% and kappa index 0.13, which demonstrates a low agreement between tests. The results showed that conjunctival swab can be a good tool in diagnose CL since is easier to collect than blood or lymphoid organs and has a good sensitivity and specificity. The three screened primers were capable to detect parasite
DNA in CS, which can be useful for epidemiological studies and for direct diagnosis of canine visceral leishmaniasis. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [948] P 540 - INTERPRETATION OF DIAGNOSTIC TEST PERFORMANCES TO IDENTIFY THE CANINE RESERVOIR OF VISCERAL LEISHMANIASIS: RELATIONSHIPS BETWEEN ANTI-LEISHMANIA ANTIBODY AND PARASITE LOADS USING FTA BLOOD SPOT TECHNIQUES. NUNES, C.M.1; BELOTI, C.C.1; CALVO-BADO, L.2; BONFIETTI, L.X.3; HIRAMOTO, R.M.3; COURTENAY, O.2 1.UNESP, UNIVERSIDADE ESTADUAL PAULISTA, FMVA, ARAÇATUBA, SP, BRAZIL; 2.UNIVERSITY OF WARWICK, COVENTRY, UNITED KINGDOM; 3.INSTITUTO ADOLFO LUTZ, SÃO PAULO, SP, BRAZIL.
Keyword:canine visceral leishmaniasis; diagnosis; fta blood spot techniques
Abstract: Brazilian MoH recommendations for reservoir control against zoonotic visceral leishmaniasis transmission is based on identification of IgG antibody in canine sera samples. This study assesses the relative performances of the diagnostic kits currently adopted (DPP rapid test, and EIE ELISA Bio-Manguinhos®) compared to crude leishmania antigen ELISA and kDNA quantitative real-time PCR, the latter tested on FTA card blood spot samples for ease of collection. 400 characterised dogs from 28 endemic municipalities in São Paulo state were tested. Results are discussed in context of field logistics and laboratory expertise requirements. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [953] P 541 - IN VITRO LEISHMANICIDAL AND IMMUNOMODULATORY EFFECTS OF NEW THIOSEMICARBAZONES DERIVATIVES SILVA, A.C.1; SANTOS, T.A.R.1; GOMES, P.A.T.2; MOREIRA, D.R.M.2; LEITE, A.C.L.2; PEREIRA, V.R.A.1 1.CENTRO DE PESQUISAS AGGEU MAGALHÃES, RECIFE, PE, BRAZIL; 2.UNIVERSIDADE FEDERAL DE PERNAMBUCO, RECIFE, PE, BRAZIL.
Keyword:leishmania amazonensis; synthetic compounds; nitric oxide
Abstract: Leishmaniasis is a chronic disease caused by kinetoplastid protozoa belonging to the genus Leishmania, which are transmitted from animals to humans by sandflies. Traditional drugs used in treatment are expensive, have many side effects and has been show patients nonresponsive because of drug-resistant parasite strains. So the need for the development of new, effective, cheap, and safe drugs for the treatment of leishmaniasis still very important. In this work was investigated the antileishmanial and immunomodulatory properties of five new molecules derived from thiosemicarbazone structure, respectively coded by PT01, PT01.2, PT01.3, PT01.4 and PT01.6. For this, Leishmania amazonensis promastigotes (106 parasites/mL) were incubated in the absence or presence of different concentrations (0,19 to 100 ug/mL) of molecules for 96h. After that, the parasite growth was determined by counting in a Neubauer chamber and the concentration that inhibited culture growth by 50% (IC50) was determined. Toxicity in mammalian cells was evaluated in J774 A.1 macrophages incubated or not with different concentrations (1 to 100ug/mL) of compounds for 48h, by MTT method. The 50% cytotoxicity concentration (CC50) was determinate. Selectivity Index (SI) was calculated as the ratio of CC50 to IC50. J774A.1 cells were incubated with the CC50 concentration for 48h and the nitric oxide (NO) levels were assayed in the supernatants by Griess reagent method. The PT01 compound showed the best result against Leishmania amazonensis with low IC50 (0.91 ug/mL) and high
CC50 (14.61ug/mL) presenting the highest SI: 16.05. The others molecules also demonstrated leishmanial activity with low IC50 values, between 1 and 5 ug/mL, and high SI, between 8 and 14. PT01, PT01.2 and PT01.4 stimulated significantly production of NO in macrophages. It is known the important role of NO in the phagocytosis of intracellular pathogens macrophage mediated and a new drug development which is able to act directly on the parasite and in the immune system stimulation, with low toxicity to host cells, can be an ideal approach to a novel treatment against leishmaniasis. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [954] P 542 - CUTANEOUS LEISHMANIASIS: COMPARATIVE STUDIES OF THE METHODS COMMONLY USED FOR DIAGNOSIS IN THE PRESENCE AND ABSENCE OF ANTIBIOTICS PASCUAL, Y.1; DELGADO, O.1; BORGES, R.2; RIVAS, M.1; ROSAS, J.1
1.SECCION DE INMUNOPARASITOLOGIA, INSTITUTO DE MEDICINA TROPICAL, UNIVERSIDAD CENTRAL DE VENEZUELA, CARACAS, VENEZUELA; 2.INSTITUTO DE BIOMEDICINA, CARACAS, VENEZUELA.
Keyword:scarification smears; intradermal test; indirect immunofluorescence assay
Abstract: Cutaneous leishmaniasis (CL) is a parasitic disease considered a public health problem worldwide, early and accurate diagnosis is required for an appropriate treatment. The presumptive diagnosis is based on epidemiological history and the clinical characteristics of lesions, which may have secondary infections, mostly bacterial. Confirmatory tests are applied, parasitological (smear, histopathology, culture and animal inoculation), cell-mediated immunity (intradermal test, IDT) and humoral (indirect immunofluorescence assay, IFA), enzyme-linked immunosorbent assay (ELISA) as well as recently the PCR use and the specific hybridization. The objective of this study was to determine the correlation between tests used in routine diagnosis of CL and if it changes with the use of antibiotics. A comparative study was performed in 114 patients attending in the Inmunoparasitología Section, with skin lesions suggestive of CL, during august/2009-september/2010. Scarification smears, IDT and IFA were performed to the patients. The data was analyzed by two statistical analysis programs SPSS v.10.0 and Microsoft Office Excel 2007. There was a good agreement (Kappa = 0.763) between scarification smears and IDT, weak between scarification smears and IFA (Kappa = 0.397) and moderate between IDT and IFA (kappa = 0.553), without the use of antibiotics. Concordance between scarification smears and IDR decreased antibiotic use (kappa = 0.491), while it was higher between scarification smears and IFA (kappa = 0.498) and between IDT and IFA (kappa = 0.948). In conclusion, the use of antibiotics improved the positivity rate of scarification smears and consequently modified the strength of agreement between it and the immunological tests. The use of antibiotics is recommended before taking samples and apply both tests to a comprehensive confirmatory diagnosis in patients with suspected clinical-epidemiological of cutaneous leishmaniasis. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [971] P 543 - ANTILEISHMANIAL ACTIVITY OF SELAGINELLA SELLOWII HIERON. (SELAGINELLACEAE) EXTRACT ON LEISHMANIA (LEISHMANIA) AMAZONENSIS AMASTIGOTES RIZK, Y.S.1; BOSQUIROLI, L.S.S.1; FISHER, A.1; DE ARAÚJO, V.C.P.1; DA COSTA, E.C.1; QUEIROZ, D.P.S.1; DE OLIVEIRA, A.N.1; MARQUES, M.C.S.2; CAROLLO, C.A.1; DE ARRUDA, C.C.P.1 1.UNIVERSIDADE FEDERAL DE MATO GROSSO DO SUL, CAMPO GRANDE, MS, BRAZIL; 2.UNIVERSIDADE CATÓLICA DOM BOSCO, CAMPO GRANDE, MS, BRAZIL.
Keyword:plant extracts; in vitro infection; cutaneous leishmaniasis
Abstract: Species of Pteridophytes of the genus Selaginella have been cited as having anti-cancer, antitrypanosomal and anti-leishmania activity. Widely found in the Pantanal of Mato Grosso do Sul, Brazil, Selaginella sellowii presents secondary metabolites with potential use in the treatment of American cutaneous leishmaniasis (ACL). This study evaluated the survival of Leishmania amazonensis amastigotes within peritoneal macrophages submitted to the action of acetone extract of S. sellowii, at concentrations of 50, 25 and 12.5 µg/mL, along 24, 48 and 72h. Untreated cells were used as control. The acetone extract (12.5 µg/mL) showed high activity against L. amazonensis amastigotes, reducing in 59,7% the infection index of infected macrophages after 72h. In the other concentrations it was not possible to estimate the infection index due to the insufficient number of cells, suggesting a possible toxicity of the extract in higher concentrations. Further studies are necessary to elucidate the cytotoxicity and characterization of the compounds responsible for the antileishmanial activity. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [975] P 544 - VALIDATION OF HIGH THROUGHPUT DIAGNOSTIC ASSAYS FOR EPIDEMIOLOGICAL SCREENING FOR LEISHMANIA CALVO-BADO, L.A.; KRAVAR-GARDE, L.; SUZANNE, G.; ORIN, C. THE UNIVERSITY OF WARWICK, COVENTRY, UNITED KINGDOM.
Keyword:qpcr; automated system; diagnostics
Abstract: Monitoring Leishmania exposure, infection and parasite loads in reservoir hosts for epidemiological evaluation involves technical, analytical and interpretational challenges. Selection of the appropriate diagnostic assay will also be detirmined by cost. This paper describes the validation of a high throughput diagnostic platform for quantification of Leishmania in buffycoat, dried blood spot-FTA cards, ear tissue and conjunctiva swab samples from endemic dog populations in Brazil and Greece. The DNA extraction, ELISA and PCR quantification was performed using a robotic arm. The sample types and diagnostic assays were compared for performance metrics, and discussed in relation to financial and logistic considerations. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [979] P 545 - FUROSEMIDE IS THERAPEUTICALLY EFFECTIVE AGAINST CUTANEOUS LEISHMANIASIS AND SHOWS INCREASED ACTIVITY AGAINST ANTIMONYRESISTANT LEISHMANIA DONOVANI PARASITES. COSTA, N.A.1; AMARAL, E.E.A.2; FERNANDES, J.R.M.1; BERGMANN, B.R.1 1.UNIVERSIDADE FEDERAL DO RIO DE JANEIRO, RIO DE JANEIRO, RJ, BRAZIL; 2.FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:furosemide; resistance; leishmania
Abstract: Intramuscularly administered pentavalent antimonials such as Glucantime® and Pentostam® are the first line therapy against cutaneous leishmaniasis despite their toxicity and drug-resistance potential. Previously, we showed that furosemide, a clinically approved anti-hypertension drug, can also inhibit the Na+-ATPase activity of Leishmania amazonensis promastigotes (De Almeida-Amaral, E.E et al., 2008). In this work, we evaluated: 1) the oral antileishmanial activity in mice; 2) the responsiveness of antimony-resistant promastigotes of L. amazonensis
and L. donovani to furosemide; and 3) the correlation between Na+-ATPase expression and sensitivity of L. amazonensis and L. chagasi (syn. L.infantum) to furosemide. For in vivo efficacy, BALB/c mice were infected in the ear with L. amazonensis promastigotes. After 7 days, the animals were daily treated with 50 mg/kg of furosemide by the oral route for 79 days. Controls received 14 doses of 20 mg/Kg of Pentostam® or PBS. The lesion sizes were measured throughout the infection with a dial calliper. On day 86 of infection, the animals were sacrificed and the parasite load was quantified by Limiting Dilution Assay. The results showed that oral furosemide prevented lesion growth in a more pronounced way than i.p. Pentostam®. To test the sensitivity of Sb-resistant parasites to furosemide, L. amazonensis and L. donovani promastigotes were rendered resistant to antimony by successive culture with increasing concentrations of antimony tartrate (SbIII). The Sb-resistant and Sb-sensitive promastigotes were cultivated for 72h at 26°C in the presence of furosemide, and growth inhibition was evaluated by MTS. The results showed that contrary to L. amazonensis in which Sb-resistance induced cross-resistance to furosemide, Sb-resistant L. donovani was found to be even more sensitive to furosemide than wild-type parasites. Finally, we correlated the sensitivity to furosemide with the expression of Na+-ATPase in normal L. amazonensis and L. chagasi as measured by qRT-PCR. The results showed that L. chagasi promastigotes expressed more Na+ATPase mRNA than L. amazonensis. However, the sensitivity of L. chagasi to the inhibitory effect of furosemide was lower than L. amazonensis (IC50= 575 µM and 147 µM, respectively). Altogether, these results show that the antileishmanial activity of furosemide is extensive in vivo against cutaneous leishmaniasis in a manner apparently unrelated to its classical Na+-ATPase inhibitory effect, and although yet to be directly demonstrated, has a therapeutic potential in the treatment of Sb-resistant L. donovani infections. Financial support: CNPq. De Almeida-Amaral, E.E; Caruso-Neves, C; Pires, V.M.P.; Meyer-Fernandes, J.R. (2008) Leishmania amazonensis: characterization of an ouabain-insensitive Na+-ATPase activity. Experimental Parasitology, 118(2):165-171. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [983] P 546 - DIAGNOSIS AND TREATMENT PROCEDURES IN A CASE OF LEISHMANIASIS WITH EXTENSIVE MUCOUS INVOLVEMENT. SILVEIRA, A.M.1; CARDOSO, R.M.1; FREIRE, G.M.1; AQUINO, T.A.1; ROSELINO, A.M.F.2; GOMES, C.M.1; JANINO, M.P.D.1; DE PAULA, N.A.2; RIBEIRO SAMPAIO, R.N.1
1.UNIVERSIDADE DE BRASÍLIA, BRASÍLIA, DF, BRAZIL; 2.UNIVERSIDADE DE SÃO PAULO, RIBEIRÃO PRETO, SP, BRAZIL.
Keyword:mucous leishmaniasis; diagnosis; treatment
Abstract: The American tegumentary leishmaniasis (ATL) has a high incidence in Brazil and a potential to determinate disfiguring lesions. A case of mucosal leishmaniasis and it's diagnostic molecular procedures are reported in this study. A 58-year-old male patient, presented nasal obstruction and corysa for 2 years, without improvement with the use of nasal corticoid. Dermatologic exam detected redness, crusting and infiltration in nasal mucosa. The leishmanin skin reaction was positive (30mm) but indirect immunofluorescence wasn`t reactive. Videonasolaryngoscopy detected bulging of the posterior wall of the right oropharynx and hypopharynx, ulceration on the posterior wall of the oropharynx, granulomatous lesion of ventricular band and left vocal fold (VF). In addition, the exam found granulomatous lesions in floor and wall of nasal vestibule, with bilaterally narrowing of the nasal cavity. Histopathological examination showed intense inflammatory histiocytic infiltrate, sometimes forming nodules with giant cell permeation, suggestive of ATL. Polymerase chain reaction-restriction fragment length polymorphism with digestion by Hae III enzyme obtained by filter paper sample and nasal swab
of nasal mucosal detected L. (V.) braziliensis species. Hospitalization was required because of the evidence of severe edema in the VF, soft palate and nasal mucosa, for a course of oral corticosteroids and begining of pentavalent antimony treatment. Mucosal involvement in LTA is uncommon and results from haematogenous or lymphatic dissemination of amastigotes from the skin to the nasal-oropharyngeal mucosa. Oral mucosa and pharyngeal sites aren`t preferentially involved in ML. The lower temperature at the nose and areas of the bolus passage offer favorable condition to installation of the parasite. The presence of the lesion in VF associated with the use of medication without hospitalization could lead to the development of asphyxia, since this finding wouldn`t be discovered in the absence of videonasolaryngoscopic routine examination. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1002] P 547 - IN VITRO AND IN VIVO ACTIVITY OF QUERCETIN AND CHALCONE CH8 IN VISCERAL LEISHMANIASIS SILVA, C.I.M.; FALCÃO, C.A.B.; BERGMANN, B.R. FEDERAL UNIVERSITY OF RIO DE JANEIRO, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:oral treatment; visceral leishmaniasis; flavonoids
Abstract: The activity of the flavonoids quercetin (QC) and chalcone CH8 in the oral treatment of cutaneous leishmaniasis has been investigated by our group in previous studies with successful results. With the aim to investigate whether such activity is extended to visceral leishmaniasis, we performed both in vitro and in vivo assays using the murine model of infection with Leishmania infantum (syn L. chagasi). Firstly, promastigotes of L. infantum were incubated with CH8 or quercetin, resulting in an IC50 of 15,4uM and 423,8uM respectively. To assess in vivo leishmanicidal activity, BALB/c mice were infected in the caudal vein with 2x107 promastigotes at stationary growth phase and, two days later, subjected to 28 daily doses of 150 mg/kg of CH8 or 16 mg/kg of QC administered by oral gavage. Animals were euthanized at the end of the treatment and the serum analyzed for quantification of the enzymes alanine aminotransferase, aspartate aminotransferase and creatinine related to hepatic, cardiac and renal toxicity respectively. Individual livers and spleens were collected for quantification of parasite burden by limiting dilution assay and in situ levels of oxide nitric and cytokines. Spleen cells were also tested for their oxide nitric and cytokine response to lipopolysaccharide and concanavalin in vitro. No signs of toxicity were found for treatment with either of the drugs. In terms of parasite burden, treatment with CH8 led to a significant reduction in both the liver and the spleen. Reduced levels of oxide nitric were found in the spleens of mice treated with CH8 which seems to be a result of their lower parasite burdens. Results also showed the efficacy of QC with a significant reduction of the parasite levels in the liver and a tendency for lower burdens in the spleen. QC appears to have led to a modulation of the host immunity as suggested by the significantly increased levels of IFN- and TNF- found in the spleens of mice at the end of the treatment. Further evidence of QC’s immunostimulatory effect is the increased production of oxide nitric and IFN- by splenic cells stimulated in vitro, when compared to the untreated control group. Altogether, these results show the safe and effective application of CH8 and QC flavonoids in the treatment of visceral leishmaniasis, with the great advantage of using a noninvasive oral route. The mode of action of each drug appears to have a distinct nature, with the former one showing mainly a direct effect on the parasite, while the later appears to also promote the establishment of an effective immune response against the parasite in the murine host. Finantial support by Fundação para a Ciência e Tecnologia (FCT), Portugal. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1007]
P 548 - ARGINASE I, WHAT IS ITS ROLE IN LEISHMANIA BRAZILIENSIS INFECTION? SANTIAGO, R.C.; COSTA, J.F.; BORGES, V.M.; DE OLIVEIRA, C.I. CPQGM/FIOCRUZ, SALVADOR, BA, BRAZIL.
Keyword:arginase ; no; leishmania braziliensis
Abstract: Arginase is an enzyme that metabolizes arginine to ornithine. The increased availability of ornithine favors the synthesis of polyamines, which is important for parasite proliferation. This work aims to advance the knowledge of cutaneous leishmaniasis (CL) caused by Leishmania braziliensis(Lb), evaluating the role of arginaseI (arg1) and nitric oxide (NO), in lesion development and parasite persistence at secondary lymphoid organs. BALB/c mice wereinfected with Lb in the ear dermis.Lesion development, parasite load, arg1 activity and NO production in the ear and in draining lymph nodes (dLN)were evaluated as well as cytokine production. Infected animals develop an ulcerated lesion that heals spontaneously atthe second month of infectionaccompanied by parasite clearance. Parasites, however, persistin the dLN, until the last point evaluated - 6 months after infection. At the infection site, arg1 activity and NO production follow parasite replication and clearance whereas in dLNs, arg1 activity is associated with parasite persistence. Inhibition of arg1 during Lb infection, using Nor-NOHA, a competitive inhibitor, led to a significantdecrease in lesion size accompanied by a reduction in parasite load in the ear and in dLN. Interestingly, this outcome was associated with higher IL-4 and IL-10 production by dLN cells.Administration of arginine, the common substrate for enzymes iNOS and arginase, during the initial phase of Lb infection exacerbatedinjury and parasite replication. Collectively, we observed that arg1 activity and NO production parallel lesion development and healing at the infection site. At sites of parasite persistence, such as the dLN, arg1 activity is increased whereas NO production is suppressed. Our data suggest that arg1 maybe involved in parasite persistenceand, possibly, immunity to re-infection with L. braziliensis. Financial support: CNPq and FIOCRUZ
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1010] P 549 - IN VITRO ACTIVITY OF FRACTIONS AND CRUDE EXTRACTS OF LIBIDIBIA FERREA AGAINST LEISHMANIA AMAZONENSIS WYREPKOWSKI, C.C.1; SANTOS, P.A.2; SOUZA, G.O.2; SOARES, F.V.1; GRAFOV, A.3; GRAFOVA, I.3; LIPIK, V.4; FRANCO, A.R.1 1.INSTITUTO NACIONAL DE PESQUISAS DA AMAZﾃ年IA, MANAUS, AM, BRAZIL; 2.UNIVERSIDADE FEDERAL DO AMAZONAS, MANAUS, AM, BRAZIL; 3.UNIVERSITY OF HELSINKI, HELSINKI, FINLAND; 4.NANYANG TECHNOLOGICAL UNIVERSITY, SINGAPORE, SINGAPORE.
Keyword:fabaceae; bioassay; natural products
Abstract: Leishmaniasis is an endemic disease in the Northern region of Brazil transmitted by a sand fly vector, when it takes a blood-meal on a mammal or human. In most cases the disease is revealed by cutaneous or muco-cutaneous lesions. Treatment of the cutaneous leishmaniasis is difficult due to a limited number of drugs capable to eliminate the intracelular parasite form. The drugs for conventional treatment (Glucantimeﾂｮ and Pentamidine) have to be injected either into a large muscle or intravenously. However, they might have various adverse effects such as: malaise, myalgia, headaches; changes in liver, kidney, and pancreas. Vegetal extracts from native Amazonian species constitute a promising source of natural bioactive products for
alternative treatment of leishmaniasis. Particularly, the genus Libidibia has become an object of detailed chemical and biological investigations. Branches, leaves and fruits of L. ferrea were collected in Manaus (AM, Brazil). The plant material was dried, grinded, and the extracts were prepared with hexane and methanol using sonication treatment for 20 minutes each, and partitioned between dichloromethane (DCM), ethyl acetate, and n-butanol. This fractions and crude extracts were filtred in milipore 0.22 µm, dissolved in complete Schneider Insect Medium, and tested in bioassay using microplates against promastigotes (106cells/mL) of Leishmania amazonensis in different concentrations to determine CI50. Pentamidine was used as the positive control, DMSO and the extraction solvent were negative controls. The number of surviving promastigotes were counted in a Neubauer’s chamber, with Trypan Blue, and compared with the controls after incubation for 24 and 48 hours at 24.9 oC. All tests were performed in triplicate. The most effective fraction (CI50< 62 µg/mL) was obtained by DCM treatment of the methanolic epicarp extract. Hexane crude extract of leaves (CI50< 125 µg/mL) showed a moderate activity, when compared to literature data on other natural plant extracts. The parasites grown in presence of the above samples and transferred into a new cultivation medium without extracts did not show any viable cell. Hence, the extracts possess a pronounced leishmanicidal activity, but the mode of action has to be an object of further investigation. Chemical studies of the samples provide additional information for understanding of the composition and the mechanism of action of the natural products in question. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1013] P 550 - IN SILICO SCREENING OF PROTEINASES INHIBITORS FOR POTENTIAL DRUGS FOR TREATMENT OF LEISHMANIA (VIANNIA) BRAZILIENSIS INFECTION. SILVA, F.S.; ALMEIDA, M.S.; RIBEIRO, M.L.; SOUZA, R.S.; PEREIRA, B.S.; CAFFARENA, E.R.; ALVES, C.R. FUNDAÇÃO OSWALDO CRUZ, RIO DE JANIRO, RJ, BRAZIL.
Keyword:l. (v.) braziliensis; proteinases; isoforms
Abstract: Currently, the standard screening of compounds to inhibit Leishmania sp. proteinases does not usually consider the following points: (i) the possibility of the compound targeting multiple proteinase classes, (ii) the possible existence of different isoforms of the target proteinase, and (iii) the synergism existing between proteinases activity. We hypothesize that if one was to take these additional points in consideration when selecting candidate-compounds, it would be possible to use as few as a single proteinase inhibitor as efficient chemotherapy drug. In this work, we propose an in silico approach to identify inhibitors acting over a broad spectrum of Leishmania (V.) braziliensis proteinase classes, an important causative agent of mucocutaneous leishmaniasis in Brasil. We have searched the L. (V.) braziliensis cysteine-, metallo-, and serine proteinases (including all their isoforms) annotated in the databank GeneDB. To this end, representative sequences of proteinases of each class were aligned to sequences in the databank using the ALIGN EMBOSS server, to identify annotated sequences with high identity scores to the representative ones. The results identified 63 cysteine-, 96 metallo-, and 22 serineproteinases. Among cysteine proteinases we were able to identify 12 isoforms and, also, 13 in metallo proteinases, all with identity > 60%. Three dimensional (3D) models representatives for each isoforms group were built by homology and the modeled proteins structures were validated by Procheck programs: ERRAT and PROVE WHAT-CHECK. In the sequence, compounds with structure similar to inhibitors of all studied proteinase classes were retrieved from in ZINC database. Only compounds with a similarity rate of at least 60% to the inhibitors were selected. This strategy was able to retrieve 790 potential inhibitors of cysteine-, 9 of metallo- and 1357 of serine-proteinases. Additionally, the chemical structures of each group of inhibitors were aligned using Ligand.info databank, to identify the main chemistry structures of inhibitors of each proteinase class. The selected compounds were subjected to molecular docking and the
results indicate that some of them can have a multivalency inhibitory affinity over distinct groups of a proteinase class. Such observation does not apply to serine-proteinases, as enzymes of this class have a higher variability making it difficult for a single inhibitor to act on multiple enzymes of this proteinase class. Financial support: CNPq, CAPES and FAPERJ.
- CLINICAL LEISHMANIASIS [1016] P 551 - CLINICAL COMPARISON OF LOW AND REGULAR ANTIMONY DOSES THERAPIES LONG TIME AFTER TREATMENT OF CUTANEOUS LEISHMANIASIS IN RIO DE JANEIRO STATE, BRAZIL VIEIRA-GONCALVES, R.1; HERINGER, J.F.1; NOGUEIRA, R.S.1; GOMES-SILVA, A.1; DACRUZ, A.M.1; OLIVEIRA-NETO, M.P.2 1.LABORATÓRIO INTERDISCIPLINAR DE PESQUISAS MÉDICAS/IOC/FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 2.IPEC/FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:leishmaniasis; antimony; therapy
Abstract: Introduction: Cutaneous leishmaniasis (CL) is the most frequent clinical form of leishmaniasis in Brazil, whereas mucosal disease (ML) is rare. Pentavalent antimony (Sb+) is used for treatment since 1912, and may cause heart and kidney adverse effects. In Rio de Janeiro State (RJ) Leishmania (Viannia) braziliensis is the specie associated with CL. The regular dose (RD) recommended by the Brazilian Health Ministry (10-15mg of Sb+/kg/day/21 days) is effective to cure CL patients from RJ. However, low Sb+ doses (LD) (5mg Sb+/kg/day/30 days) are also effective. If LD therapy increases relapses or evolution to mucosal leishmaniasis (ML) long time after therapy is not known. Our goal was to show if LD is as effective as RD even long time after therapy. Methods: 310 CL healed patients from Rio de Janeiro (IPEC/FIOCRUZ) and Paraty (SMS Paraty) municipalities previously treated with LD (n=175) and RD (n=135) were included. Thirty-nine field visits were done for patient’s clinical and laboratorial evaluation. Results: 52 CL-LD patients and 30 CL-RD patients were found. The groups were examined 1125 (LD) and 10-27 (RD) years after treatment. During active disease, all patients had typical CL lesions and positive Montenegro Skin Test. The age ranged from 5-76 years old in LD and from 5-69 years old in RD groups. After treatment, 48 LD patients (92%) cured and 4 patients (7.5%) needed a second treatment with RD. All patients treated with RD had cured. Two patients, one in each group, developed ML one year after treatment and were cured with 20mg Sb+/kg/day and amphotericin-B. The mean total Sb+ accumulated dose in the LD patients was 8,574 mg/patient. If they were treated with RD it would be 18,479 mg/patient. Conclusion: no drug resistance was observed in patients treated with RD after an initial LD. The cure was reached in 92% of cases with less than half of the recommended dose. Besides the easier administration, lower dose cause less adverse effects. A mean economy of $93.00/patient was achieved. One LD patient developed ML. Anyhow, this form occurs in 3-5% of all CL cases. We believe that CL resistance to Sb+ may occur independent of the used dose since RD therapy may occasionally fail. These results showed that in RJ patients, LD Sb+ therapy was as efficient as RD to clinically cure CL even long time after therapy (11-25 years), at least with L. braziliensis strains that circulate in Rio de Janeiro State. Support: IOC/FIOCRUZ, CNPq, FAPERJ. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1017] P 552 - PCR SCREENING OF VISCERAL LEISHMANIASIS BY CONJUNCTIVAL SWAB IN DOMICILED DOGS OF NORTH REGION OF BELO HORIZONTE, MINAS GERAIS STATE, BRAZIL. LEITE, R.S.; BARBOSA, A.D.; FERREIRA, A.L.C.; ANDRADE, A.S.R.
CENTRO DE DESENVOLVIMENTO DA TECNOLOGIA NUCLEAR, BELO HORIZONTE, MG, BRAZIL.
Keyword:canine visceral leishmaniasis; conjunctival swab; molecular diagnosis
Abstract: PCR assays have greatly improved the sensitivity of visceral leishmaniais (VL) diagnosis in dogs. Various canine tissues (including blood, urine, skin biopsies, lymph node, bone marrow and spleen) have been used for PCR detection of the parasite. However, the non invasive samples assume great importance in this context because they are simpler, painless and more easily allowed by the dog-owners. Non invasive samplings would represent an essential tool in mass-screening survey for interventional programs. An interesting approach in this context is the conjunctival swab (CS), a method for sample collection that uses a sterile swab for sampling the dog conjunctivas. This method was shown to be highly sensitive when used for diagnosis of symptomatic and asymptomatic dogs. The purpose of this study was to evaluate the molecular diagnosis of canine visceral leishmaniasis using conjunctival swab (CS) samples in dogs living in a highly endemic area and to compare the results with those obtained by the conventional serological tests used by the Brazilian VL control program. Eight hundred and seventy-seven dogs domiciled in the North Region of Belo Horizonte city (Minas Gerais state, Brazil) were screened. In parallel to the annual canine serological survey, in which blood samples were collected in filter paper, CS were also collected from both dogs conjunctives. The dogs were also clinically evaluated by a veterinarian. The DNA purification from CS was carried out by the phenol/chloroform method and after extraction DNA from both conjunctivas of the same dog were mixed constituting a single sample for the PCR assay. Leishmania infantum indirect fluorescent antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) and real time PCR of CS samples were performed and the results were compared. The real time PCR was performed using Sybr Green and primers addressed to kDNA minicircles. Sixty-six percent (579/877) of dogs showed at least one symptom related to VL, being skin changes the main symptom. In the serologic assays 11% (97/877) of animals were ELISA positive and 5.2% (46/877) IFAT positive. In real time PCR assay 25% (222/877) were positive. The real time PCR using CS samples was highly sensitive and able to detect Leishmania infection in symptomatic or assymptomatic dogs with negative or positive serological diagnosis. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1020] P 553 - QLOGP INFLUENCE IN ACTIVITY ANTILEISHMANIAL BY BENZOPHENONE DERIVATIVES ALVES, K.F.; DE ALMEIDA, L.; MACIEL-REZENDE, C.M.; PIRES, F.R.; DOS SANTOS, M.H.; MARQUES, M.J. UNIVERSIDADE FEDERAL DE ALFENAS, ALFENAS, MG, BRAZIL.
Keyword:leishmania; benzophenones; lipophilicity
Abstract: Leishmaniasis is a group of tropical diseases caused by species of protozoan parasites of the genus Leishmania. The drugs currently available for treatment of leishmaniasis are unsatisfactory because of their limited efficacy, frequent side effects, and increasing drug resistance. As a result of previous studies reveals that some natural benzophenones exhibit action on parasites such as Leishmania and Trypanosoma cruzi. The aiming of this work was correlate the leishmanicidal activity in promastigote forms of Leishmania (Leishmania) amazonensis with the calculated logP (QlogP) from benzophenone derivatives besides their starting compounds (CMA, CMB, CMC, LFQM-116 to LFQM-123). To evaluated the antileishmanial activity, benzophenones solubilized in DMSO were added to promastigotes of L. (L.) amazonensis (1x106 cells/ml ) in Schneider medium, performed in triplicate on three
separate occasions. Results were expressed as percentage inhibition compared to control. Lipophilicity values were estimated by determining the theoretical QlogP (octanol/water partition coefficient) using QikProp program. This software calculates the QlogP from regression equations using the experimental data and molecular descriptors (physical count of hydrogen bond, atom types and charges, etc.) through statistical mechanical simulations of Monte Carlo. The concentrations of 50% inhibition of growth (IC50) promastigotes with QlogP of starting benzophenones were CMA (29.00 µg/ml, 3.46); CMB (40.92 µg/ml, 3.07) and CMC (121.36 µg/ml, 2.71). The results presented by benzophenone derivatives were LFQM-115 (4.90 µg/ml, 3.79), LFQM-116 (9.80 µg/ml, 5.04), LFQM-117 (7.05 µg/ml, 5.29), LFQM-118 (5.05 µg/ml, 3.40), LFQM-119 (8.56 µg/ml, 4.60), LFQM-120 (7.82 µg/ml, 4.73), LFQM-121 (21.30 µg/ml,3.57), LFQM-122 (23.80 µg/ml, 5.71) and LFQM-123 (5.94 µg/ml, 6.21). From the results we can see that the QlogP of the compounds and the IC50 values show that the inclusion of groups resulted in derivatives with lipophilic profile more pronounced than their respectively precursors moreover, the benzophenone derivatives leishmanicidal activity on promastigotes forms, was similarly, most promising than the precursors. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1023] P 554 - HEMATOGENIC SPREAD OF LEISHMANIA (VIANNIA) IN NEUTROPHILS AND MONOCYTES ABREU, H.C.N.; CONTER, C.C.; PEDROSO, R.B.; SILVEIRA, T.G.V.; ARISTIDES, S.M.A. UNIVERSIDADE ESTADUAL DE MARINGÁ, MARINGÁ, PR, BRAZIL.
Keyword:leishmania; hematogenic spread; mononuclear cell
Abstract: After inoculation of Leishmania promastigotes in the host, the establishment of infection occurs involving phagocytosis and parasitism of neutrophils and macrophages. Since the persistence of Leishmania was already demonstrated, the possibility of finding Leishmania DNA in neutrophils and monocytes enables a better understanding of the pathogenesis of cutaneous leishmaniasis (CL). The objective was evaluating the presence of Leishmania (Viannia) DNA in polymorphonuclear and mononuclear blood cells from patients suspected of CL, using the Polymerase Chain Reaction (PCR). In order to achieve the cells, 6 mL blood with EDTA were collected and the reagent Mono-Poly Resolving Medium (MP Biomedicals, LLC) was used as prescribed by the manufacturer. After polymorphonuclear and mononuclear separation and washing, DNA was extracted by guanidine isothiocyanate and phenol. Were used MP3H and MP1L primers for amplification. Were studied 94 patients, 63.83% were men and, 36.17% women. The majority of patients (86-91.49%) had cutaneous lesions, with a predominance of single lesion (58.51%), and 8 (8.51%) presented mucosal lesion. From 94 patients, 71 (75.53%) had primary lesion and no history of CL and, 23 (24.47%) reported previous infection. From 71 patients with primary lesions and no history of LT, 30 (31.91%) were diagnosed with CL, according to the Health Ministry (Brazil). From 23 patients who reported previous infection, 7 (30.43%) presented reactivation of the primary lesion, 2 (8.69%) presented new skin lesion and, 14 (60.86%) returned for treatment control. PCR was positive for two patients who presented active primary lesion with 3 to 6 months of time evolution; these patients did not present history of CL and were diagnosed according to tests recommended by the Health Ministry (Brazil). The presence of Leishmania (Viannia) DNA in mononuclear and polymorphonuclear cells demonstrates the parasite persistence, considering the lesion time in both patients. This persistence indicates that, in some patients, the parasite is able to escape to the macrophage elimination mechanisms and remain in the body and, hematogenic spread can occur with development of mucosal lesion.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1026] P 555 - A NOVEL FLUORESCENT PROBE FOR THE STUDY OF LEISHMANIA SMALLMOLECULE PHARMACOLOGY BATISTA, A.J.S.1; OLIVEIRA, S.D.1; DUVAL, R.2; COUTINHO-SILVA, R.1; ROSSIBERGMANN, B.1 1.UFRJ, RIO DE JANEIRO, RJ, BRAZIL; 2.UNIVERSITÉ TOULOUSE III, TOULOSE, FRANCE.
Keyword:leishmaniasis; fluorescent probe ; pharmacology
Abstract: We have devised a novel fluorescent probe derived from antiparasitary natural products, in order to study the small-molecule pharmacology of Leishmania in vitro. The new probe penetrates rapidly and with elevated specificity in L. amazonensis promastigotes, and allows stable fluorescent labeling of the treated cells. This probe thus demonstrates optimal features for the biological screening of natural and synthetic small molecules by fluorescence microscopy. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1027] P 556 - TOPICAL TREATMENT USING A CHLOROALUMINUM PHTHALOCYANINE (CLALPC)-NANOEMULSION AGAINST LEISHMANIA (VIANNIA) BRAZILIENSIS INFECTED BALB/C MICE ESCOBAR, P.1; LEAL, S.M.1; CARREÑO, H.1; JUNIOR, E.R.2; CORREA, J.R.3; DE AZEVEDO, R.B.3 1.UNIVERSIDAD INDUSTRIAL DE SANTANDER, BUCARAMANGA, COLOMBIA; 2.UNIVERSIDAD FEDERAL DE RIO DE JANEIRO, RIO DE JANEIRO, RJ, BRAZIL; 3.UNIVERSIDAD DE BRASILIA, BRASILIA, DF, BRAZIL.
Keyword:topical treatment; chloroaluminum phthalocyanine; nanothechnology
Abstract: Nanoemulsions(NE) as delivery system is an interesting option for cutaneous leishmaniasis (CL) ensure better solubility and biodistribution of hydrophobic compounds. We determinate the efficacy of photodynamic therapy (PDT) using ClAlPc-NE topically in BALB/c mice infected with L(V).braziliensis. ClALPc-NE was prepared by ultrasonication and size, polydispersity index (PDI) and Zpotential were determined. Female BALB/c mice 8-10 weeksold were intradermically infected at the base of the tail with 2x106 L.(V)braziliensis stationary promastigotes. Forty five days after, animals were randomly separated. In Exp1 (two groups, 3 mice each), animals were treated with 100 µM of ClPcAl-NE/day at interval of 2 days, 4 times. Control mice were leaving without treatment. Fifteen minutes after each dose mice were anesthetized and irradiated for 20 min using a red lightemitting diode (LED). In Exp2 (5 groups, 4 mice each) mice were treated as followed: Group1, 200 mg/kg/day Glucantime/20 days intramuscularly; group2, 100 µM ClPcAl-NE/day, 10 times, two days interval; group3, 300 µM ClAlPc in DMSO:Tween 80:type I water, 5 times, two days interval; group4, NE without ClAlPc; and group5, without treatment. Three hour after each treatment, mice from 2-4 groups were irradiated as above. Animals were sacrificed 20 days after last dose. Lesion sizes were estimated weekly using a dial caliper and parasitological efficacy was determined microscopically. The ClAlPc-NEs form Exp 1 and 2 showed: size 129nm, PDI 0.1, Zpotential -13.3mV and size 311.4nm, PDI 0.403, Zpotential -23.5mV respectively. In Exp1, the lesion size before treatment was 22.51±11.08mm2 and at the end of experiment was 18.81±7.92 mm2 and 19.97±19.25 mm2(control). In Exp2 lesion areas before and at the end of treatment were: Grup1, 31.97±8.92mm2 and 107.61±7.80mm2 (nodule)/44.17±4.80mm2 (ulcer); group2, 62.06±8.16mm2 and 211.74±23.14mm2 (nodule)/107.90±16.02mm2 (ulcer); grup3,
20.25±6.57 mm2 and a necrosis in tail and leg at the end. Control groups (4 and 5) lesion areas were 161.23±53.65mm2 (nodule)/63.80±17.74mm2(ulcer) at the end. In all stained slides abundant intracellular and extracellular parasites were observed in skin preparation but not in liver or spleen. The lack of ClAlPc-NE efficacy reaffirm the difficulties for development of CLtopical treatment by L.(V)braziliensis. More dosages are recommended however a precaution in order to avoid necrosis or deleterious reactions is recommended.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1028] P 557 - STRUCTURAL AND FUNCTIONAL STUDIES OF LEISHMANIA MAJOR NUCLEOSIDE DIPHOSPHATE KINASE DE OLIVEIRA, A.H.C.1; DIAS, F.C.2; WARD, R.J.1; PINTO, M.R.1; VIEIRA, P.S.1 1.FFCLRP-USP, RIBEIRÃO PRETO, SP, BRAZIL; 2.FMRP-USP, RIBEIRÃO PRETO, SP, BRAZIL.
Keyword:enzyme; mutagenesis; overexpression
Abstract: The nucleoside diphosphate kinase (EC 2.7.4.6; NDK) is ubiquitous in both prokaryotes and eukaryotes catalyzing the transfer of the gama-phosphoryl group from a nucleoside triphosphate to a nucleoside diphosphate by a ping-pong mechanism. Besides the involvement of this enzyme in maintaining the nucleotides cellular pool, it was described in other cellular processes, and is considered a multifunctional enzyme. Studies have demonstrated that NDKb is secreted by intracellular pathogens during infection, including by Leishmania, preventing ATP-induced cytolysis of macrophages, thereby preserving the integrity of host cells to the benefit of the parasite. Our group has previously shown NDKb as an abundant component of the microsomal fraction of L. major promastigotes by subproteomic analysis (de Oliveira et al., Comp. Biochem. Physiol. Part D, vol. 3, 2006). Thus, the characterization of L. major NDK (LmNDK) may contribute to understand the functions of this protein in parasite/host interactions. The LmNDK coding sequence was amplified genomic DNA, cloned into pET28a and modified by sitedirected mutagenesis. The recombinant LmNDK and mutants R17A, E28A, P95S, P100S- 5 and 5C-term were efficiently expressed as soluble form in E. coli and purified by affinity chromatography. The solution characterization was carried out with the recombinant LmNDK and the P95S, P100S- 5 and 5C-term mutants. Circular dichroism and intrinsic fluorescence of tryptophan emission experiments showed LmNDK is more stable than P95S and P100S- 5 mutants. The catalytic activity analysis showed the P95S had a similar LmNDK activity, but other mutants (R17A, E28A, P100S- 5 and 5-Cterm) exhibited a decreased activity. All proteins presented a cytotoxic effect in the ATP presence, but this effect was reduced in the acceptor presence. Polyclonal antibodies raised against the native protein from the promastigote extracts and the subcellular localization, using fluorescence microscopy, demonstrated that the native NDK was localized in the promastigote cytoplasm. L. major (LV39) transfectant overexpressing LmNDK presented partial virulence attenuation in the BALB/c footpad lesion. Preliminary comparative analysis of 2D-gels protein profile of transfectants revealed differences of promastigote protein expression, including the NDK overexpression. Supported by FAPESP, CNPq, PRP-USP.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1031] P 558 - ENCAPSULATION IN LIPID CORE NANOCAPSULES INCREASES THE ORAL EFFICACY OF QUERCETIN AGAINST CUTANEOUS LEISHMANIASIS
SOUSA-BATISTA, A.J.1; POLETTO, F.2; POHLMANN, A.R.2; GUTERRES, S.S.2; ROSSIBERGMANN, B.1 1.UFRJ, RIO DE JANEIRO, RJ, BRAZIL; 2.UFRGS, PORTO ALEGRE, RS, BRAZIL.
Keyword:nanotecnology; leishmaniasis ; chemoterapy
Abstract: Our group has previously described the in vivo activity of quercetin (QC) against Leishmania amazonensis when administered by the oral route (Muzitano, M.F., et. al., 2008). With the purpose to improve antileishmanial efficacy we encapsulated QC and its pentacetylated derivative (PQC) in lipid core poly(epsilon-caprolactone) nanocapsules (LNC), prepared by interfacial deposition of preformed polymer. To test those formulations, BALB/c mice were infected in the ear with 2x106 L. amazonensis GFP-promastigotes. After 7 days of infection the oral treatment was initiated daily for 52 days with either the free drugs (QC and PQC) or their LNC formulations (LNC-QC e LNC-PQC, respectively). The ear lesion sizes were measured throughout the infection with a dial caliper. On day 59 of infection, the animals were sacrificed and the parasite load in the lesions was quantified both by Limiting Dilution Assay and by fluorimetry. We observed that nanoencapsulation increased drug effectiveness by 40-fold in relation to free drugs. To evaluate if the formulations induced cardiac, liver or kidney toxicity, the concentrations of aspartate aminotransferase (TGO), alanine aminotransferase (TGP) and creatinine were measured in the serum. None of the formulations tested showed detectable toxicity. These results show that the nanocapsules raised the efficacy of free QC and PQC in the treatment of cutaneous leishmaniasis through a non-invasive oral administration. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1032] P 559 - THE ANTILEISHMANIAL EFFECT AND SKIN ABSORPTION STUDIES OF LIPID-CORE POLY- €-CAPROLACTONE NANOCAPSULES ENTRAPPING THE CHALCONE CH8 USING BIOLUMINESCENT MICROSCOPY. LOPES, M.V.1; POLETTO, F.2; FERRARINI, S.R.2; MEDEI, E.H.1; GUERRA, B.1; GUTERRES, S.S.2; POHLMANN, A.R.2; GOMES, A.M.O.1; BERGMANN, B.R.1 1.UFRJ, RIO DE JANEIRO, RJ, BRAZIL; 2.UFRGS, PORTO ALEGRE, RS, BRAZIL.
Keyword:tegumentar leishmaniasis; topical treatment; nanocapsules
Abstract: No effective topical treatment exists for cutaneous leishmaniasis in many parts of the world. This is mainly due to the difficulty of an active drug to cross the tickened skin barrier. Over the last years, new drug delivery systems have been investigated to promote increased skin permeation. In this study we evaluated the efficacy of lipid-core poly-€-caprolactone nanocapsules entrapping the antileishmanial chalcone CH8 (LNC-CH8) to treat cutaneous leishmaniasis by the subcutaneous (sc) route. Also, by using fluorescent rhodamine-labeled LNC-CH8 (LNCF-CH8), we conducted bioluminescence studies to evaluate macrophage internalization and skin absorption across the skin. The LNC-CH8 and LNCF-CH8 nanoformulations measuring 210 nm were prepared by interfacial deposition of rhodaminecoupled preformed polymer. The antileishmanial activity of LNC-CH8 was first evaluated in vitro against L. amazonensis promastigotes cultured with varying concentrations of LNC-CH8, free CH8 or empty LNC. The number of parasites was counted after 48h of culture at 27oC. The results showed that like free CH8, LNC-CH8 inhibited promastigote growth at IC50 = 1.2 µg/mL. For in vivo efficacy, BALB/c mice were infected in the ear and from day 7 were treated twice a week for 3 weeks with 5 µg of CH8 in LNC or in its free form. LNC-CH8 s.c. treatment effectively controlled parasite growth in the infected ears as compared to untreated and free CH8-treated mice. To evaluate macrophage uptake, non-infected and L. amazonensis-GFPinfected macrophages were incubated for 2 hours with LNCF-CH8. Images of red-fluorescent
particles and green-fluorescent parasites were taken by intravital multiphoton microscopy, showing LNCF-CH8 co-localized with the parasites inside macrophage vacuoles. To evaluate skin absorption, LNCF-CH8 was topically applied onto shaved dorsum of mice. At different times after application, bioluminescent images were taken with IVIS LUMINA, showing a decreased fluorescence indicative of permeation at 120 min of LNCF-CH8 topical application. In all, these results demonstrated that lipid-core LNC-CH8 nanocapsules are effective against L. amazonensis in vitro and in vivo, can deliver the lipophilic drug CH8 into the parasitophorous vacuoles of macrophages, and can permeate mouse skin, indicating potential use in topical treatment of cutaneous leishmaniasis. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1033] P 560 - EX VIVO PERMEATION AND RETENTION OF CHOLOALUMUNINUM PHTHALOCYANINE (CLALPC) NANOEMULSION IN HEALTHY HUMAN SKIN LAYERS, FOR OPTIMIZATION OF TOPICAL FORMULATIONS FOR CUTANEOUS LEISHMANIASIS ESCOBAR, P.; OSPINA, V.E.; CONDE, C.A.; MANTILLA, J.C. UNIVERSIDAD INDUSTRIAL DE SANTANDER, BUCARAMANGA, COLOMBIA.
Keyword:franz diffusion cells; topical treatment; nanothechnology
Abstract: The ClAlPc, a phototosensitizer used in photodynamic therapy (PDT), constitutes an important alternative for cutaneous leishmaniasis (CL) treatment. The ClAlPc topically applied must penetrate skin layers and be retained in dermis, place where Leishmania-infected macrophages reside. The aim of this study was to determine the permeation and retention of ClAlPc, incorporated in a nanoemulsion (NE) delivery system, in healthy human skin membranes .The ClAlPc-NE (oil-to-water) was prepared by ultrasonication techniques. The average size was 195.2 nm, polydispersity index 0.235, zeta potential -18.8mV, ClAlPc concentration 256.96 ¾M and phospholipids concentration 4788 nmol/mL. Abdominal skin was obtained from healthy women under gone cosmetic surgery (Ethics Committee approval). The skin was washed, cleaned for subcutaneous fatty tissue and storage at -20°C up to 2 months. Skin permeation studies were performed on full-thickness human skin using Franz diffusion cells with exposure to ClAlPc-NE for 0, 4, 8, 10 12 and 24 h. PBS 7.4 SDS 2% was used as receptor fluid. Two different skin donors were used. Six assays (6 cells each) were made. Skin integrity before and after experiments were analyzed from skin biopsies stained with hematoxylin-eosin. ClAlPc retained in stratum corneum (SC) was analyzed by skin surface stripping with adhesive tape (15 tapes). Each tape was placed for 30 seconds on the membrane and transferred to ClAlPc extraction-solvent for 24 h. ClAlPc retained in epidermis and dermis (E+D) was analyzed from a macerate of the remaining membrane tissue transferred for extraction as above. The ClPcAl concentration was analyzed by spectrofluorimetry and results were expressed as nM/cm2. The ex vivo treatment of human membranes with ClAlPc-NE in Franz diffusion cells was unable to detect, at any point of time, ClAlPc concentration in the receptor medium indicating that ClAlPc could not completely through the skin. The ClAlPc was retained mainly in SC with average values of 42.03 nM, and in E+D with values of 7.3 nM. Histological changes were noted in the skin after testing such as: SC detachment, collagen fragmentation and changes in the conformation of the epidermis stratified epithelium. The results showed that although ClAlPc contained in a NE could not diffuse across the skin, it could penetrate and be retained in the different parts of the skin (SC, E, D). The greater retention SC compared with E+D sugested than NE should be improved to provide a greater flow of the compound into deeper layers of the skin, promoting greater ClAlPc retention in the dermis.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1041] P 561 - ANTI-LEISHMANICIDAL ACTIVITY OF THE ANTIMICROBIAL PEPTIDE DRS01 OBSERVED IN LEISHMANIA INFANTUM (SYN. LEISHMANIA CHAGASI) CELLS BY ATOMIC FORCE MICROSCOPY SILVA, V.C.1; EATON, P.2; BITTENCOURT, C.R.3; VERAS, L.M.C.3; COSTA, C.H.N.1; FEIO, M.J.F.2; DE ALMEIDA LEITE, J.R.S.3
1.LABORATÓRIO DE PESQUISAS EM LEISHMANIOSES DO INSTITUTO DE DOENÇAS TROPICAIS NATAN PORTELLA, TERESINA, PI, BRAZIL; 2.REQUIMTE, DQB, FACULDADE DE CIÊNCIAS DA UNIVERSIDADE DO PORTO, PORTO, PORTUGAL; 3.NÚCLEO DE BIODIVERSIDADE E BIOTECNOLOGIA DA UNIVERSIDADE FEDERAL DO PIAUÍ, PARNAÍBA, PI, BRAZIL.
Keyword:antimicrobial peptide; leishmania infantum; atomic force microscopy
Abstract: Background: Leishmaniasis is one of the most serious diseases in the world, with inadequate medication, especially for visceral leishmaniasis, commonly caused by Leishmania (L.) infantum. A recently described antimicrobial peptide DRS 01 has been reported to kill L. infantum promastigotes, but nothing is known about its mode of action or effect on the cell. Methods: The Dermaseptin 01 (DS01) isolated and identified from frog skin secretions of Phyllomedusa hypochondralis was synthesized in an automated peptide synthesizer (PSSM 8, Shimadzu) by solid-phase synthesis according to the F-moc procedure and purified by RPHPLC. Leishmania infantum live cells were treated for 24 h with 16, 64 and 128 µg/mL of DRS 01, concentrations chosen to be below, around and above LC50, and samples were prepared for microscopic observation. Results: In the AFM images of the treated cells, considerable differences were found compared to the control. In general disturbances were found on the membranes of the cell body, and often the cells appeared less elongated after treatment. The surface roughness increased with treatment with the antimicrobial peptide. The roughness, as measured by Rq, almost doubled from 3.4 ± 0.8 nm for the control cells to 7.1 ± 2.1 nm for the cells treated with 128 µg/mL DRS 01. Conclusions: For the first time, high-resolution microscopy has been carried out on promastigote forms of Leishmania infantum, and revealed important morphological details of the parasite. The results suggest than the mode of action of DRS 01 is directly on the membrane, presumably leading to cell lysis. The implication of a membrane-directed attack is that resistance will be difficult for the parasite to develop, and that the specificity towards the target cells is likely to be high. Supported by FCT/CNPq Bilateral project Brasil/Portugal, FCT project PEst-C/EQB/LA0006/2011 and Nanobiomed Network CAPES/Brasil.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1043] P 562 - GOLD NANOROD-BASED LOCALIZED SURFACE PLASMON RESONANCE BIOSENSOR FOR SENSITIVE DETECTION OF ANTIBODY ANTI-LEISHMANIA INFANTUM IN CANINE SERUM SESANA, A.M.; DE JESUS, A.C.; ANDRADE, H.M.; LADEIRA, L.O.; GAZZINELLI, R.T. UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL.
Keyword:nanotechnology; visceral leishmaniasis; diagnosis
Abstract: Up to date, immunoassay plays a prominent role in the early diagnosis of infectious disease. As the current gold standard, enzyme-linked immunosorbent assay (ELISA) has been one of the
most extended methods for the serological diagnosis in immunogenic protein detection. It still has some shortcomings, such as long testing time needed, indirect format of detection and multiple washing steps. Efforts have been made to increase their detection limits, however, progress has been disappointing especially in quantitative analysis. Recently, there have been extensive efforts to develop nanoparticles-enhanced analytical technologies for the highly sensitive and selective detection of protein biomarkers that can be powerfully utilized to diagnose various types of diseases. Gold nanorods (GNRs) functionalized with antibodies can also be used in sandwich assays to greatly enhance the sensitivity of measurements for the ultrasensitive detection of protein biomarkers, based on the surface plasmon resonance (SPR). Functionalization of the GNRs with antibodies or other biomolecules allows their specific attachment to any target cell. This is a useful attribute for biomedical diagnostic applications. In this study, it has been taken as the example for the antibody detection, a hypothetical protein of Leishmania infantum labelled on the surface of the GNRs through covalent adsorption. A hypothetical protein has previously chosen interesting results in terms of sensitivity and specificity in ELISA tests for the diagnosis of disease in canine serum. Some characterizations have been conducted to access the nature of the GNRs after biological modification. In addition, a validation experiment also has been designed in a 96-well microplate based on ELISA to evaluate the influence of steric effects on the binding affinity of the antibody–antigen pair. The use of the hypothetical protein of Leishmania infantum associated with the gold nanorods enable up to a further 102 times increase in sensitivity of the diagnosis test compared with the ELISA method. The technique presented in this study can play a key role in developing tunable sensors for sensitive and precise monitoring of different biological interactions.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1044] P 563 - ANTILEISHMANIAL ACTIVITY OF NEW QUINONE DERIVATIVES OF LAPACHOL REMPEL, S.S.1; GOMES, S.2; CANTO-CAVALHEIRO, M.M.1; DA SILVA, A.J.M.2; COSTA, P.R.R.2; CUNHA-JÚNIOR, E.F.1; CAMPOS-SANTOS, E.C.1 1.FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 2.UFRJ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:chemotherapy; leishmania amazonensis; quinone
Abstract: Considered by World Health Organization (WHO) a neglected disease, leishmaniasis affects over 12 million people in the world per year. Treatment receives low investment from the pharmaceutical industries, requires long periods of administration and presents low efficacy, resistance, toxicity and high cost. Quinones represent a very important class of molecules which is directly related to many metabolic processes in the cell and presents trypanosomicidal effect. The purpose of this study is to evaluate the activity of 12 derivatives from lapachol (LQBs) in promastigotes and intracellular amastigotes of L. amazonensis and their toxicity in murine macrophages. Promastigotes (1x106/ml) were incubated with different concentrations of LQBs or none by 72 h and the parasite viability was analyzed by MTT assay. The activity on intracellular amastigotes was evaluated by light microscopy in L. amazonensis-infected murine macrophages after incubation with several concentrations of LQBs for 72 h. Toxicity was evaluated in murine macrophages by MTT assay after 72 h of incubation. The differences on the structure of each substance were related to some aspects of the drug action to the cells. The closing of the ring in "3" rendered low toxicity and antiamastigote activities, while the addition of a diethylformamide in "2" gives higher activity to intracellular amastigotes but also higher toxicity. On the other hand, when both modifications took place in the same compound, a series with selective antiamastigote activity was originated, from which LQB-270 presented the lowest IC50 (1.5 µM) on intracellular amastigotes and selectivity index high. This structure-activity
study pointed LQB-270 as a promising candidate to be evaluated in experimental cutaneous leishmaniasis. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1051] P 564 - NEW THIOSEMICARBAZONES DERIVATIVES: IN VITRO EFFECTS ON LEISHMANIA AMAZONENSIS AND IMMUNE SYSTEM CELLS SANTOS, T.A.R.1; SILVA, A.C.1; SILVA, E.B.2; MOREIRA, D.R.M.2; LEITE, A.C.L.2; PEREIRA, V.R.A.1 1.CENTRO DE PESQUISAS AGGEU MAGALHÃES, RECIFE, PE, BRAZIL; 2.UNIVERSIDADE FEDERAL DE PERNAMBUCO, RECIFE, PE, BRAZIL.
Keyword:thiosemicarbazones; leishmania amazonensis; immune system
Abstract: Thiosemicarbazones derived molecules are known for its remarkable biological effects, with studies showing its antitumoral, antibacterial and antiprotozoal activities. Several drug screenings with tripanossomatidae family members have been conducted, demonstrating these molecules to have special properties against parasites, particularly in Trypanossoma cruzi. The aim of this work was to assess the impact of five new thiosemicarbazone derived molecules (coded AT1, AT2, AT3, AT4 and AT6) on immune system cells, and its effects on Leishmania amazonensis. For this purpose, BALB/c splenocytes were collected and incubated in 96 wellplate with different molecules concentrations (1 to 100 µg/mL) and 1 µCi of tritiated thymidine was add to each well. After 18 hours, cells were harvested and the incorporation of triatiated thymidine was measured with a beta counter. Cell viability after exposure to the compounds was evaluated in J774A.1 macrophages by MTT assay. The 50% cytotoxicity concentration (CC50) values were determined to splenocytes and macrophages. To analyze the direct effect of compounds in L. amazonensis, promastigotes (106 parasites/mL) were incubated with molecules in different concentrations (0.19 to 100 µg/mL) and after 96 hours cells were counted in Neubauer chamber and the concentration that inhibited culture growth by 50% (IC50) was calculated. Selectivity Index (SI) were determined by the ratio of CC50 (Macrophages) to IC50 (Promastigotes). Among the analyzed compounds, AT2 and AT6 showed the lowest IC50 values (1.40 and 1.99 µg/mL respectively) and the highest CC50 values either in splenocytes (18.28 and 14.49 µg/mL) or macrophages (12.73 and 12.97 µg/mL). Consequently these two molecules presented the highest SI values (9.09 and 6.52 respectively) demonstrating more selectivity to the parasites than the mammalian cell. AT1, AT3 and AT4 also showed antiparasite activity, with IC50 values varying from 1.49 to 3.11 µg/mL and SI values from 2.29 to 4.54. The leishmanial activity in relatively low level and fewer effects on immune system cells endorse this series of molecules as promising candidates to drugs against leishmaniasis, stimulating new studies. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1053] P 565 - LEISHMANIA (L.) AMAZONENSIS PROMASTIGOTE MUTANT SELECTED IN VITRO FOR RESISTANCE TO SB(III) CONFERS RESISTANCE TO PENTAVALENT ANTIMONIAL THERAPY IN VIVO. REIS, P.G.1; MONTE NETO, R.L.D.2; BEZERRA, F.M.B.V.1; NEVES, F.T.P.1; MELO, M.N.3; FRÉZARD, F.1
1.DEPARTAMENTO DE FISIOLOGIA E BIOFÍSICA, UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL; 2.CENTRE DE RECHERCHE EN INFECTOLOGIE, UNIVERSITÉ LAVAL, QUÉBEC-, CANADA; 3.DEPARTAMENTO DE PARASITOLOGIA, UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL.
Keyword:l. (l.) amazonensis; antimony resistance ; in vivo
Abstract:
Leishmania promastigote mutants selected in vitro for resistance to Sb(III) have been used extensively as a model of investigation of the mechanisms of resistance in antimonial chemotherapy. However, this model is often questioned with respect to its relevance to in vivo condition, as it is based on the assumption that Sb(III) is the final active form in antimonial chemotherapy and that resistance to pentavalent antimonials results from resistance of Leishmania parasite to Sb(III). Support to this hypothesis was obtained from in vitro assays using the macrophage model, showing cross-resistance to Sb(V) of intracellular Leishmania strains initially selected for resistance to Sb(III). However, the macrophage model is still far from the in vivo condition and it would be important to investigate in vivo whether Leishmania promastigote mutants selected in vitro for resistance to Sb(III) confer resistance to pentavalent antimonial therapy. As a first step, the infectivity of Sb(III)-susceptible (WTS) and SbIIIresistant (SbR) populations of L. (L.) amazonensis was determined by inoculation of 1 × 106 metacyclic promastigotes from the sixth day of the stationary phase into the footpads of Balb/c mice. After appearance of the lesion, groups of six animals infected by each strain were treated with meglumine antimoniate for 30 days and other groups remained without treatment. The lesion growth was measured, and after the treatment, the animals were sacrificed and parasite load was determined using a quantitative limiting-dilution assay. The rates of increase of footpad lesion were similar in WTS and SbR groups, demonstrating the maintenance of infectivity of the resistant strain after in vitro selection. Pentavalent antimonial treatment promoted the stabilization of lesion growth in WTS group (3.8 mm) as opposed to SbR group, whose lesions evolved similarly to untreated SbR group (6 to 8mm). The in vivo resistance was also confirmed by parasite loads in the lesion, with SbR group showing 1028 and 1028 parasites with or without treatment, respectively (WTS showed 104 and 1012 with and without treatment). In conclusion, L. (L.) amazonensis promastigote mutant selected in vitro for resistance to Sb(III) confers resistance to pentavalent antimonial therapy in vivo. This in vivo model should be useful in the search of reversion strategies for antimonial resistance in L. (L.) amazonensis. Supported by : CNPq; Capes; Fapemig
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1059] P 566 - FLOW CYTOMETRY ON THE DIAGNOSIS OF PATIENTS WITH AMERICAN TEGUMENTARY LEISHMANIASIS (ATL) THROUGH RESEARCH ON IGG ANTIBODIES OF L. (V.) BRAZILIENSIS DE OLIVEIRA, A.P.; DE OLIVEIRA, B.C.; CASTRO, M.C.A.B.; DE ALMEIDA, A.F.; SOUZA, M.A.; DE ALMEIDA, T.M.; SILVA, A.C.; DE BRITO, M.E.F.; PEREIRA, V.R.A. CPQAM-FIOCRUZ, RECIFE, PE, BRAZIL.
Keyword:atl; igg; flow cytometry
Abstract: ATL is a serious public health issue in Brazil. Besides the socioeconomic challenges, it is known that the disease’s diagnosis does not present a laboratorial method which can be used as gold standard. Until now, ATL’s diagnosis is based on clinic, epidemiologic and laboratorial factors, so the association of several elements is frequently necessary to reach the definitive diagnosis. Therefore, the development of new diagnostic methods is essential, and flow cytometry is a new approach which is utilized on the research on antibodies with superior applicability than different conventional protocols of detection and development. Taking flow cytometry’s applicability into consideration, this study brought an alternative through the research on IgG antibodies of L.(V.)braziliensis, which is applicable to ATL’s diagnosis. Serum from 20 patients before treatment (BT) were diluted (1:64 to 1:8192), incubated with L.(V.)braziliensis parasites and marked with human anti-IgG conjugated with fluorescein
isothiocyanate - FITC.The preview of the results was accomplished on the flow cytometer FACScalibur using the software CELL QuestTM, having the results expressed on levels of IgG’s reactivity, through the percentage of positive fluorescent parasites (PPFP).They were analyzed using the statistics program MedCalc. Using 20% of PPFP, cut-off suggested by Martins-filho et al, 1995, the results pointed that at the 1:128 cut-off, it is the reactivity region which corresponds to the first titer of differential reaction from BT individuals. Thus, it was shown 80% (16/20) of BT individuals located on a high reactivity region, in other words, positive for ATL; and 20% (4/20) of after treatment (AT) individuals with restricted PPFP values from a low reactivity region, meaning negative for the flow cytometry test. However, flow cytometry proved to be an applicable methodology to ATL’s diagnosis, suggesting a greater explanation of the disease, identifying cases of active ATL. In addition, further studies will elucidate the role of IgG antibodies on the hummoral immune response on ATL’s diagnosis and cure criterion. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1060] P 567 - COMPARISON OF THREE SEROLOGICAL TESTS FOR THE DIAGNOSIS OF THE CANINE VISCERAL LEISHMANIASIS IN THE CITY OF PORTEIRINHA, AN ENDEMIC AREA IN THE STATE OF MINAS GERAIS, BRAZIL. FRANÇA-SILVA, J.C.1; HORACIO, A.M.L.2; ROCHA, A.M.S.2; REIS, V.W.2; REGINA-SILVA, S.3; MICHALSKY, E.M.3; DIAS, E.S.3; MACHADO-COELHO, G.L.2 1.UFMG, BELO HORIZONTE, MG, BRAZIL; 2.UFOP, OURO PRETO, MG, BRAZIL; 3.CPQRR/FIOCRUZ, BELO HORIZONTE, MG, BRAZIL.
Keyword:canine visceral leishmaniasis; kala-azar detect; porteirinha
Abstract: The canine visceral leishmaniasis (CVL) is a serious public health and veterinarian problem in Brazil. The disease control is extremely complex and with considerable cost. Canine diagnosis and euthanasia are very discussed in terms of effective disease control. The present study aim to determine the incidence of CVL in the city of Porteirinha (Minas Gerais state) and started after a first canine census survey performed in 2008. The total number of dogs included in that study was 1,108. Canine serum collection was performed along time at 73, 162, 252, 342, 437 and 497 days and those sera were assayed by three different methods for VL diagnosis: indirect immunofluorescence (IFA, with 1:40 dilution cut off), ELISA (expressed by A450nm) and kalaazar detect rapid (KD-rK39) assays. According to the results, five groups of dogs were defined: 1. Confirmed (all three tests positive); 2. Undetermined (only ELISA positive); 3. Negative (all three tests negative); 4. Suspected (ELISA and IFA or KD positive); 5. Gray zone (A450nm 20% of the ELISA cutoff). The positivity rates were expressed as percentages and the incidence rates as new cases per 1,000 dogs per year. In group 1, the highest incidence was 3.5 at 342 days and the lowest one was 1.0 at 437 days. For group 2, the highest incidence was 18.9 after 252 days and the lowest one was 6.4 at both 162 and 437 days. The highest incidence observed in group 4 was 6.2 after 252 and 497 days and the lowest one (3.0) occurred at the 437th days. For the gray zone, the highest incidence was 4.4 at 342 days and the lowest one was 1.1 after 497 days. In group 3, the positivity rates displayed a uniform tendency to decrease, starting with 84% (73 days) and ending with 39.9% (497 days). The sensitivity of the KD test was 48% and the specificity was 81% (p<0.001) as calculated by McNemar test taking the parasitological (gold standard) test as reference. The Youden (29.3%) and Kappa (26.2%) indexes were significantly discordant (P>0.0001). Although the KD test is extremely useful in the field, in an overall analysis with all the times together it displayed a low sensitivity (31%). This data indicates that the test needs improvements for better performance.
Financial support: FAPEMIG, FIOCRUZ, UFMG, HERTAPE-CALIER, CNPq. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1065] P 568 - DIAGNOSTIC OF VISCERAL CANINE LEISHMANIASIS BY SEROLOGICAL METHODS IN NON-ENDEMIC REGION OF MINAS GERAIS. JUNIOR, J.G.C.1; FREIRE, M.L.1; CAMPOS, S.P.1; CUPOLILLO, E.2; DE ALMEIDA, R.P.2; SCOPEL, K.K.G.1; COIMBRA, E.S.1 1.UFJF, JUIZ DE FORA, MG, BRAZIL; 2.FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:visceral canine leishmaniasis; diagnosis ; serology
Abstract: Introduction: Human visceral leishmaniasis constitutes a public health problem that affects millions of people throughout the world. In Brazil, visceral leishmaniasis (VL) is an endemic zoonotic disease caused by L. infantum. In general, VL is a rural disease, but nowadays this disease has spread to the urban centers. Dogs play an important role in the maintenance of this disease in the human environment serving as reservoirs for this infection. This study aimed to carry out a serological survey on canine visceral leishmaniasis (CVL) at Juiz de Fora, Minas Gerais. Methods: Blood was collected from 401 dogs allocate in the public kennel from municipality of Juiz de Fora. Diagnosis of CVL was performed using the colloidal gold-based immunochromatography TR DPP® kit, and EIE-leishmaniose-visceral-canina® kit, both produced by FIOCRUZ (Bio-Manguinhos, RJ, Brazil). All the procedures were performed according to the manufacturer’s instructions. In parallel, questionnaire with animal clinical aspects was filled out by a veterinarian. Dogs were scored for asymptomatic, oligosymptomatic and symptomatic. Results and Conclusion: From 401 dogs examined, 3.74% showed positive by TR DPP® kit, and 1.74% were positive by EIE-LVC. However, only 0.25% were positive for both diagnostics methods. Among the positive dogs only 1% was classified as oligosymptomatic. This is a first study about CVL, caused by L. infantum, in Juiz de Fora municipality and the results reinforce the idea that this disease presents a process of expansion and urbanization in Brazil. Furthermore, DPP technology represents a breakthrough in the detection of positive dogs from indene areas of CVL. These results show the importance of diagnosing CVL in non-endemic area and reinforce the necessity of epidemiological survey in Juiz de Fora. Supported by FAPEMIG, CNPq and UFJF. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1073] P 569 - LEVELS OF ANTI-LEISHMANIA IGG3 CAN BE ASSOCIATED WITH THERAPEUTIC PROGNOSTIC OF VISCERAL LEISHMANIASIS IN CHILDREN. OLIVEIRA NASCIMENTO, J.M.1; SILVA, G.A.F.2; DORVAL, M.E.C.1; OSHIRO, E.T.1; FRAGA, T.L.1; BRUSTOLONI, Y.M.1; SILVA, A.G.2; DA-CRUZ, A.M.2; DE OLIVEIRA, A.L.L.1 1.UNIVERSIDADE FEDERAL DE MATO GROSSO DO SUL, CIDADE UNIVERSITÁRIA, 79070-900, CAMPO GRANDE, MS, BRAZIL; 2.FUNDAÇÃO INSTITUTO OSWALDO CRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:visceral leishmaniasis; antibodies; humoral response
Abstract: The clinical expression of visceral leishmaniasis (VL) depend on factors such as species, parasite strain's virulence, individual, genetic and environmental characteristics and immunologic system responsiveness. The presence of antibodies reactive to Leishmania chagasi antigen by polyclonal activation of B cells is reported in literature. The analyses of IgG subclasses can be useful in monitoring the immunologic response and promoting relevant information in therapeutic conduct. The present study aimed for the identification of
immunoglobulin isotypes in the IgG class associated with the active disease and clinical remission and evaluate the possibility of using the established parameter in prognostic identification. Nineteen children with confirmed diagnostic of visceral leishmaniasis in the Pediatric Service of the Hospital Universitario of UFMS were studied. The serological samples were obtained in three distinct time periods: (P1) before treatment, (P2) one month after treatment and (P3) six months after treatment. The control group was formed by ten healthy children paired according to sex and age. For the ELISA test was used soluble L. chagasi antigen to detection of IgG isotypes (total IgG, IgG1, IgG2, IgG3 and IgG4). The data were expressed in median and standard deviation, considering significant p<0.05. Of nineteen children studied the age average was 3,9 years old (1 to 8 years old), 12 of those children being boys (63.1%). Sixty per cent of the children were from Campo Grande. Fever was a symptom in 100% of the cases, visceromegaly was present in 94.7% (18/19), pallor in 84.2% (16/19), weight loss in 57.8% (11/19) and diarrhea in 31.5% (6/19). The immunoglobulin study pointed high significant levels of total IgG (2730±781.0), IgG1 (1022±796.0), IgG2 (180.0±267.8) and IgG3 (865.0±807.9) in comparison with the negative control group. After six months of treatment, the IgG3 levels decreased significantly (45.5±552.1) compared with the negative control group. The analyses of subtype IgG4 did not present significant differences in the studied groups, showing absence or low levels of IgG4. The presence of IgG subclasses in VL can serve as a prognostic marker due to the subclasses connection to the individual's protection and susceptibility. In this study, the levels of IgG3 indicate this subtype to be a complementary biomarker in monitoring the post-therapeutic cure in human VL. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1085] P 570 - THE USE OF PHOTODYNAMIC THERAPY WITH PHTHALOCYANINE ALUMINUM CHLORIDE IN THE TREATMENT OF EXPERIMENTAL CUTANEOUS LEISHMANIASIS IN C57BL/6 MICE. CARNEIRO, A.L.; ALMEIDA, N.A.; SAMPAIO, R.N.R. UNIVERSIDADSE DE BRASÍLIA, BRASILIA, DF, BRAZIL.
Keyword:word1; word2; word3
Abstract: Considering the clinical and technical challenges in the treatment of cutaneous leishmaniasis (CL), the objective of this study was to evaluate the in vivo efficacy of photodynamic therapy using topical aluminum phthalocyanine chloride in the treatment of experimental CL. Twenty four C57BL/6 mice were infected with metacyclic promastigotes of Leishmania (L.) amazonensis in the concentration of 3x106 and divided into 3 groups: one treated with photodynamic therapy using aluminum phthalocyanine chloride gel (AlPhCl) in a concentration of 20 g (application for 15 minutes, wavelength 640-710nm, 150 J/cm2, 10 daily sessions), a group infected and treated with N-methylglucamine (NMG), an infected control group without treatment (INT) and a group without infection (NINT). The evaluation was performed by measuring infected paws, culture (in Novy-Nicolle-McNeal medium), smears and limiting dilution followed by Thiazolyl Blue Methil colorimetric method (MTT). Statistical analysis was made by the statistical program ELIDA® and SPSS® version 17.0, with the Shapiro-Wilk test, ANOVA followed by the Tukey test. In the photodynamic therapy group the mean diameter of the legs fell from 0.25mm to 0.20mm after treatments. In N-methyl glucamine group it increased from 0.25mm to 0.30mm and in control untreated infected group it increased from 0,25mm to 0.30mm (p <0.05). The evaluation of leishmanial viability by MTT showed that in NMG group, the viability reduction was 50% compared to the INT group reduction, and that the mean viability in photodynamic therapy group was 80% of the INT group viability (p <0.05). We conclude that this specific therapy was effective in the treatment of experimental cutaneous leishmaniasis.
KEYWORD:Cutaneous Leishmaniasis, , photodynamic therapy, treatment. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1093] P 571 - STUDY OF LEISHMANICIDAL ACTIVITY OF DERIVATIVES FROM COMBRETASTATIN A-4 ARAÚJO, M.V.1; AMARAL, D.N.D.2; DA MATTA, C.B.B.1; CAVALCANTE-SILVA, L.H.A.1; BARREIRO, E.J.2; LIMA, L.M.2; ALEXANDRE-MOREIRA, M.S.1
1.FEDERAL UNIVERSITY OF ALAGOAS, MACEIÓ, AL, BRAZIL; 2.FEDERAL UNIVERSITY OF RIO DE JANEIRO, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:combretastatin a-4; leishmaniasis; promastigotes
Abstract: Leishmaniasis is considered a public health problem, affecting millions of people around the world.Considering that so far there is no ideal therapeutic for leishmaniasis, this study aims investigate the leishmanicidal activity of derivatives from combretastatin A-4. The cell line J774 viability was determined with MTT assay. Leishmanicidal effects against promastigotes of Leishmania baziliensis (MHOM/BR/87/BA125), Leishmania amazonensis (MHOM/BR/77/ltb0016) and Leishmania infantum chagasi (MCAN/BR/89/BA262) were evaluated. Data obtained from experiments were expressed as the mean ± standard error of mean (S.E.M). of triplicate cultures of representative assays. Statistical differences between the treated and the vehicle groups of in vitro experiments were evaluated by ANOVA and Dunnett hoc tests and considered significant when p <0.05. The derivatives LASSBio 1586, 1587, 1588, 1589, 1590, 1592, 1594, 1595 and 1716 not affected the viability of macrophages at concentration of 100 µM by evidenced MTT assay. Moreover, the derivatives LASSBio DNA 1586, 1594, , 1714, 1715, 1716, 1738, 1739, 1740, 1741, 1742 and 1744 exhibited high antileishmanial activity against promastigotes of L. braziliensis. Already in assay against the growth of promastigotes of L. amazonensis, the derivatives LASSBio 1589, 1593, 1738, 1740 and 1741 showed activity against the parasite. However, against promastigotes of L. Infantum Chagasi the derivatives LASSBio DNA 1586, 1586, 1587, 1588, 1589, 1590, 1591, 1592, 1593, 1596, 1715, 1738 and 1740 showed significant activity against the parasite. The derivatives from combretastatin A-4 LASSBio 1586, 1587, 1588, 1589, 1590, 1592, 1594 and 1716 are strong candidates for antileishmanial prototypes drug because since they exhibit presented pronounced leishmanicidal activity against promastigotes without citotoxity effect against host cells. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1094] P 572 - CLINICAL CHALLENGES IN THE MANAGEMENT AND TREATMENT OF LEISHMANIA/HIV COINFECTION IN YOUNG PATIENT: A CASE REPORT ARAUJO NETO, A.R.1; MOURA, V.A.1; PARREIRA, B.L.1; SÁ, V.M.H.1; JANOTTI, A.2; COUTINHO, O.M.V.C.1; MILAGRES, F.A.P.1 1.UNIVERSIDADE FEDERAL DO TOCANTINS, PALMAS, TO, BRAZIL; 2.HOSPITAL GERAL PÚBLICO DE PALMAS DR. FRANCISCO AYRES, PALMAS, TO, BRAZIL.
Keyword:hiv; vl; coinfection
Abstract: Introduction: The HIV infection increases hundreds of times the risk of visceral leishmaniasis (VL) developing in areas of endemicity, as Tocantins, the third state in confirmed cases of VL in Brazil.VL still cooperates for the development of AIDS defining conditions. Both diseases had an important synergistic detrimental effect for patient´s health.Case report: S.B.S., Female, 24 years old, HIV positive since 2004 and abandonment of highly active antiretroviral therapy (HAART) for 8 months, antecedent of chronic alcoholic, present at admission complaintsof
anorexia, generalized myalgia, fever and malaise for 15 days. Physical examination with painful abdomen palpation and hepatosplenomegaly.Was suspected pneumonia being prescribed ceftriaxone,clindamycin, symptomatic and prophylaxis for pneumocystis with trimethoprimsulfamethoxazole. In the laboratorial tests, there was pancytopenia (Ht: 24%; Hb: 8 g/dL; leukocytes: 2,500/mm³; platelets: 71,500/mm³), AST of 334 U/L, ALT of 118U/L, urea 18 and creatinine 0.6 mg/dL. The patient has developed mental confusion, chest pain, mild cough, eyelid and lower limb edema, and later generalized edema. Abdominal ultrasound showed an enlarged and homogeneous spleen.Aspiration biopsy of bone marrow was inconclusive and a new procedure was refused by the patient due the intense local pain complaint. It’s started HAART (tenefovir, lamivudine, ritonavir and atazanovir) and empirical therapy with amphotericin B for VL, with improvement in symptoms and fever. There was, however, a progressive worsening of general condition with jaundice, nausea, abdominal pain and distension and mild dyspnea, keeping the pancytopenia, associated with severe elevation of liver transaminasis (AST of 1,457 U/L, ALT of 2,384 U/L).Was suspected drug-induced hepatitis,with suspension of Amphotericin B and HAART.Began Meropenem and Vancomycindue probable bacterium infection associated. Were transfused packed red blood cells, initiated Vitamin K and started Liposomal Amphotericin B. The resulted of the urine culture showed Kleibsiella pneumoniae multiresistant.The therapy was continued and the patient had progressive improvement of symptoms, increased platelets, decreased bilirubin, albumin increased. Vancomycin and Meropenem was discontinued and the patient conclude the treatment with 1.5g of Liposomal Amphotericin and remaining in monitoring outpatient. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1098] P 573 - SKIN PERMEATION AND IN VIVO BIODISTRIBUTION OF CHOLOALUMUNINUM PHTHALOCYANINE (CLALPC) AFTER TOPICAL TREATMENT IN WISTAR RATS ESCOBAR, P.; RICO, C.I.; CONDE, C.A.; RODRIGUEZ, J.; MANTILLA, J.C. UNIVERSIDAD INDUSTRIAL DE SANTANDER, BUCARAMANGA, COLOMBIA.
Keyword:topical ttreatment; franz diffusion cells; wistar rats
Abstract: Photodynamic therapy (PDT) using a topical ClAlPc formulation in cutaneous leishmaniasis (CL) should ensure its dermis accumulation, place where Leishmania-infected macrophages are located. This study determined the skin permeation and biodistribution of ClAlPc after topical application in rats with healthy and surgically damaged skin. Two formulations were used (F1: ClAlPc in DMSO:Tween 80:type I water; and F2: ClAlPc, incorporated in a nanoemulsion (NE) delivery system). Skin permeation studies were performed on full-thickness rat skin using Franz diffusion cells with exposure to ClAlPc-F1 and F2 for 0, 4, 8, 10 12 and 24 h. PBS 7.4 SDS 2% was used as receptor fluid. ClAlPc retained in stratum corneum (SC) was analyzed by tape stripping method and retained in epidermis and dermis (E+D) was analyzed from a macerate of the remaining membrane tissue. Biodistribution of ClAlPc was performed after topical application in the dorsal region (aprox area of 3 cm2) analyzing ClAlPc concentration in skin layers and rat organs by fluorometry. For induction of surgical injury, animals were anesthetized and two cuts with dissection scissors were made in the dorsal region. The lesions measured approximately 5 mm in diameter. The results were expressed as concentration of ClAlPc nM/mg of tissue. Skin biopsies were taken in order to determine their histological characteristics. All two formulations were stable and preserved ClAlPc spectroscopic properties. The ex vivo treatment using rat membranes did not permit any detection of ClAlPc in the receptor fluid. The ClAlPc was retained mainly in SC and E+D with results dependent on formulation type, time of the assay and skin layers; The averages of ClAlPc were 75,716 and 58,905 nM/cm2 in SC and E+D, respectively. In animals, the ClAlPc
was distributed mainly in the skin (site of application). “Skin healthy” animal showed ClAlPc 19.15 in SC and 117.79 nM/mg tissue in E+D. “Skin injury” animals showed 3.47 nM/mg tissue in SC and 40.46 nM/mg tissue in E+D. The ClAlPc distribution in organs was observed only in “skin injury” animals after F1 application with values in lung, liver, spleen, heart, brain and kidney between 1.58 and 20.33 nM/mg tissue. The no ex vivo diffusion of ClAlPc across the rat membranes was considered a positive effect in relation to the study aims. ClAlPc distribution depended on its concentration, skin state and type of formulation. The ClAlPc;NE (F2) showed a better ClAlPc skyn retention capacity, even in “skin injured” animals. The findings provide important knowledge about ClAlPc localization in skin layers a and its potential distribution in a body after topical application.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1099] P 574 - ENCAPSULATION EFFECTIVENESS OF AN ACTIVE CHALCONE CH8 IN LIPOSOMAL SYSTEMS ON THE MACROPHAGE MICROBICIDAL MECHANISMS. DE OLIVEIRA, D.E.; CHAVES, S.P.; BERGMANN, B.R. UFRJ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:chalcone; macrophage; lipossomes
Abstract: Cutaneous leishmaniasis is a tropical disease caused by protozoa of genus Leishmania which proliferate within the macrophage`s phagolysosome. The conventional chemotherapy is based on multiple antimony injections or other toxic drugs, moreover, with no topical treatment for the species transmitted in Brazil. In our previous studies, we demonstrated the effectiveness of a synthetic injectable chalcone CH8, much higher (60x) than the antimony Glucantime. In this study, we evaluated the effect of liposomal encapsulation of CH8 with conventional phosphatidylcholine (CH8-LC) and pegylated liposomes (CH8-LP) in vitro, over the macrophages and over the parasite, in order to clarify its mechanism of action in the controlling of the disease. To evaluate the in vitro effect, peritoneal macrophages from BALB / c mice were infected with L. amazonensis and incubated for 48 hours at 37oC with free CH8 , with the liposomal formulations (CH8-LC and LP-CH8) or with empty liposomes (LC and LP). To investigate the role of free CH8 and LC-CH8 in the innate immune response, we quantified the production of NO by the Griess reagent, the ROS production by fluorimetry using the dye H2DCFDA and intracellular pH by flow cytometry using acidic dye Lysotracker Red . The production of ROS in promastigotes of L. Amazonensis was evaluated by fluorimetry. Infected Macrophages had their parasite load decreased 49% and 36% when compared to the control when treated with free CH8 (15 mM) and CH8-LC (15 mM CH8 free), respectively. Both free CH8 as CH8-LC were able to increase the production of NO (5x and 6x) and ROS (2x and 5x), respectively. The free CH8 increased 1.3 xo intracellular pH and CH8-LC decreased 1.5 x. All formulations induced the production of ROS by promastigotas .In summary, as well as serving as excellent carriers for transdermal CH8, conventional liposomes LC, but not pegylated liposomes LP, activated oxidative mechanisms of macrophage and its pH reduced within the fagolisossomo, which might contribute to the antiparasitic effect of the CH8. Therefore, this formulation CH8-LC has a great potential for the development of a topical noninvasive formulation for cutaneous leishmaniasis.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1104] P 575 - BIOLOGICAL CHARACTERIZATION OF LEISHMANIA AMAZONENSIS STRAIN TRANSFECTED WITH FLUORESCENT PROTEINS GONDIM, F.C.; GUEDES, H.L.M.; BERGMANN, B.R. UFRJ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:leishmania amazonensis; fluorescent protein; transfection
Abstract: In the last decade, green fluorescent Leishmania parasites have appeared as interesting tools in drug screening and parasite imaging. However, red-fluorescent parasites may more accurately be detected in intravital microscopy and in situations where green-fluorescent probes are used. In this work, our research group produced a strain of Leishmania amazonensis expressing a red fluorescent protein (La-RFP). Hence, we are attesting the applicability of this newly produced La-RFP for anti-drug testing in vitro and in vivo in order to evaluate the protein-expression stability, growth rate in culture, and their infectivity to macrophages and mice compared with La-GFP and La-WT strains, which have been validated in our laboratory routine. - CLINICAL LEISHMANIASIS [1118] P 576 - SERUM PROFILE OF SOLUBLE CD40 LIGAND AND MMP-9 IN HUMAN VISCERAL LEISHMANIASIS BEFORE AND AFTER TREATMENT DE OLIVEIRA, F.A.1; DOS SANTOS, P.L.1; SANTOS, M.L.B.1; BARRETO, A.S.1; OLIVEIRA SILVA, C.V.1; DAMASCENA, N.P.1; PASSOS, R.O.1; DUTHIE, M.2; GUDERIAN, J.2; BATHIA, A.2; DE MOURA, T.R.1; REED, S.2; DE ALMEIDA, R.P.1; DE JESUS, A.R.1 1.UNIVERSIDADE FEDERAL DE SERGIPE, ARACAJU, SE, BRAZIL; 2.INFECTIOUS DISEASE RESEARCH INSTITUTE SEATLE, SEATLE, UNITED STATES.
Keyword:soluble cd40 ligand; metalloproteinase 9; visceral leishmaniasis
Abstract: Introduction: Soluble CD40 ligand (sCD40L) and matrix metalloproteinase 9 (MMP- 9) are Inflammation markers and have been poorly described in infections disease. In this prospective study we describe the sera kinetics of these two molecules in the course of treatment follow up in human visceral leishmaniasis (VL). Methods and results: Sera from VL patients were collected before and during follow up of regular Antimony treatment. sCD40L and MMP-9 were measured by Luminex assay. While sCD40L and MMP-9 were not observed in sera from healthy control subjects at low risk of Leishmania chagasi infection, elevated levels were observed in sera from VL patients, and an increase in sCD40L and MMP-9 levels were detectable during the follow-up of VL patients undergoing antimony treatment. sCD40L levels were also high in individuals living in endemic settings at high risk of infection. Additionally, negative correlations were found between spleen sizes and MMP-9 before treatment and sCD40L at day 15 of treatment.Conclusion: Our findings suggest that elevated sCD40L and MMP-9 levels are associated with resistance to infection and resolution of VL. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1127] P 577 - APPLICATION OF PCR-RFLP METHODS AS ROUTINE IN A TERTIARY HOSPITAL IN SÃO PAULO, BRAZIL. SATOW, M.M.1; KANASHIRO, E.H.Y.1; ROCHA, M.C.1; MAIA, R.1; SOLER, R.C.2; OYAFUSO, L.K.2; COTRIM, P.C.1; LINDOSO, J.A.L.2 1.INSTITUTO DE MEDICINA TROPICAL, SÃO PAULO, SP, BRAZIL; 2.INSTITUTO DE INFECTOLOGIA EMILIO RIBAS, SÃO PAULO, SP, BRAZIL.
Keyword:tegumentary leishmaniasis; pcr-rflp; diagnosis
Abstract: Mucosal leishmaniasis (ML), which main causative agent in Brazil is Leishmania (V.) braziliensis, is the severe form of tegumentary leishmaniasis. Initial clinical manifestation of ML is often misdiagnosis with cutaneous leishmaniasis (CL) and other dermatologic diseases, leading to inadequate treatment. The technique of polymerase chain reaction (PCR) with analysis of restriction fragment length polymorphism (RFLP) has been reported as a useful tool for identification of the Leishmania’ species, but it still not used as a routine technique in Brazilian hospitals due lack of standardization of the techniques. In our study, 160 patients with suspect clinical manifestation of CL or ML were submitted to traditional diagnosis methods and two PCR-RFLP methods: kDNA-HaeIII and ITS1-HaeIII. The positivity of traditional diagnosis tests observed was: 19.4% by in vitro culture, 35.6% by direct investigation and 38.8% by Montenegro’ skin test. Molecular methods were more sensitive than traditional tests showing positivity of 81.3% by kDNA-PCR and 75% by ITS1-PCR. ITS1 and kDNA PCR results were in agreement for 82.5% of the DNA samples. Species identification by PCR-RFLP was done in PCR-positive samples by both primers: ITS1 and kDNA. The L. (V.) braziliensis electrophoretic pattern was observed in 89.3% of the samples tested by kDNA-HaeIII. ITS1-HaeIII products presented electrophorectic patterns for L. (V.) braziliensis in 66.1% samples and for L. (L.) amazonensis in 25.9% samples. 62.5% of the tested samples were identified as L. (V.) braziliensis by both techniques. This preliminary study indicates that PCR based techniques using kDNA or ITS1 primers can contribute for an early diagnosis of suspected leishmaniasis patients and also indicates the probable infecting Leishmania’ species (ITS1-HAEIII). Applications of the techniques were considered viable for routine use in public Brazilian hospitals of endemic areas and should be considered in order to improve the actual leishmaniasis diagnosis practice. Supported by CAPES, FAPESP, LIM 48- FMUSP
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1132] P 578 - ACTIVATION OF SPLENIC MACROPHAGE BY LEISHMANIA INFANTUM INFECTION AND MODULATION BY CERAGENINS RODRIGUEZ, O.L.1; ORTIZ, R.1; RODRIGUEZ, C.1; MARTINEZ, E.1; SAVAGE, P.2; SANCHEZ, M.A.1 1.INSTITUTO DE BIOMEDICINA, CARACAS, VENEZUELA; 2.DEPARTMENT OF CHEMISTRY AND BIOCHEMISTRY, BRIGHAM YOUNG UNIVERSITY, PROVO, UNITED STATES.
Keyword:experimental visceral leishmaniasis; macrophage activation; csa-8
Abstract: Macrophages according to classical or alternative activation exerts a function of effector cell or just as host cell of the Leishmania parasite, The Ceragenin formerly known as Cationic Steroid Antibiotic 8 (CSA-8) derivative of cholic acid with amine groups on a cross-steroidal core was synthesized to mimic the properties of natural antimicrobial peptides. The anti-Leishmania effect of this antibiotic has been demonstrated "in vitro", however, its immunomodulatory action has not been clarified. The aim of the study was to determine the activation of splenic macrophages after infection of BALB/c mice with L. infantum and "in vitro" modulation by different doses (5, 14, and 20 g) of CSA-8, by flow cytometric determination of NOS-2 molecules, Arginase-1, IL-12, IL-23, IL-10 and TNF- . The results show that macrophages
obtained at the beginning of the infection (day 2) showed an increased expression of by arginase and IL-1-23 by 75.3% and 94.2% respectively compared to control (Day 0) (p< 0.01). While macrophages obtained on day 53 (end of the infection) expressed more NOS-2 and IL-12 that macrophages from days 0, 2 and 21 pi (p<0.05). We found a significant increase on the expression of IL-10 and TNF- from day 2 to 53 post infection (p.i.) with respect to day 0 (p<0.01). At 2h after treatment with 14 and 20 mg/ml of the ceragenin, the expression of NOS-2 in macrophages from day 2 p.i. increased 45% (p = 0.02) and 54% (p = 0.0004), respectively, when compared to untreated macrophages. On day 21 (peak of infection and highest parasite load), the increase was 61% (p< 0.001) with 5 ug/ml. Meanwhile Arginase-1 by day 2 p.i. decreased (p <0.01) and by day 21 with 20 ug/ml decreased in 88% (p<0.001). the cytokines analysis by, day 21 with different doses of CSA-8, showed an 80% increase of IL-12, meanwhile the cytokines IL-10 and TNF- showed a reduced expression of 60% and 70% respectively (p<0.001),. The key cytokine IL-23 decreased on day 2 p.i. 65% (p<0.001) in CSA8 treated macrophages (14 g) while increasing 21 and 53 pi. This suggests that this antibiotic acts as an immunomodulator, which enhances macrophage activation to NO production and cytokines involved in the elimination of the parasite. Supported by grants: FONACIT G2005000375, CDCH-PSU 09-7878/2009 - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1134] P 579 - DIAGNOSIS OF VISCERAL LEISHMANIASIS IN INDIVIDUALS WITH HIV / AIDS IN CAMPO GRANDE-MS, BRAZIL DRUZIAN, A.F.1; MATOS, V.T.G.1; JUNIOR, M.G.H.2; BRUSTOLONI, Y.M.2; OLIVEIRA, S.M.D.V.L.2; DRIEMEIER, M.1; GUIMARÃES, L.N.A.1; SANTABARBARA, M.C.A.2; OLIVEIRA, P.O.1; DORVAL, M.E.C.3; JUNIOR, M.S.C.L.4; SANTA BARBARA, M.C.A.2; PANIAGO, A.M.M.2 1.HOSPITAL UNIVERSITÁRIO, UNIVERSIDADE FEDERAL DE MATO GROSSO DO SUL, CAMPO GRANDE, MS, BRAZIL; 2.FACULDADE DE MEDICINA, UNIVERSIDADE FEDERAL DE MATO GROSSO DO SUL, CAMPO GRANDE, MS, BRAZIL; 3.LABORATÓRIO DE PARASITOLOGIA CLÍNICA, UNIVERSIDADE FEDERAL DE MATO GROSSO DO SUL, CAMPO GRANDE, MS, BRAZIL; 4.INSTITUTO FEDERAL DE MATO GROSSO DO SUL, NOVA ANDRADINA, MS, BRAZIL.
Keyword:leishmaniasis; hiv; co-infection
Abstract: Approximately 90% of Visceral Leishmaniasis (VL) cases occur in three regions of the world: the Indian subcontinent (India, Bangladesh and Nepal), Sudan, and Brazil. VL / HIV coinfection makes it difficult to determine if the Leishmania infection is new or a relapse reactivated by the immunosuppression caused by HIV / AIDS. VL-HIV / AIDS co-infection has been shown to worsen clinical symptoms, elevate lethality and limit the diagnosis of VL in patients. The objective of the present study was to evaluate the clinical and laboratory manifestations of visceral leishmaniasis in individuals with HIV / AIDS. The epidemiological study analyzed 77 patients, all over 18 years old, that were treated in the Professor Esterina Corsini Hospital / UFMS, from August 2011 to December 2012. Two groups of patients were clinically analyzed: Group I (patients with VL that are HIV positive) and Group II (patients with VL that are HIV negative). Seventy percent of the study participants were male. The average age of HIV positive patients was 39.5 years and 48 years old for HIV negative patients. Leishmaniasis was diagnosed by looking for Leishmania amastigotes in bone marrow aspirate and rapid test rK39. For patients with unknown HIV serology, diagnosis of infection followed the criteria established by the Ministry of Health. The laboratory evaluation was carried out by analyzing hemograms, markers of hepatocyte lesions and cholestasis. The results of the analysis of the bone marrow aspirate in Group I was 91% positive and 56.1%positive in Group II, (p<0,01). The rapid test for LV in HIV positive patients was 50% and 95% in HIV negative patients (p<0.01). The clinical characteristics found were: fever, weight loss, weakness, hepatomegaly, cough, diarrhea, edema, and bleeding, without statistical significance between the groups. In the laboratory analyses, only the number of leukocytes (p<0,05) and ALT (p<0,01) was significant. Analysis of bone marrow aspirate was indispensable for the diagnosis
of VL in patients with HIV / AIDS because the clinical criteria and the immunological test were not enough to diagnose VL in patients with HIV / AIDS. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1135] P 580 - 5’-DEOXY-5’-METHYLTHIOADENOSINE PHOSPHORYLASE: DEMONSTRATION OF THE ACTIVITY IN CRUDE EXTRACTS OF LEISHMANIA INFANTUM PROMASTIGOTES. MEJRI, T.; KAFFELA, M.; BORCHANI, L.; ABID, H.; GUIZANI, I. INSTITUT PASTEUR TUNIS, TUNIS, TUNISIA.
Keyword:mtap; polyamine pathway; leishmania
Abstract: Various forms of clinical manifestation of human leishmaniasis have been described. Development of new drugs against leishmaniasis constitutes a research priority. The characterization of potential drug targets is our general objective. MTA-Pase (5’-Deoxy-5’-methylthioadenosine phosphorylase) plays a critical role in purine and methionine salvage pathways and constitutes their initial enzyme. MTA-Pase, in presence of phosphate, recycles MTA (5’-Deoxy-5’-methylthioadenosine), a toxic by-product of the polyamine pathway, to generate adenine and methylthioribose-1-phosphate (MTR). MTR is recycled to methionine, while adenine is redirected to nucleoside synthesis. Leishmania genome sequencing revealed the existence of one gene coding for a putative MTA-Pase that is annotated in TDR target database as highly druggable (with an index of 0.8). In line with this, MTA-Pase constitutes a drug target against a number of diseases including some cancers and human African Trypanosomiasis, with known potent inhibitors. The objectives of the study are to demonstrate and to characterize the existence of this activity in crude extracts of Leishmania infantum promastigotes. For this purpose, the gene was amplified, sequenced and analyzed using a panel of 26 Leishmania species and strains, and an enzymatic test was established. The activity was measured on 4 different strains of Leishmania infantum. The predicted protein on the different Leishmania parasites was highly conserved with few mutations according to species. The crude extracts presented an MTA-Pase activity measured by the amount of adenine released in the reaction using an enzymatic assay. It was sensitive to the absence of phosphate, and dependent on concentration of MTA. Although, the assays contained the same amount of total proteins, differences in the activity were observed among the 4 Leishmania infantum strains extracts tested. This difference in activity despite identity of the primary sequence can be explained by many hypotheses including: (i) a difference in the amount of enzyme present in the extract; (ii) differential expression of the protein according the developmental stages in vitro; (iii) different levels of post-translational modifications. Work is in progress to characterize further the protein (as a recombinant) and its expression and to genetically validate its essentiality. This work received support from Ministry of Higher Education and Scientific Research in Tunisia (LR00SP04/LR11IPT04). - OTHER SPECIAL TOPICS [1138] P 581 - COMPARATIVE STUDY ON PROTOCOLS OF DIAGNOSIS OF CANINE VISCERAL LEISHMANIASIS, LACEN-PE, 2012. FRANÇA, T.M.C.; DA SILVA, J.C.; SILVA, M.D.C.F.; DA SILVA, A.; TAVARES, V.B.; SILVA, M.B.A. LABORATÓRIO CENTRAL DE PERNAMBUCO, RECIFE, PE, BRAZIL; ; .
Keyword: canine visceral leishmaniasis; immunoassay; enzyme immunoassay
Abstract: Introduction: Although visceral leishmaniasis (VL) is considered a rural endemic, it has been reported in large urban centers, remaining as an important public health problem in many countries worldwide. According to WHO are recorded annually 500 thousand new cases of VL, with 59 thousand deaths. As in other states in the Northeast, the LV is historically endemic in Pernambuco. In Brazil, especially in the state of Pernambuco, until 2001, the canine serological diagnosis was performed by Indirect Imunofluorescence Reaction (RIFI), considering all samples reagents from the 1:40 dilution. In 2002 the Ministry of Health began to recommend the Immunosorbent Assay (ELISA) for screening and RIFI for confirmation of positive results for canine visceral leishmaniasis (CVL). From 2012, (Technical Note joint NÂş 01/2011 "Clarifications on replacing the protocol diagnosis of canine visceral leishmaniasis" - CGDT - / CGLAB / DEVIT / SVS / MS) recommended a new protocol with the performance of screening by Rapid Test Platform Double (TR DPP) and confirmation of positive results by ELISA. The aim of this study was to compare the results of serum samples confirmed as reagents in the previous protocol (ELISA x RIFI) with the current protocol (TR DPP x ELISA) in Central Public Health Laboratory (LACEN-PE). Materials and Methods: Were examined in the LACEN-PE 1473 sera of dogs from various localities of the state. They were subjected to a screening with ELISA and confirmed by RIFI. For comparative study, the samples were used in the first stage reagents tested with new methodology using TR DPP and confirmed by ELISA. Results: From the 1473 sera examined, 42 samples were reactive to ELISA, with 42 samples confirmed by RIFI. For a comparative study, the 42 samples were submitted to the DPP TR, where 38 (90,5%) were positive. Conclusions: These results demonstrated that the DPP TR along with the ELISA may improve the diagnosis of canine visceral leishmaniasis (CVL) avoiding a false-positive result will represent the unnecessary euthanasia of healthy animal.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1148] P 582 - CLONING, EXPRESSION AND PURIFICATION OF L. DONOVANI SPECIFIC ANTIGEN FOR SERODIAGNOSIS OF VISCERAL LEISHMANIASIS KUAMAR, D.1; TIWARY, P.1; DUBE, A.2; CHAKRAVARTY, J.1; SUNDAR, S.1 1.BHU, VARANASI, INDIA; 2.CDRI, LUCKNOW, INDIA.
Keyword:vl; elisa; wb
Abstract: Microscopic observation of amastigotes in splenic or bone marrow biopsies and serology including rK39 dipstick test are tools commonly used for diagnosis of visceral leishmaniasis (VL). However, bone marrow and splenic aspiration are painful and risky procedures and serology with rK39 dipstick can yield false positive responses in 20-32% of endemic healthy individuals. Identification of additional Leishmania donovani antigens could increase the specificity of noninvasive serologic testing for active VL. Therefore, we screened promastigotes soluble proteins using western blotting with a series of serum specimens from patients with acute VL. Western blots revealed a protein of molecular weight 70kDa (BHUP1), recognized by sera from VL patients but not healthy controls. Mass spectrometry of the gel-purified protein revealed the antigen as L. donovani HSP70. The full length hsp70 gene of 1959 nucleotides was determined, cloned and expressed as a His-tagged fusion protein, purified, and retested.
Antibody against this protein were detected in more than 96% of serum samples from patient with VL but not detected in sera from the endemic and non endemic control persons. Crossreactive responses of sera from subjects with different disease like malaria and tuberculosis revealed the BHUP1 antigen test is highly sensitive for VL, but specificity was too low to differentiate from other infectious diseases
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1149] P 583 - MUCOSAL LEISHMANIASIS - DESCRIPTION OF NINE CASES FROM BRAZILIAN AMAZON REGION ASSOCIATED LEISHMANIA (VIANNIA) BRAZILIENSIS AND LEISHMANIA (VIANNIA) GUYANENSIS. PRESTES, S.R.1; CARVALHO, R.S.1; FERNANDES, M.A.2; SILVA, M.S.2; OLIVEIRA, L.V.; CARVALHO, D.M.3; PEIXOTO, S.A.T.4; MAGALHÃES, L.K.1; BARBOSA, M.G.V.1; SILVEIRA, H.M.C.1; GONZALEZ, Y.P.3; ALENCAR, A.A.5; PEREIRA, C.A.4; PIRANGY, F.R.F.6; GUERRA, J.A.O.1
1.FUNDAÇÃO DE MEDICINA TROPICAL HEITOR VIEIRA DOURADO, MANAUS, AM, BRAZIL; 2.HOSPITAL GETÚLIO VARGAS, MANAUS, AM, BRAZIL; 3.FUNDAÇÃO HOSPITAL ADRIANO JORGE, MANAUS, AM, BRAZIL; 4.UNIVERSIDADE FEDERAL DO AMAZONAS, MANAUS, AM, BRAZIL; 5.HOSPITAL GETULIO VARGAS, MANAUS, CE, BRAZIL; 6.UNIVERSIDADE FEDERAL DO AMZONAS, MANAUS, AM, BRAZIL.
Keyword:mucosal leishmaniasis; amazon region; leishmania
Abstract: Introduction: Mucosal Leishmaniasis (LM) is defined by impaired mucosal lining of the upper airway, but the mucosal of the nasal septum hardest hit. Leishmania (Viannia) braziliensis is the species most prevalent cases of LM in the Americas, however cases associated with L. (V.) guyanensis have been demonstrated to be significant in the Amazon region. Objectives: To describe nine cases of LM from Amazon, clinical aspects relating to the species and their distribution characterized based on the places of origin of cases. Materials and methods: Patients treated at the outpatient clinic of the Fundação de Medicina Tropical Doutor Heitor Vieira Dourado (FMTHDV) in the period January to November 2012, diagnosed, the clinical and laboratory, and treated with LM. The species were characterized by Polymerase Chain Reaction (PCR) from biopsies of nasal mucosa and pharynx of the patient. Results: We recorded nine cases of LM of which 05 were caused by L. (V.) braziliensis (55.55%) and 04 by L. (V.) guyanensis (44.44%). Of the 03 patients worked with plant extraction (33.33%), 05 were male (50%), 03 had a history of cutaneous leishmaniasis not properly treated (33.33%). The most common symptoms were epistaxis in 06 (66.66%) and removal of crusts in 07 patients (77.77%), 02 had nasal septum perforation (22.22%), 03 infiltration (33.33% ) and 04 with nasal mucosal ulcers (44.44%). All underwent drug treatment and are outpatients in FMTHVD. Conclusion:
The LM has great impact on the patient, it affects your life in the economic and social. Important correlate symptoms and disease progression in both species analyzed, thereby linking the species with prognosis and cure of mucosal leishmaniasis. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1151] P 584 - A MECHANISTIC INSIGHT INTO ARABINOSYLATED LIPOARABINOMANNAN (ARA-LAM) MEDIATED MODULATION OF IFN- SIGNALING AND DIFFERENT TRANSCRIPTION FACTORS DURING EXPERIMENTAL VISCERAL LEISHMANIASIS MAJUMDAR, S.; MAJUMDER, S.; PAUL CHOWDHURY, B. BOSE INSTITUTE, KOLKATA, INDIA.
Keyword:lipoarabinomannan; interferon; antigen presentation
Abstract: Visceral Leishmaniasis (VL), commonly known as kala-azar is one of the most widespread infectious diseases affecting the third world countries. The hallmark of VL, caused by Leishmania donovani, is a strong suppression of the host-protective anti-leishmanial responses in a susceptible host. Arabinosylated Lippoarabinomannan or Ara-LAM isolated from an avirulent strain Mycobacterium smegmatis is a TLR2 ligand and has potent immunomodulatory properties such as up-regulation of pro-inflammatory cytokines, NO production and induction of TLR-2 mediated signaling in macrophages. In the present study, we have demonstrated that AraLAM confers protection against leishmanial pathogenesis via the modulation of IFN- Receptor expression as well as its downstream signaling. By inhibiting the ceramide generation, AraLAM maintained the cell membrane integrity of the parasitized macrophages and increased the MHC-II expression. Along with this, Ara-LAM, significantly upregulated the phosphorylation level of JAK1 and JAK2 following IFN- -stimulation in infected macrophages, which ultimately induced the nuclear translocation of STAT1. Not only that, Ara-LAM was also found to differentially regulate Interferon Reulatory Factors (IRFs), among which IRF8 is directly involved in IFN- signaling . Therefore, our study demonstrates that by modulating IFNresponsiveness in L. donovani infected macrophages, Ara-LAM helps in the improved antigen presentation as well as better parasite clearance. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1154] P 585 - SAMPLE QUALITY CONTROL SYSTEM FOR LEISHMANIASIS DIAGNOSIS IN RODENTS BY GLICERALDEHYDE-3-PHOSPHATE DEHYDROGENASE GENE AMPLIFICATION GONÇALVES, S.C.; TRAJANO, L.A.M.; DE MORAIS, R.C.S.; SILVA, R.P.E.; BRITO, L.P.S.; MARINHO-JÚNIOR, J.F.; BRANDÃO-FILHO, S.P.; DE PAIVA-CAVALCANTI, M. AGGEU MAGALHÃES RESEARCH CENTER, RECIFE, PE, BRAZIL.
Keyword:sample quality control; pcr; g3pd
Abstract: The difficulties in controlling leishmaniasis may be related to the complex epidemiology of this disease, which involves the participation of different wild and domestic hosts, humans, vectors, and their interaction with different environmental characteristics. The validation of sensitive tests contributes to the early treatment of patients also subserving the design of disease control actions. Molecular biology methods, as PCR, have become increasingly relevant to the diagnosis and control of infectious diseases, such as leishmaniasis. The possibility of combining multiple primer systems in the same assay, in both conventional and real-time PCR protocols, enables the identification of parasites, associated with other targets to sample quality assurance (internal
controls – IC). In this context, this study aimed to design primer systems able to detect the gene G3PD of mammals, allowing the evaluation of the DNA quality on samples for real-time PCR (qPCR) diagnostics in different rodent species involved in leishmaniasis cycle. Based on the results obtained by Castilho et al. (2008), an alignment of gene G3PD for different mammals involved in the cycle of leishmaniasis was made. Three detection systems were designed: G3PD1, G3PD2, G3PD3 with products 441, 567 and 621 base pairs (bp), respectively. As a preliminary evaluation of the systems, DNA extracted from blood samples of 6 rodent species captured in an leishmaniasis endemic area of Pernambuco, Brazil were tested. The reproducibility of results was evaluated by duplicates intra-test. Amplification of the gene was obtained for all species tested with the three detection systems, indicating good performance of oligonucleotides designed. The system G3PD1 showed reproducibility, as well as homogeneity between duplicates. We also observed that melting curve analysis may allow the differentiation of the rodent species by PCR products. The G3PD1 will be further adapted to compose a duplex qPCR protocol for leishmaniasis diagnosis with sample quality control, by TaqMan probe technology. Standardization of a duplex qPCR with internal control amplification simultaneously with the main target will limit the possibility of false-negative results. The technique will allow correct diagnosis and monitoring of rodent species, contributing to the enhancement of knowledge related to parasite load and parasite-host interaction, providing useful information to improve the treatment and control measures in endemic areas. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1162] P 586 - DEVELOPMENT AND EVALUATION OF A LOOP-MEDIATED ISOTHERMAL AMPLIFICATION ASSAY FOR RAPID DETECTION OF LEISHMANIA INFANTUM CHAOUCH, M.1; MHADHBI, M.2; SCHOONE, G.J.3; LIMAM, S.2; ADAMS, E.R.3; DARGHOUTH, M.2; GUIZANI, I.1; BEN ABDERRAZAK, S.B.1 1.INSTITUT PASTEUR DE TUNIS, TUNIS, TUNISIA; 2.ECOLE NATIONALE DE MÉDECINE VÉTÉRINAIRE, SIDITHABET/TUNIS, TUNISIA; 3.ROYAL TROPICAL INSTITUTE, AMSTERDAM, NETHERLANDS.
Keyword:lamp, pcr, serology, microscopy; leishmania infantum; diagnosis, tunisia
Abstract: The proposal aims at improving means of molecular diagnosis of Leishmania infections and diseases by developing and assessing potential of Loop Mediated Isothermal Amplification Method (LAMP) assays, a simple, rapid, reliable and affordable DNA assays. Moreover, results can be visually detected by the Real Time Turbidimeter but also using different type of dye such Hydroxy-naphtol staining (HNB). Accurate and rapid diagnosis of Canine Leishmaniasis is an important issue that allows early treatment and prevents transmission. We developed and validated a L. infantum specific LAMP assay was carried out using a set of six primers targeting the cysteine protease B multi copy gene of L. infantum. The reaction conditions were setup and the specificity and sensitivity of the assay assessed. The specificity experiment showed that LAMP primers amplified L. infantum DNA successfully, while no amplification was seen for L. major, L. tropica, L. turanica, L. aethiopica, L. tarentolae, L. gerbilii and T. cruzi as well as for human genomic DNA. When compared to conventional cpb based PCR, the sensitivity of LAMP assay was higher with a detection limit of 50 fg/ l of genomic L. infantum parasite DNA. The colorimetric visualization shows a good correlation between the results obtained using the Real Time Turbidimeter and those relying on the HNB. Finally, this LAMP assay was applied on seventy five (75) DNAs extracted from suspected Leishmania infected dogs blood obtained from the field. Classical PCR, serology and microscopy were used as gold standards. Compared to molecular methods, the serology (IFI Indirect Immunofluorescence) test presents the best sensitivity (88.57%) with however, a much lower specificity (52.5%) due to a relatively high number of false-positive results (22 animals). The PCR assay shows a low sensitivity 37.14 % and specificity around 82.5%. Performed on
blood samples, our LAMP assay shows a suitable sensitivity (54%) and a good specificity (80%), with however, positive (70%) and negative (66%) predictive values. In contrast to existing LAMP, our developed assay do not present cross reactions with T. cruzi, and as previously reported, is more resistant to inhibitors that prevent conventional PCR. In a brief cost evaluation, the LAMP assay compares favorably with other molecular diagnostic tests. The cost (including transportation and fluorescent detection reagent but excluding staffing costs) is $3.5 per 25 L reaction; the reduction of the reaction volume by half present no adverse consequences and will allows a cost reduction of 50%. This is less or comparable to other molecular diagnostics such as real time-PCR, estimated at $12 and PCR-restriction fragment length polymorphism (PCR-RFLP) estimated at $2.5 per reaction. Moreover, the relative stability of LAMP reagents at 25°C and 37°C promotes the possibility of field deployability of the techniique. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1167] P 587 - EFFICACY OF PAROMOMYCIN, MILTEFOSINE AND MEGLUMINE ANTIMONIATE IN COMBINATION FOR THE TREATMENT OF EXPERIMENTAL CUTANEOUS LEISHMANIASIS CAUSED BY LEISHMANIA (VIANNIA) BRAZILIENSIS TEIXEIRA, E.M.1; AGUIAR, M.G.1; FERREIRA, L.A.M.2; RABELLO, A.L.T.1 1.CENTRO DE PESQUISAS RENÉ RACHOU - FIOCRUZ, BELO HORIZONTE, MG, BRAZIL; 2.UNIVERSIDADE FEDERAL DE MINAS GERAIS - FACULDADE DE FARMÁCIA, BELO HORIZONTE, MG, BRAZIL.
Keyword:combination therapy; cutaneous leishmaniasis; experimental
Abstract: Combined therapy may represent an alternative to monotherapy for the treatment of cutaneous leishmaniasis (CL), to increase efficiency, reduce total drug doses and preventing the resistance, with consequent reduction of cost and toxicity. In the case of CL, topical and oral administrations are desirable options. The objective of this study was to evaluate the efficacy of binary combinations between: topical paromomycin-gel at 10% (PA) and Glucantime® 50mg/kg/day intramuscular (Glu), PA and oral solution of miltefosine 25mg/Kg/day (Milt), and Milt + Glu in experimentally infected animals. Hamsters (Mesocricetus auratus) were inoculated subcutaneously at the base of tail with 1x105 amastigotes of L. (V.) braziliensis (WHOMHOM/BR/75/M2903). After ulceration of lesions, the animals were divided into seven groups (n = 5): 1) untreated control 2) PA, 3) Milt, 4) Glu, 5) Glu + PA, 6) Milt + PA and 7) Glu + Milt. The animals were treated for 20 consecutive days (Glu and PA, once daily), and/or 10 alternate days (Milt, once daily). The lesions sizes were determined weekly. After the end of treatment, animals were euthanized, and parasites recovered from skin lesions and spleens were evaluated by limiting-dilution assays. Significant reduction in lesions sizes was observed for all combined treatments as compared to the control group (p<0.05). Lesion size was statistically smaller in animals treated with the combination Glu + PA or Milt + PA as compared with those treated with Glu, Milt or PA in monotherapy (p<0.05). There was a significant reduction of viable parasite recovered from the skin lesions in all combined treatments as compared to the control group and monotherapy (p<0.05). In the spleen, significant reduction in the number of viable parasites was observed for all groups in combined treatments as compared to the control group and monotherapy regimes except for animals receiving Glu alone. These results suggest that the combinations of systemic therapy (Glu or Milt) with topical treatment (PA) were more effective in reducing the parasitic load and lesion size compared to monotherapy with these drugs. These combinations may be an interesting alternative for the treatment of CL caused by L. (V.) braziliensis. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE
[1169] P 588 - EVALUATION OF EFFICACY OF ORAL FEXINIDAZOLE IN PRE-CLINICAL STUDY OF THE CUTANEOUS LEISHMANIASIS CAUSED BY LEISHMANIA (VIANNIA) BRAZILIENSIS AGUIAR, M.G.1; TEIXEIRA, E.M.1; ALVES, F.P.2; CORRÊA, I.A.A.Q.2; RABELLO, A.L.T.1 1.CENTRO DE PESQUISAS RENÉ RACHOU - FIOCRUZ, BELO HORIZONTE, MG, BRAZIL; 2.DRUGS FOR NEGLECTED DISEASES INITIATIVE, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:fexinidazole; leishmania braziliensis; experimental
Abstract: In the search for new alternatives, the Drugs for Neglected Diseases initiative (DNDi) has rediscovered the fexinidazole (Fex), an oral antimicrobial that is under development for sleeping sickness and has demonstrated leishmanicidal activity. This study evaluated the in vivo activity of oral Fex in animals experimentally infected with L. (V.) braziliensis, causative species of cutaneous leishmaniasis (CL) in New World. Hamsters were inoculated subcutaneously at the base of tail with 1x105 amastigotes of L. (V.) braziliensis (WHO-MHOM/BR/75/M2903). After ulceration of lesions, the animals were divided into six groups (n=5): untreated control, diluent, Glucantime® 200mg/kg/day intramuscular (Glu), Fex50mg/Kg/day (Fex50), Fex200mg/Kg/day (Fex200) and Fex300mg/Kg/day (Fex300). The size of the lesions (millimetres–mm) was comparable among groups at treatment onset. The animals were treated for 20 consecutive days (once daily), lesion sizes were measured on days D1, D7, D14 during treatment and D+3 post treatment. After this period, animals were euthanized, and parasites recovered from skin lesions and spleens were evaluated by limiting-dilution assays. Significant reduction in lesion size was observed for the group Glu in the D14 (5.0±2.5) as compared with control group (11.4±3.2) (p<0.05). In the D+3 there was significant reduction for groups: Glu (1.2±2.7), Fex200 (4.2±3.9) and Fex300 (4.1±4.0) as compared with control (11.0±1.9) and diluent (13.0±2.9) groups (p<0.05), and there was no statistically significant difference between the groups treated with Glu and Fex200 and Fex 300 in D+3 (p>0.05). There was significant reduction of viable parasite recovered from the skin lesions (control:1x103, diluent:1x103, Glu:1x101, Fex50:1x102, Fex200:1x102, Fex300:1x102), and spleen (control:1x103, diluent:1x103, Glu:0, Fex50:1x101, Fex200:1x101, Fex300:1x101), for the all groups treated as compared to control and diluent groups (p<0.05). However, the animals treated with Glu showed a greater reduction of parasite load in the lesion as compared to animals treated with all doses of Fex (p<0.05). There was no statistically significant difference among Fex groups in reduction of parasite load in the lesion and spleen (p>0.05), but the lowest dose of Fex has not led to a reduction in lesion size (p>0.05). These results showed that Fex at doses of 200 and 300 mg/kg/day were active in the treatment of CL caused by L. (V.) braziliensis. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1170] P 589 - EVALUATION OF THE ANTI-LEISHMANIAL ACTIVITY IN ANTIINFLAMMATORY AND WOUND HEALING DRUGS SELECTED BY BIOINFORMACTIC MESA, C.V.1; BLANDON, G.A.2; OCHOA, R.A.2; MUÑOZ, D.L.2; RESTREPO, A.M.2; DAZA, J.A.2; JURADO, D.S.2; MUSKUS, C.E.2; VELEZ, I.D.2; ROBLEDO, S.M.2 1.PECET, UNIVERSITY OF ANTIOQUIA AND CIDEPRO, MEDELLIN, COLOMBIA; 2.PECET, UNIVERSITY OF ANTIOQUIA, MEDELLIN, COLOMBIA.
Keyword:bioinformatic; leishmaniasis; leishmanicidal activity
Abstract: Currently few drugs are available to treat cutaneous leishmaniasis (CL) that although effective they have important concerns regarding to their toxicity, way of administration and cost.
Bioinformatic represents a useful tool that allows a fast and effective way to indentify new drugs with potential anti-leishmanial activity. Using these tools 21 drugs exhibiting anti-inflammatory and wound healing properties were identified and then evaluated in vitro for their cytotoxicity and anti-leishmanial activity against intracellular and axenic amastigotes of L. (V) pananensis. The therapeutic response was also determined in the hasmter model for CL. Four of the selected drugs showed high in vitro activity against axenic amastigotes with EC50 values < 50 g/ml and three compounds were active against intracellular amastigotes. Four of these 7 drugs were tested in vivo for their therapeutical response. Of the three compounds active against intracellular amastigotes two produced cure in 3 of 6 animals after three months of the end of treatment. Of the four drugs that were active against axenic amastigotes, two of them only induced clinical improvement but not cure of the lesion in 4 of 5 and 3 of 5 animals, respectively. These results suggest that by modifying the therapeutic scheme or the pharmaceutical formulation the therapeutic response could be optimized. The results also confirm that bioinformatic is a promising methodology to identify effective potential drugs with anti-leishmanial activity yielding reliable results in short time. - CLINICAL LEISHMANIASIS [1173] P 590 - EFFECT OF INSULIN-LIKE GROWTH FACTOR-I ON ARGINASE ACTIVITY AND PROLIFERATION OF STRAINS OF LEISHMANIA (VIANNIA) BRAZILIENSIS FROM PATIENTS WITH DIFFERENT CLINICAL FORMS OF AMERICAN CUTANEOUS LEISHMANIASIS SOUZA, L.D.1; VENDRAME, C.M.V.1; GUIMARﾃウS, L.H.2; CARVALHO, E.M.2; GOTO, H.1 1.UNIVERSIDADE DE Sﾃグ PAULO, Sﾃグ PAULO, SP, BRAZIL; 2.UNIVERSIDADE FEDERAL DA BAHIA, SALVADOR, BA, BRAZIL.
Keyword:leishmania braziliensis; arginase; insulin-like growth factor i
Abstract: American tegumentary leishmaniasis (ATL) is caused mostly by Leishmania (Viannia) braziliensis in Brazil and cutaneous (CL), mucosal (ML) and disseminated (DL) forms of the disease are known. The diversity of clinical manifestations has been attributed to differences in the host immune response, but recently it has also been related to intraspecific variability of L. (V.) braziliensis. In previous studies using Leishmania (Leishmania) amazonensis IGF-I was shown to increase the activity of arginase generating polyamines and to decrease the synthesis of nitric oxide (Vendrame et al, Scand J. Immunol. 66:287, 2007). Recently the effect of IGF-I on L. (V.) braziliensis was also studied and we showed that IGF-I induced higher basal arginase activity in promastigotes of isolates of L. (V.) braziliensis derived from patients with CL and DL. In isolates from ML the arginase activity was already increased in basal conditions with no further increment upon IGF-I stimulus being higher in those from patients with ML (Vendrame et al, Trans Roy Soc Trop Med Hyg 104: 566, 2010). In the present study, we evaluated the characteristics of individual isolates, derived from patients with CL (N = 8), with ML (N = 7) and DL (N = 7) from an endemic area in the Northeastern Brazil, as amastigote within human macrophage cell line THP-1 with and without IGF-I stimulation (50ng/mL). Adherent phorbol 12-myristate 13 acetate differentiated-THP-1 cells were infected with promastigotes (parasite/macrophage ratio = 10:1) and after 24 hour culture with or without IGF-I stimulus parasitism and arginase activity were evaluated. In amastigotes derived from in isolates of DL
the basal arginase activity was lower than in those of CL, respectively 33.4 and 42.7 (median) mU of arginase/106 cells. The effect of IGF-I on parasitism was evaluated in relation to the basal infection and we observed that occurred an increase of parasitism upon stimulus in 7 of 8 (87.5%) isolates from CL and in 5 of 7 (71.4%) from ML while a decrease in most of isolates from DL (only 1 in 7 thus in 14.3% showed an increase). The data suggest that differences in the biological characteristics of parasites may contribute to the diversity of clinical presentation of ATL. Supported by FAPESP, FAPESB, CNPq, LIM-38 (HC-FMUSP)
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1176] P 591 - ELECTRON MICROSCOPIC STUDIES OF ORGANELLE CHANGES OF LEISHMANIA TROPICA PROMASTIGOTE; SENSITIVE AND RESISTANT TO MEGLUMINE ANTIMONIATE DABIRI, S.1; BAHRIANI, M.1; BOULORIZAEDH, M.2; SHARIFI, I.1 1.AFZALIPOUR MEDICAL SCHOOL, KERMAN, IRAN, ISLAMIC REPUBLIC OF; 2.HOWARD HUGHESMEDICAL INSTITUTE, WASHINGTON, UNITED STATES.
Keyword:electron microscope; lesihmania tropica; drug sensitive, resistant
Abstract: Electron microscopic studies of organelle changes of Leishmania tropica promastigote; sensitive and resistant to meglumine antimoniate Mitra Bahriani1,MS;Mehdi Bahriani3, PhD;Shahriar Dabiri1, MD; Iraj Sharifi2, PhD 1Patholology ,2Parasitology Departments, Afzalipour Medical School,Leishmaniasis Research Center,Kerman Iran.3Howard Hughes Medical Institute,USA This study was carried out in the city of Bam ,after devastating earthquake, between 2008 to 2012,. The samples were taken from 66 patients who were referred to the Cutaneous Leishmaniasis (CL) treatment center. Diagnosis was confirmed by smear, culture media, while identification of species was carried out by nested-PCR (NPCR) and sensitive and resistant patients by clinical follow-up and MDR1 sequencing. Ten cultured promostigotes of sensitive and resistant patients and also one standard strain of L.tropica were selected for transmission electron microscopic (TEM) examination.This work was the first TEM presentation from Iran. We found that ultrastructural result in drug-sensitive promastigote revealed a significant change in vesicles size (multivesiculated bodies),nucleus was normal and located posterior to kinetoplast at the short distance. Cytoplasmic mitochondrion which was enlarged and elongated. No change in cytoplasmic membrane of sensitive promastigote was observed. The location of microtubules were changed from margin of promastigotes to middle in resistance strain compared to promastigote in standard strain. The mitochondrion was circular and located around the nucleus.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1178] P 592 - EVALUATION OF THE ANTIGENICITY OF FOUR NOVEL ANTIGENIC PROTEINS OF LEISHMANIA CHAGASI/INFANTUM AS ALTERNATIVES FOR THE DIAGNOSIS OF HUMAN AND CANINE VISCERAL LEISHMANIASIS DE CASTRO NETO, A.L.1; NASCIMENTO, M.B.1; MAGALHAES, F.B.2; DE MELO NETO, O.P.3 1.UFPE, RECIFE, PE, BRAZIL; 2.CENTRO DE PESQUISA AGGEU MAGALHAES - FIOCRUZ, RECIFE, PE, BRAZIL; 3.CENTRO DE PESQUISA AGGEU MAGALHÃES - FIOCRUZ, RECIFE, PE, BRAZIL.
Keyword:visceral leishmaniasis; leishmania infantum; diagnosis
Abstract: Developing methods for early diagnosis and effective vaccination or immunotherapy are essential conditions for an effective combat against Visceral Leishmaniasis (VL). For these reasons, our group has been seeking the improvement of serological techniques using highly immunogenic proteins able to identify early infections in dogs and humans. Through serological screenings of DNA expression libraries, 13 new antigenic proteins from Leishmania chagasi/infantum were previously identified. Four of these (Lci1, Lci10, Lci12 and Lci13) were further evaluated in this study. The Lci10 and Lci12 display rich tandem repetitive sequences, whereas the Lci1 and Lci13 are stress related. Their coding sequences were cloned into expression vectors and the corresponding his-tagged recombinant proteins produced in Escherichia coli. These were purified through affinity chromatography using a nickel column and quantified through the Bradford assay. Immunoassays (ELISA) were then performed with infected sera from humans (50 positive and 50 negative) and dogs (46 positive and 15 negative), with the positive ones confirmed through prior parasitological examination. The CUT-off of the reaction was derived from the average of the negative controls, from healthy individuals, plus three standard deviations. The tested proteins (Lci1, Lci10, Lci12 and Lci13) displayed 93, 67, 91 and 97% sensitivities in the dogs’ sera, respectively. With human sera, the same proteins displayed 72, 48, 46 and 44% sensitivities. Following the individual assays, the Lci1, Lci12 and Lci13 were combined into a mix, which displayed 91 and 84% sensitivities in the dogs and humans’ sera, respectively. The differential recognition of these proteins by the sera from VL infected humans and dogs highlight the difference in immune response between these two hosts and the need for optimization of current serological tests. Nevertheless, these preliminary tests also demonstrate that the chosen proteins are highly immunogenic in dogs and could potentially contribute as part of canine specific serological tests or vaccines against canine VL. The higher sensitivity of the mix with both sets of sera may also be a good alternative for improvement in view of a single diagnostic test for both humans and dogs. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1182] P 593 - LEISHMANICIDAL ACTIVITY OF NOVEL LAWSONE DIMERS ARAÚJO, M.V.1; OLIVEIRA, P.S.2; DA MATTA, C.B.B.1; QUEIROZ, A.C.1; ALEXANDREMOREIRA, M.S.1; CAMARA, C.A.2; SILVA, T.M.S.2
1.FEDERAL UNIVERSITY OF ALAGOAS, MACEIÓ, AL, BRAZIL; 2.FEDERAL UNIVERSITY RURAL OF PERNAMBUCO, RECIFE, PE, BRAZIL.
Keyword:lawsone dimers; promastigotes; leishmania braziliensis
Abstract: Leishmaniasis is considered a public health problem. The World Health Organization (WHO) estimates that 350 million people are exposed to the risk. Considering that the treatment adopted has serious adverse effects, high toxicity and various strains of Leishmania are resistant to therapy, this study aims to investigate the leishmanicidal activity of novel lawsone dimers obtained by one-pot high-yield procedure from the condensation of lawsone and suitable
aldehydes. The cell line J774 viability was determined with MTT assay. Leishmanicidal effects against promastigotes of Leishmania braziliensis (MHOM/BR/87/BA125) were evaluated. Data obtained from experiments were expressed as the mean ± standard error of mean (S.E.M) of triplicate cultures of representative assays. Statistical differences between the treated and the vehicle groups of in vitro experiments were evaluated by ANOVA and Dunnett hoc tests and considered significant when p <0.05. The compounds pentamidine and DE at concentration of 100 µM showed activity deleterious to the host cell evidenced by MTT assay, but was not cytotoxic at 10 µM. Furthermore, the compounds DA, DB, DF, DH, DI, DN and DP not affected the viability of macrophages at concentration of 100 µM. Moreover, the compounds DN, DF, DP, DH, DA, DE and DI exhibited high antileishmanial activity against promastigotes of L.braziliensis, with maximal effect of 91.7 ± 1.3, 88.7 ± 0.3, 88.4 ± 0.9, 93.0 ± 0.1, 61.6 ± 7.5, 90.4 ± 0.7 and 91.7 ± 0.3%, respectively. The compounds DB did not show significant activity against the growth of promastigotes of L. braziliensis at concentration of 100 µM. The compounds DN, DF, DP, DH, DA and DI are strong candidates for antileishmanial prototypes drug because exhibit pronounced leishmanicidal activity against promastigotes of L. braziliensis without citotoxity effect against host cells. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1183] P 594 - LEISHMANICIDAL ACTIVITY OF GEOPROPOLIS COLLECTED BY STINGLESS BEE MELIPONA SUBNITIDA AGAINST LEISHMANIA AMAZONENSIS DIAS, T.L.M.F.1; LEITE, A.B.1; SOUSA, S.A.2; QUEIROZ, A.C.1; ARAÚJO, M.V.1; DA MATTA, C.B.B.1; SANTOS, M.S.1; TENÓRIO, A.P.M.1; DA SILVA, A.E.1; SILVA, T.M.S.2; ALEXANDRE MOREIRA, M.S.1 1.LABORATÓRIO DE FARMACOLOGIA E IMUNIDADE - ICBS - UFAL, MACEIÓ, AL, BRAZIL; 2.LABORATÓRIO DE BIOPROSPECÇÃO FITOQUÍMICA - DCM - UFRPE, RECIFE, PE, BRAZIL.
Keyword:leishmaniasis; antileishmanial activity; geopropolis
Abstract: Leishmaniasis is a neglected disease, that affects mostly poor populations living in tropical and subtropical regions of the world and presents withhigh incidence in Brazil. The current treatment of this disease includes limitations in terms of toxicity, variable efficacy, high costs and inconvenient doses and treatment schedules. Continuing the research who seek alternatives to developing new therapies for leishmaniasis, this study aims to evaluate the antileishmanial activity of geopropolis collected by a stingless bee (Melipona subnitida Ducke) native to Northeastern Brazil. Six sample (BP-March2010, BP-April2010, BP-June2011, BPJanuary2012, BP-June2012 and BP-July2012) were collected from semi-arid region of Paraiba, Brazil. The cell line J774 viability was determined with MTT assay. Leishmanicidal effect against promastigotes of Leishmania amazonensis (MHOM/BR/77/LTB0016) were evaluated at concentration of 100 g/mL – 0.1 g/mL to calculate LC50 (concentration required to give 50% of death). Data obtained from experiments were expressed as the mean ± S.E.M. of triplicate cultures of representative assays. The results showed that all products at concentration of 100 g/mL showed deleterious activity to the host cell evidenced by MTT assay and BP-June2011 was also citotocity at 10 g/mL. Moreover, BP-March2010, BP-April2010, BP-June2011, BPJanuary2012 and BP-July2012 exhibited high antileishmanial activity against promastigotes of L. amazonensis, with maximal effect greater than 65%, without large seasonal variation. The reference drug pentamidine presented maximum effect against promastigotes of 93.0 ± 1.0%. All compounds tested were lower potent than pentamidine (IC50 of 1.1 ± 0.1µg/mL) to dead promastigotes. This is the first detailed study of antiprotozoal effect of geopropolis collected by Melipona subnitida bee, a natural product with pronounced leishmanicidal activity which can originate new prototypes useful for leishmaniasis treatment.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1186] P 595 - LEISHMANICIDAL ACTIVITY OF THE ETHYL AND BENZYL ESTERS OF NISOPROPYL OXAMATE ON PROMASTIGOTES , EXTRACELLULAR AND INTRACELLULAR AMASTIGOTES OF LEISHMANIA MEXICANA MONROY OSTRIA, A.M.1; HERNANDEZ RIVERA, M.P.2; HERNANDEZ MONTES, O.2; WONG RAMIREZ, C.2; BAEZA RAMIREZ, I.3; NOGUEDA TORRES, B.2
1.ENCB, INSTITUTO POLITÉCNICO NACIONAL, MEXICO, MEXICO; 2.ENCB, INSTITUTO POLITECNICO NACIONAL, MEXICO, MEXICO; 3.ENCB, INSTITUO POLITECNICO NACIONAL, MEXICO, MEXICO.
Keyword:cutaneous leishmaniasis; n-isopropyl oxamato; leishmania mexicana
Abstract: In Mexico CL is the most important form of Leishmaniasis. It is distributed in several States, the mainly endemic areas are located in the South-East of the country. In Mexico CL can be produced by L. mexicana and L braziliensis Complex members, the clinical forms present can be primary localized skin lesions (LCL) some cases can self-heal, but parasites can disseminate to the nasopharyngeal mucosa and cause mucocutaneous leishmaniasis (MCL), or disseminated to the entire body as nodular lesions in diffuse cutaneous leishmaniasis (DCL). Meglumine antimoniate (Glucantime) is the principal drug used for treatment of CL and Amphotericine B (Amph B), the response to therapeutic drugs can vary among different species present in the same area. In this study was evaluated the leishmanicidal activity of the ethyl and benzyl esters of Nisopropyl oxamate (NIPOX-B AND NIPOX-Et) on promastigotes in exponential phase of growth, finding leishmanicidal activity of LD50 at 16 mM for NIPOX B and 8 mM for NIPOXET. In axenic extracellular amastigotes was found a LD50 at 2mM for NIPOX-B and at 8 mM for NIPOX-ET. In macrophages (J774) infected with intracellular amastigotes of Leishmania mexicana it was found 100% of parasite elimination in a dose of 64 mM for NIPOX-B and 32 mM for NIPOX-ET, for Glucantime it was found 80% of parasite elimination at 64 Mm, Amph B I a dose 32mM eliminated 100% of parasites. The more toxic to macrophages were Amph B and glucantime, NIPOXs showed low toxicity. In conclusion the NIPOXs are good candidates for CL therapeutic treatment. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1190] P 596 - DNDI-VL-2098: A NITROIMIDAZOOXAZOLE DERIVATIVE AS A POTENTIAL CLINICAL CANDIDATE TO TREAT VISCERAL LEISHMANIASIS LAUNAY, D.1; MARTIN, D.1; BRAILLARD, S.1; VISHWAKARMA, P.2; GUPTA, S.2; PURI, S.2; YARDLEY, V.3; RAO, M.4; RAMANATHAN, V.4; KRISHNAPPA, H.4; PATI, H.4; THOMPSON, A.5
1.DRUGS FOR NEGLECTED DISEASES INITIATIVE, GENEVA, SWITZERLAND; 2.CENTRAL DRUG RESEARCH INSTITUTE, LUCKNOW, INDIA; 3.LONDON SCHOOL OF HYGIENE AND TROPICAL MEDICINE, LONDON, UNITED KINGDOM; 4.ADVINUS THERAPEUTICS, BANGALORE, INDIA; 5.AUCKLAND CANCER SOCIETY RESEARCH CENTRE, UNIVERSITY OF AUCKLAND, AUCKLAND, NEW ZEALAND.
Keyword:nitroimidazooxazine; in vivo efficacy; preclinical development
Abstract: DNDi (Drugs for Neglected Diseases initiative) is a collaborative, patients’ needs-driven, notfor-profit organization whose mission is to develop new drugs for the most neglected tropical diseases, including Visceral Leishmaniasis (VL). To this purpose, our research effort focused on the nitroimidazole chemical class, well known for its antimicrobial activity, and quickly led to the identification of DNDI-VL-2098 as a potential candidate for VL. This compound is indeed likely to fulfill DNDi’s Target Product
Profile, such as: a) effectiveness against L. donovani in East Africa and India (including drug resistant strains), b) 95 % clinical efficacy, c) no contraindications, d) oral formulation, e) dosing once a day for a maximum of 10 days, f) cost under $10 per course. DNDI-VL-2098 is a chiral nitroimidazooxazole analog prepared in 8 steps from commercially available materials. Its in vitro activity (IC50) against different strains of Leishmania donovani ranges from 0.3 to 1 µM. In VL in vivo models, DNDI-VL-2098 showed the following efficacies: - Total cure at 12.5 mg/kg single dose or 3 mg/kg over 5 days in the acute mouse model - Total cure at 50 mg/kg over 3 days or 25 mg/kg over 5 days in the chronic hamster model The oral bioavailability of DNDI-VL-2098 varied from 70 % (rat) to 100 % (mouse, hamster). Its volume of distribution ranged from 2.0 to 2.5 L/kg (about 3-fold the total body water volume) and the mean elimination half-life ranged from 1.2 (hamster) to 6.0 h (dog). DNDI-VL-2098 moderately inhibited the hERG channel (in transfected Chinese Hamster Ovary cells), with an IC50 of 10.5 µM. However, a telemetry study in male Beagle dogs showed no modification of QTc up to the highest dose tested (250 mg/kg). DNDI-VL-2098 was shown to be neither mutagenic nor clastogenic. In mice and rats, after single dose oral administration, the No-Observed-Effect Level (NOEL) was > 2000 mg/kg. According to allometric scaling predictions for human PK, the minimum efficacious dose in human beings (60 kg) is expected to be in the range of 200 to 600 mg, with a once a day oral administration. In conclusion, DNDI-VL-2098 so far shows promising properties to be developed as a clinical candidate for the treatment of visceral leishmaniasis.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1191] P 597 - A COMPARATIVE EVALUATION OF DIAGNOSTIC ACCURACY OF COMMERCIALLY AVAILABLE IMMUNOCHROMATOGRAPHIC TEST FOR VL ON WHOLE BLOOD AS WELL AS SERUM AT BOTH BED SITE AND LABORATORY SITE. KUAMAR, D.; TIWARY, P.; M, S.L.; SUNDAR, S. BANARAS HINDU UNIVERSITY, VARANASI, INDIA.
Keyword:vl; rdts; senstivity,specificity
Abstract: Background: Leishmaniasis is a fatal and one of the most neglected disease which is caused by a protozoan parasite Leishmania donovani. Diagnosis of leishmaniasis is still a challenge in front of investigator. A firm diagnosis of visceral leishmaniasis (kala azar) requires demonstration of the parasite in organ aspirates or tissue biopsy samples. The aim of this prospective study was to assess the diagnostic usefulness of non-invasive testing for antibody to the leishmanial antigen by means of antigen-impregnated nitrocellulose paper strips adapted for use under field as well as laboratory conditions. There are so many rapid diagnostic tests (RDTs) of different brands available and published report of variable accuracy. Methods: This study of evaluation includes six commercially available VL-RDTs of four different manufacturers. These VL-RDTs were evaluated using whole blood as well as serum at
two different conditions (At bed site and laboratory site). In this study total 135 confirmed VL cases, 85 endemic healthy with no kala azar history, 15 malaria patients and 20 tuberculosis patients were enrolled. All tests were performed against blood and serum according to the manufacturer’s protocol. The interpretation was recorded into a data sheet. At the laboratory site two individuals were performed the tests by randomised the samples and cross check the result of each other and note their interpretation. All the data were entered in duplicate by two technicians to overcome the error. Result: This study is the comparison of performance of VL RDTs of different manufacturers and first time reports the diagnostic accuracy of all VL RDTs with whole blood and serum both. Overall, our finding illustrate that different RDTs demonstrated varied sensitivity in case of VL which ranged from 94% to 100% in case whole blood and 95%to 100% in case of serum respectively. However the specificity range from 80% to 100% for blood while 78% to 100% for serum. The other findings are under analysis.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1192] P 598 - ANOMERIC FORMS OF SUGARS AND SUGAR-PHOSPHATES IN GLYCOSOMAL CARBOHYDRATE METABOLISM OF TRYPANOSOMATIDS GUPTA, D.S.1; MICHELS, P.A.2 1.HERITAGE INSTITUTE OF TECHNOLOGY, KOLKATA, INDIA; 2.DE DUVE INSTITUTE, BRUSSELS, BELGIUM.
Keyword:trypanosomatids; glycosomes; glucose phosphate epimerase
Abstract: Trypanosoma brucei is a flagellated parasite of the order Kinetoplastida causing sleeping sickness in human beings and a similar disease called ‘nagana’ in cattle. Trypanosomes contain unique peroxisome-like organelles designated as glycosomes, which contain enzymes involved in a variety of metabolic processes such as the glycolytic and pentosephosphate pathways and routes of lipid anabolism and catabolism. Trypanosomatids possess a hexokinase (HK) inside the glycosomes. In addition, a glucokinase (GlcK) was found in glycosomes of Trypanosoma cruzi and Leishmania species, but not in those of Trypanosoma brucei. The crystal structure of TcGlcK with bound glucose shows that the sugar is present in its beta-anomeric form. In contrast, all known HK-glucose structures possess alpha-glucose, in line with results from kinetic studies that HKs have a preference for this latter anomer. The enzymes following the sugar-kinases in the glycolytic and pentosephosphate pathways, phosphoglucose isomerase and glucose-6-phosphate dehydrogenase (G6PDH), are highly specific for respectively the alpha- and beta-form of glucose 6-phosphate. Although both forms can spontaneously anomerize, the rate of this interconversion may be too low if a high activity of the pentosephosphate pathway is required (for NADPH production) and only a HK is present that produces essentially the alpha-anomer. In T. brucei, where GlcK is absent, a glucose-6phosphate-1-epimerase (G6PE) (also known as hexose-6-phosphate mutarotase) may therefore be required for the anomerization. A candidate G6PE gene was found in the trypanosomatid genome databases. The predicted amino-acid sequence contains a typical peroxisome-targeting signal (PTS1) that suggests a glycosomal localization of the enzyme. The G6PE activity of the enzyme was confirmed after its expression in Escherichia coli as a recombinant protein. Knocking down its expression in bloodstream-form trypanosomes has no effect on the growth of the parasites in regular growth
medium. However, the RNAi-induced cells display an increased susceptibility to oxidative stress in non-reducing medium, similar to results obtained for trypanosomes in which G6PDH has been depleted. These results suggest a role of glucose-6-phosphate-epimerase in the interconversion of alpha- and beta-anomers inside glycosomes of Trypanosoma brucei making glucose 6-phosphate available for the pentosephosphate pathway and consequent NADPH production. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1197] P 599 - CHARACTERISATION OF TRYPAREDOXIN ISOFORMS OF L. DONOVANI AND DEVELOPMENT OF DIAGNOSTIC MARKER ALI, V.; SUMAN, S.S.; EQUBAL, A.; TOPNO, R.K.; PANDEY, K.; BIMAL, S.; DAS, P. RAJENDRA MEMORIAL INSTITUTE OF MEDICAL SCIENCES, PATNA, INDIA.
Keyword:trypanothione; tryparedoxin; diagnosis
Abstract: The trypanothione metabolism, a low molecular mass dithiol, present in trypanosomatids is an appealing source of drug target, since it is absent in mammals. Many vital functions are either directly fulfilled by trypanothione or catalyzed by trypanothione dependent enzymes in kinetoplastids. An important mediator for the trypanothione fuelled redox reaction is the tryparedoxin cascade containing tryparedoxin and tryparedoxin peroxidase enzymes. The biological function of TXNPx is to regulate oxidative stress. The reduced tryparedoxin (TXN) interacts with and passes on a reducing equivalent to tryparedoxin peroxidase, allowing it to catalyze the reduction of hydrogen peroxide and organic hydro peroxides to water or alcohol, respectively. We have cloned and expressed several proteins of thiol metabolism (TryS, TryR, TXN, and TXNPx) either containing GST-tag or His-tag successfully in soluble condition. The recombinant proteins were purified by affinity columns chromatography. Polyclonal antibodies were raised in rabbit against purified recombinant proteins which showed high titre. TXN and TXNPx interact to each other proved by in vitro approach using different tag proteins. It was found that cTXN interacts with cTXNPx, and mTXN interacts with mTXNPx and make stable complex confirmed by co purification and western blot analysis. The purified antigens were subjected to immunoblot analysis and ELISA with VL-patients, HIV-VL, asymptomatic cases, and healthy control sera showed that the Leishmania antigens recognized antibodies in VL and HIV-VL patients. Our results showed very high specificity and above 80% sensitivity. These results suggest that these proteins have the potential for the development of diagnostic tool for detection of VL.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1201] P 600 - 1ST IN-VIVO RESULTS OF A TOPICAL FORMULATION OF MEGLUMINE ANTIMONIATE IN THE TREATMENT OF CUTANEOUS LEISHMANIASIS BLAYNEY, B.1; COLLAVERI, J.2; HERNANDEZ, C.3; PACIENZA-LIMA, W.4; GONDIM, F.4; ROSSI-BERGMANN, B.4 1.ACCESS TO MEDICINES, SANOFI, PARIS, FRANCE; 2.INDUSTRIAL DEVELOPMENT & INNOVATION, SANOFI, PARIS, FRANCE; 3.TECNOLOGIA DE PRODUTO, SANOFI, SUZANO, SP, BRAZIL; 4.INSTITUTO DE BIOFÍSICA CARLOS CHAGAS FILHO, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:meglumine antimoniate; topical formulation; in vivo
Abstract: Background: Meglumine antimoniate (MA) (Glucantime®), is a pentavalent antimonial, which remains one of the recommended 1st line treatments for cutaneous Leishmaniasis1. Pentavalent antimonials are only available as parenteral formulations, most frequently administered intramuscularly , or intralesionally, to the discomfort of the patients, many of whom are children. The availability of a topical formulation of MA could obviate the need for parenteral treatment in many instances, and thus improve the comfort and acceptability of treatment for patients. In 2011, the Access to Medicines department of Sanofi initiated a topical MA development programme with formulation and pre-clinical development being carried out in Latin America, in collaboration with Sanofi R&D in France. Methods: Studies were performed on BALB/C mice, using the infected ear model, with L amazonensis (strain WHOM/BR/75/JOSEFA). 4 types of ointments were tested (simple gel, gel in liposomes, oil in water, and water in oil) and these were compared to intralesional (IL) injection of MA, and a MA lanette cream formulation. 7 days post infection, mice were treated once daily with 20mg of topical ointment, or twice weekly by IL injection of 200 micrograms, for 30 days. Ear thickness was measured every 3-4 days, and 9 days post end of treatment, animals were sacrificed, and parasite load estimated by limiting dilution assay. Results: Only the simple gel, and the IL injection reduced lesion growth, as measured by difference in thickness of the infected versus the uninfected ear. The gel with liposomes produced skin irritancy, and increased ear thickness, while with the water in oil cream, there was initial control of lesion growth, but unexpected growth in the days of treatment and post treatment. Only treatment with the simple gel, and the IL injection, resulted in a significant decrease in parasite loads compared to the untreated group, with the IL injection producing a greater reduction. Conclusion: In this BALBc infected ear model study, a simple MA containing gel, applied once daily for 30 days, significantly reduced L amazonensis parasite load and lesion growth as compared to non-treatment, and was well tolerated. Surprisingly, a liposomal formulation of MA proved to be non-efficient and excessively irritable. Refinement of the MA gel is ongoing, prior to further pre-clinical and toxicity testing. Reference: 1. WHO - Technical report 949. Control of the leishmaniases ; 2010.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1206] P 601 - EVALUATION OF THREE DIFFERENT SEROLOGICAL METHODS AMONG LEISHMANIA (L.) INFANTUM NATURALLY INFECTED DOGS IN A CANINE VISCERAL LEISHMANIASIS ENDEMIC AREA IN NORTHWESTERN REGION OF SÃO PAULO STATE, BRAZIL. ARAÚJO, M.F.L.1; CUTOLO, A.A.2; PEREIRA-CHIOCCOLA, V.L.1; MEIRA, C.S.1; TANIGUCHI, H.H.1; HIRAMOTO, R.M.1; GAVA, R.1; OLIVEIRA, E.B.1; CASTELÃO, K.G.1; TOLEZANO, J.E.1; MENZ, I.3 1.INSTITUTO ADOLFO LUTZ, NÚCLEO DE PARASITOSES SISTÊMICAS, CENTRO DE PARASITOLOGIA E MICOLOGIA, SÃO PAULO, SP, BRAZIL; 2.DEPTO DE VIGILÂNCIA À SAÚDE, SMS, PREFEITURA MUNICIPAL DE MONTE MOR, MONTE MOR, SP, BRAZIL; 3.DEPTO DE BIOLOGIA ANIMAL, INSTITUTO DE BIOLOGIA - UNICAMP, CAMPINAS, SP, BRAZIL.
Keyword:canine visceral leishmaniasis; serodiagnostics; qpcr
Abstract: In Brazil, in spite of a continuous effort towards Visceral Leishmaniasis (VL) control, the disease is spreading to new areas. The official control strategy includes serodiagnostics of dogs in endemic areas and culling of positive cases. Therefore the use of serological methods that decrease positive or negative false results is highly recommended. In a longitudinal study 110 healthy dogs, originally brought from a non VL transmission area, were evaluated after 6 months exposure period to a natural L. infantum challenge in an endemic northwestern region in S達o Paulo state. Blood and bone marrow were collected from dogs before and after 6 months transference to VL transmission area. Serology was performed by the following methods: ELISA with crude parasite L. major soluble antigen and two different immunochromatographic tests (Dual Path Platform DPP速 - for detection of K28 antibodies and Speed Duo Leish/Ehrli速 L. infantum and Ehrlichia canis antigens - SD). Parasitological diagnosis was performed on bone marrow smears, and DNA detection by real time PCR (qPCR) for L. infantum. Comparisons were made between serological and parasitological techniques. Leishmania infection was demonstrated in 26.4% (29/110) dogs, where 79.3% (23/29) were qPCR positive, some of them were positive for amastigote forms in smear samples; 20.7% (6/29) were only amastigote smear positive. From the qPCR positive dogs 30.4% (7/23) were positive in all serological methods; 21.7% (5/23) were positive in ELISA and DPP; 17.4% (4/23) were positive only in DPP and SD; others 17.4% (4/23) were positive only at DPP and 13.0% (3/23) dogs were positive only for ELISA. From the non-infected dogs 49.4% (40/81) were negative in all serological techniques; 14,8% (12/81) were positive for both DPP and SD; 13.6% (11/81) were positive for the three serological tests; 9.9% (8/81) were positive only in DPP; 4.9% (4/81) were positive only for ELISA; 3.7% (3/81) animals were positive only for SD; 2,5% (2/81) were positive for DPP and ELISA and 1,2% (1/81) positive for ELISA and SD. Currently, in Brazil, the official surveillance and control of VL program indicates the use of DPP as screening test and ELISA as confirmatory one. In conclusion: 1.The study demonstrates that using three different serological techniques is more efficient that only one or two to assure positive dogs, although DPP plus ELISA could be a good option in the field; 2.The great force of infection in the study region was confirmed. - CELLULAR AND MOLECULAR BIOLOGY [1222] P 602 - BIOCHEMICAL CHARACTERIZATION OF LEISHMANIA CRN12 SAHASRABUDDHE, A.A.1; SRIVASTAVA, R.1; KAJILURU, L.P.1; PATHAK, N.2; GUPTA, C.M.1 1.CENTRAL DRUG RESEARCH INSTITUTE, LUCKNOW, INDIA; 2.INTEGRAL UNIVERSITY, LUCKNOW, INDIA.
Keyword:coronin; actin-binding protein; higher order assembly
Abstract: Coronins (CRNs) constitute a family of highly conserved actin-binding proteins that play role in several actin-based cellular functions in eukaryotes. Leishmania parasites contains CRN12, the phylogenetically oldest clade of coronin proteins, which plays critical role in cell division process, especially during daughter cell separation, and is essential for the survival of these parasites in culture. However, very little, if any, information is available on the biochemical nature of CRN12. We have purified full length Leishmania CRN12 in a homologous expression system of L. tarentolae and characterized its biochemical properties in vitro. We found that in contrast to other CRNs reported so far, Leishmania CRN12 forms higher order assembly, possibly by virtue of its C-terminal coiled-coil region, which has 2 prominent leucine zipper motifs. The quarternary structural attributes were experimentalized through size-exclusion chromatography and glutaraldehyde cross-linking methods. Whereas the former showed one major peak position corresponding to a molecular size of ~398 kDa suggesting it to be a possible hexamer, the later revealed a band at ~280 kDa on SDS-PAGE suggesting it to be a
possible pentamer. Furthermore, sedimentation assay of CRN12 with Leishmania actin (LdACT) showed its binding with polymeric actin thereby confirmed functional integrity of the purified protein. Together, these results show that Leishmania CRN12 exists both in monomeric and as well as pentameric forms and binds with the filamentous form of actin to regulate actinnetwork based cellular functions in Leishmania parasites. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1233] P 603 - CELLULAR LOCALIZATION AND TRANSIENT INTERACTION BETWEEN LDISCS AND LDISCU PROTEINS FOR ASSEMBLY OF FE-S CLUSTERS IN LEISHMANIA DONOVANI SINGH, K.P.; ZAIDI, A.; EQUBAL, A.; DAS, P.; ALI, V. RAJENDRA MEMORIAL RESEARCH INSTITUTE OF MEDICAL SCIENCES, PATNA, INDIA.
Keyword:leishmania donovani; iron-sulfur cluster assembly pathway; iscs, iscu
Abstract: Leishmania donovani cause Visceral Leishmaniasis, one of the neglected tropical diseases listed by WHO. Rapidly developing drug resistance to first and second line drugs further adds to its menace. Iron-Sulfur Cluster (ISC) assembly is a housekeeping pathway essentially required for all aerobic or facultative anaerobic living organisms. Leishmania has a complete ISC system but it lacks other systems for Fe-S cluster biogenesis. The catalytic component (IscS) requires PLP as cofactor for the cysteine desulfurase activity (EC 2.8.1.7) and the scaffold component (IscU) serves as transient cluster platform for biogenesis of Fe-S cluster in ISC system. We have expressed and purified recombinant fusion proteins L. donovani IscS (LdIscS) and L. donovani IscU (LdIscU) with GST- and His-tag, respectively. Polyclonal antibodies were raised in rabbit against these purified proteins. In vitro experiments using E. coli lysate expressing these two proteins with different tags showed that these proteins interact with each others. However, experiments using recombinant protein bound to affinity matrix and L. donovani lysate did not pulled down the partner protein. To further confirm this result, we also performed immunoprecipitation experiments using polyclonal antibodies either anti-LdIscS or anti-LdIscU and each of them were unable to pull down their putative partner protein from L. donovani cell lysate. Hence, it is suggested that LdIscS and LdIscU interact only transiently for Fe-S cluster assembly in this organism. In yeast and human, interaction between these two proteins is very stable. Thus it appears that ISC assembly machinery of L. donovani is different from other organisms. Subcellular localization studies using differential digitonin fractionation and indirect immunofluorescence analyses showed that both of these proteins are co-localized in the single reticulated mitochondria of L. donovani promastigotes.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1237] P 604 - NESTED-PCR CONJUNCTIVAL SWAB FOR THE DIAGNOSIS OF LEISHMANIA INFANTUM INFECTION IN CATS VERONESI, F.1; FIORENTINO, E.2; DI MUCCIO, T.2; SCALONE, A.2; GRAMICCIA, M.2 1.DEPARTMENT OF BIO-PATHOLOGICAL SCIENCES, UNIVERSITY OF PERUGIA, PERUGIA, ITALY; 2.UNIT OF VECTORBORNE DISEASES AND INTERNATIONAL HEALTH, ISTITUTO SUPERIORE DI SANITÀ, ROME, ITALY.
Keyword:conjunctival swab n-pcr ; feline leishmaniasis; leishmania infantum
Abstract: Dogs are the domestic reservoir of zoonotic visceral leishmaniasis (ZVL) caused by Leishmania infantum, however the role of other hosts in the parasite life cycle cannot be ruled out. Cats may suffer from feline leishmaniasis (FL), a syndrome less frequent and severe than canine leishmaniasis (CanL). Several FL serosurveys performed in Mediterranean countries and the recent evidence of transmissibility of feline parasite to the phlebotomine vector have led to propose cats as secondary hosts for ZVL. In general, cats exhibit faint antibody responses which does not help to elucidate the complexity of the disease. Therefore more accurate and reliable approaches are necessary to estimate FL infection prevalences. The present work aimed to investigate on the spreading of L. infantum infection in a feline population resident in a CanL endemic focus of Central Italy, by using a n-PCR assay on conjunctival swabs (CS), a lowinvasive sample successfully proposed for CanL diagnosis (Di Muccio T et al, 2012, J Clin Microbiol, 50:2651). From April through August 2010, 98 stray cats of both genders, were randomly selected. Cats were examined for signs suggestive for FL, and peripheral blood and CS samples were obtained during the sterilization/castration sessions of the regional straying’s program. Sera were tested for anti-Leishmania IgG by IFAT. Total genomic DNA extracted from buffy coat (BC) and CS eluted material was submitted to the molecular assay. Crosssectional prevalence of Leishmania infections was calculated as the rate of animals positive to each test. No cats showed clinical signs of FL. IFAT revealed low-titre anti-Leishmania antibodies in 48 animals (49.0%). Thirty-eight cats were found positive to CS n-PCR (38.8%), whereas none of the animals was positive by BC n-PCR. CS n-PCR positives only were 38.0%, and IFAT-positive only 29.6%, with fair agreement between the 2 tests (K=0.4). The rate of Leishmania positives to at least one test was 68.4%, and that of positives by both tests 39.6%. Results pointed out the limited diagnostic value of BC n-PCR in cats. The absence of suggestive FL clinical signs confirms the low clinical susceptibility of cats to L. infantum infections. This study was carried out in the frame of EDENext, a collaborative project of the 7th FP (20112014) funded by the European Commission under the DG Health; Contract Number 261504. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1238] P 605 - RANDOMIZED CLINICAL TRIAL COMPARING ITRACONAZOLE, KETOCONAZOLE AND MEGLUMINE ANTIMONATE IN THE TREATMENT OF HUMAN CUTANEOUS LEISHMANIASIS IN GUATEMALA RIZZO, N.R.; ARANA, F.E.; ARANA, B.A. CENTRO DE ESTUDIOS EN SALUD, UNIVERSIDAD DEL VALLE DE GUATEMALA, GUATEMALA, GUATEMALA.
Keyword:cutaneous, leishamaniasis; treatment; itraconazole
Abstract: Objective: To compare the efficacy of itraconazole with ketoconazole and meglumine antimonate for the treatment of Cutaneous Leishmaniasis (CL) in Guatemala Methods: A total of 165 Guatemalans with parasitological proven CL were randomly assigned to one of three treatments groups: itraconazole (200 mg po twice a day/28 days); ketoconazole (600 mg po once a day/28 days) and meglumine antimonate (20 mg/kg body weight iv once a day/20 days). All patients were followed-up for 12 months. Diagnosis of CL was made by thin smears of lesion scrapings or culture of lesion aspirates. Leishmania isolates were characterized by isoenzyme electrophoresis. Final cure was defined as a patient whose lesions had completely reepithelialized and had no evidence of inflammation or indurations by the 13-week follow-up examination and had no reactivation during the 52-week follow-up period. Results: Of the 165 enrolled subjects, 118 (71.5%) had their diagnoses confirmed by both culture and smear; 19 (11.6%) only by culture, and 28 (16.9%) by thin smear of lesions alone.
Of the 137 subjects with positive cultures, we were able to speciate the infecting parasite for 113 (82.4%); where 86 (76.1%) were L. braziliensis, and 27 (23.9%) were L. mexicana. Only five patients (3%) were lost to follow-up over 52 weeks. No serious adverse effects were encountered in any of the subjects. The cure rate at 52-week examination were the following: meglumine 84.9%, ketoconazole 75.9% and itraconazole 52.8%. Since there is evidence that treatment outcome depended on the infecting species, results also presented by Leishmania infecting specie. For infections caused by L. braziliensis, meglumine proved to be significantly more effective than either ketoconazole or itraconazole. At the 52-week examination the clinical cure rate in the three treatment groups were: meglumine, 26 (86.6%) of 30; ketoconazole, 17 (65.3%) of 26; and itraconazole, 8 (29.6%) of 27. For infections caused by L. mexicana, meglumine was inferior to both itraconazole and ketoconazole. At the 52-week examination the clinical cure rates were: meglumine, four (57.1%) of seven; ketoconazole, nine (81.8%) of 11; and itraconazole, seven (77.7%) of nine. Conclusion: These data indicate that ketoconazole is better than itraconazole in the treatment of CL in Guatemala, and confirm previous reports from Guatemala that antimonials are effective against infections caused by L. braziliensis.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1239] P 606 - ASSOCIATION OF LIPOSOME-ENCAPSULATED MEGLUMINEANTIMONIATE AND ALLOPURINOL IN TREATMENT OF CANINE VISCERAL LEISHMANIASIS: TOXICITY AND CLINICOPATHOLOGICAL FINDINGS DA SILVA, S.M.1; AMORIM, I.F.2; RIBEIRO, R.R.3; AZEVEDO, E.G.2; DE PAULA, R.C.4; DEMICHELI, C.5; MELO, M.N.2; TAFURI, W.L.2; GONTIJO, N.F.2; MICHALICK, M.S.2; FREZARD, F.2 1.INSTITUTO DE CIÊNCIAS BIOMÉDICAS, FEDERAL UNIVERSITY OF UBERLÂNDIA, UBERLÂNDIA, MG, BRAZIL; 2.INSTITUTO DE CIÊNCIAS BIOLÓGICAS, FEDERAL UNIVERSITY OF MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL; 3.CENTRO DE CIÊNCIAS AGRÁRIAS, AMBIENTAIS E BIOLÓGICAS, FEDERAL UNIVERSITY OF RECONCAVO DA BAHIA, CRUZ DAS ALMAS, BA, BRAZIL; 4.FACULDADE DE FARMACIA, FEDERAL UNIVERSITY OF MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL; 5.INSTITUTO DE CIÊNCIAS EXATAS, FEDERAL UNIVERSITY OF MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL.
Keyword:liposome-encapsulated meglumine antimoniate ; allopurinol; canine visceral treatment
Abstract: Recently, we reported a new treatment protocol which promoted for the first time parasitological cure in dogs with CVL. This protocol was based on the association of six doses of liposomeencapsulated MA (6.5mg Sb/kg/96h/i.v.) with allopurinol (20mg/kg/24h/per os/140 days) (LMA+Allop). Here, we demonstrate benefits of this association to improve hematological and serum biochemistry parameters and its absence of toxicity. Hemogram, proteinogram, hepatic and renal functions were evaluated in treated dogs (n=6) prior to (T0), at day 140 (T140) and 60 days after treatment (T200). At T0 the red blood count (RBC) was bellow to the reference values (5,295,000.0±867,565.6/dL). After treatment starts the RBC reached the normal values at T140 (6,591,667.0±604,761.7/dL) (p< 0.05). This tendency was maintained at (T200) (6,848,334.0±347,529.4/dL) (p<0.05). As expected, concentration of hemoglobin and hematocrit presented similar results. At T0 these values were bellow the reference (hemoglobin:11.9±2.2g/dL; hematocrit: 36.8±6.2%) and treatment leaded to an increasing (p < 0.05) in both values at T140 (14.7±1.2g/L; 46.5±3.4%) and T200 (15.6±0.9g/L; 49.0±3.3), reaching the normal values. Also, the number of platelets increased at T140 (270,833.3±113,740/dL) and T200 (269,166.0±117,302/dL) in comparison to T0 (204,666.70±53,787.2) (p<0.05). Total serum proteins (8.7±1.5g/dL) and globulins
(6.3±1.3g/dL) were above the reference values at T0 and returned to normal at T140 (7.2±1.1g/dL; 4.2±1.2d/dL; respectively) and T200 (6.1±1.4g/dL; 3.3±0.9g/dL) (p<0.05). In consequence, A/G ratio also increased (T0:0.45±0.2; T140: 0.8±0.2; T200: 0.9±0.2) (p<0.05). Serum urea was slightly above the normal parameters (T0:34.7±13.4mg/dL; T140: 43±14mg/dL; T200: 35±55mg/dL) and creatinin did not suffer significant variations (T0: 0.60±0.2mg/dL; T140: 0.7±0.2mg/dL; T200: 0.68±0.2mg/dL). It was also observed to alanine aminotransferase (T0:17.7±1IU/L; T140: 25.3±18.8IU/L; T200: 22.5±16IU/L), aspartate aminotransferase (T0:27.8±13IU/L; T140: 44.7±30IU/L; T200: 27.7±13IU/L) and alkaline phosphatase (T0:109.7±72IU/L; T140: 81±48.2IU/L; T200: 53.8±34 IU/L). Evaluations by ultrasound (US) of liver, spleen and kidneys during the experiment period did not showed significant variations in size, shape or signs of disorders, such as urolithiasis by prolonged use of allopurinol. Our results demonstrated that the LMA+allop association leaded to an improvement of clinicopathological parameters and showed absence of hepatic and renal toxicity. As a major advance, the present study displays for the first time a new, safe and highly effective therapeutic alternative for CVL, based on the combination of nanotechnology and conventional therapy, with the prospect of achieving parasitological cure. Financial support: CNPq, FAPEMIG and CAPES. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1246] P 607 - NINE YEARS OF DATA COLLECTION AND ANALYSIS FOR THE LEISHMANIA DIAGNOSTIC LABORATORY AT WALTER REED ARMY INSTITUTE OF RESEARCH MENDEZ, J.; BRASOV, I.E.; DIAO, L.Y.; TALLY, J.D.; GROGL, M.; WEINA, P.J. WRAIR, SILVER SPRING, UNITED STATES.
Keyword:leishmaniasis; clinical; diagnosis
Abstract: Abstract Leishmaniasis is a disease complex caused by 42+ species of protozoan parasites belonging to the genus Leishmania. Successful culturing of Leishmania parasites requires several techniques to insure proper identification of the species involved in the disease manifestation. An accurate and verifiable final diagnosis is the most important tool in finding the correct treatment for the affected individuals. Finding the proper diagnosis requires a series of laboratory techniques such as culturing and expanding the parasites and later identifying the species of Leishmania involved via molecular techniques. Our diagnostic laboratory is the only known certified laboratory by the College of American Pathologist Laboratory, approved by the Clinical Laboratory Improvement Program for high complexity testing of Leishmania disease, under the Department of Defense (DoD). This laboratory has seen a significant number of cases over the last 9 years, with our involvement in the Middle East and elsewhere that Leishmania is endemic. We have reviewed nine years of data collected from submissions to this laboratory not only from DoD but from civilian sources as well. This data tabulation and interpretation provides to any interested party and most importantly to laboratory personnel who may encounter the need to assist in the diagnosis of the disease, a clear view of the past and present problems associated with diagnosing the disease, monitoring the incidence of exposure and infectivity in mapping different population groups, and discovering patterns of sample submission. This valuable information will hopefully help us to predict future problems and help us learn how to avoid them. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1252]
P 608 - EVALUATION OF USEFULNESS OF SWAB AS AN AUXILIARY METHOD OF COLLECTION TO AMERICAN CUTANEOUS LEISHMANIASIS DIAGNOSIS IN PERNAMBUCO STATE, BRAZIL ARAUJO, A.I.F.1; VIANA, J.B.M.1; BRITO, M.E.F.1; ALEXANDRE, J.L.A.1; ALMEIDA, E.L.1; WERKHAUSER, R.P.1; MEDEIROS, A.C.R.2; ALMEIDA, A.P.1; BRANDÃO-FILHO, S.P.1; RODRIGUES, E.H.G.1
1.CENTRO DE PESQUISAS AGGEU MAGALHÃES, RECIFE, PE, BRAZIL; 2.HOSPITAL UNIVERSITÁRIO OSWALDO CRUZ, RECIFE, PE, BRAZIL.
Keyword:american cutaneous leishmaniasis; swab; pernambuco
Abstract: Among the many problems associated with american cutaneous leishmaniasis (ACL), the difficulty in obtaining the diagnosis has highlighted due to the various methods associated to its conclusion. The collection of biological material is fundamental to this process, in view of the severity of injuries coupled with common secondary infections observed, especially the bacterial ones. The collection method employed routinely in the health service is based on obtaining a biopsy of the injured site, being used for this purpose the surgical punch. This procedure is characterized by offer risks to patients such as increased exposure of injury to new infection or reactivation of the lesion. This work aims to standardize and evaluate the effectiveness of the method for biological sample collection based on the use of swab associated with the molecular diagnosis of ACL. Between the years 2011 and 2012 were selected 88 patients with the disease, from which were collected samples of biopsy and skin exudates through swab. The biopsy samples and exudates were subjected to molecular diagnostics, initiated by the technique of DNA extraction using a commercial kit and then the polymerase chain reaction (PCR) targeting to the kinetoplast minicircle DNA (kDNA) of Leishmania. The epidemiological profile of the patients revealed the predominance of the disease in males (57.8 %) and the age groups affected were predominantly young and adult, being 40.36 years the average age. Most patients were from the municipality of Moreno, metropolitan area of Recife (70/ 79.54 %). Were observed mostly ulcerated lesions with exudate (90.9 %) and these were concentrated in the lower limbs (71.6 %). By comparing the molecular diagnostic result among the samples was obtained the convergence of 86.36 % and this value was statistically significant (p-value = < 0.001). The results suggest that the method of collection of biological sample through bacteriological swab for molecular diagnosis of ACL shows excellent efficacy when compared with the biopsy method and can be deployed as an assist in the health service due to its simplicity and ease of execution, offering yet no secondary risks to the patient. Financial support: CNPq, FACEPE, CAPES. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1254] P 609 - CLINICAL AND EPIDEMIOLOGICAL EVALUATION OF AMERICAN CUTANEOUS LEISHMANIASIS PATIENTS AT A HOSPITAL OF RECIFE, PERNAMBUCO, BRAZIL BRITO, M.E.F.1; ALVES, N.M.E.2; DA CONCEIÇÃO, W.M.2; FREITAS, A.I.1; SILVA, C.J.3; VIANA, J.B.M.1; ALEXANDRE, J.L.A.1; ALMEIDA, E.L.1; WERKHAUSER, R.P.1; RODRIGUES, E.H.G.1; BRANDÃO-FILHO, S.P.1; MEDEIROS, A.C.R.2 1.CENTRO DE PESQUISAS AGGEU MAGALHÃES, RECIFE, PE, BRAZIL; 2.HOSPITAL UNIVERSITÁRIO OSWALDO CRUZ, RECIFE, PE, BRAZIL; 3.NÚCLEO DE VIGILÂNCIA EM SAÚDE E AMBIENTE, MORENO, PE, BRAZIL.
Keyword:american cutaneous leishmaniasis; epidemiology; treatment
Abstract: American cutaneous leishmaniasis (ACL) is an infectious and parasitic disease records with an average of 35,000 cases per year in Brazil, occurring in almost States, and, in Pernambuco, it predominates at Rain Forest area. The aim of the present study is to evaluate the clinical and
epidemiological profile of patients with ACL treated in outpatient dermatology of Hospital Universitårio Oswaldo Cruz of the Universidade de Pernambuco (HUOC/ UPE) for 10 months. This is an observational and prospective study carried in outpatient dermatology clinical, through of assessment clinic, epidemiological and laboratory in patients with suspected ACL. Of the 48 patients involved, six were excluded of the study by present other dermatological infectious. The majority was male and farmers coming from endemic areas. The age range between zero and 18 years old, with 33.33% (14/42) and the group between 19 and 55 years old predominated with 59.52 % (25/42) of the total of patients. Ten patients were younger than 12 years old, and there is evidence that they were infected in domiciliary area. The predominant clinical presentation was unique ulcers in the lower limbs. A six-year-old showed mucosal form after two years of treatment of cutaneous lesions with N-methylglucamine. Laboratory tests were performed for diagnosis in 33 patients, and the direct examination was positive in 60.6 % (20/33). The polymerase chain reaction specific to Leishmania (Viannia) spp. was positive in 65.52 % (19/29) and Leishmania spp. was isolated from 36 samples. Nine patients received the N-methyl glucamine at the recommended dose. Five patients developed spontaneous healing of the lesion, 1 did not return to the hospital, and 1 chosen intralesional treatment with N-methylglucamine due to contraindications to conventional treatment. Financial support: CNPq, FACEPE, FIOCRUZ. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1259] P 610 - EVALUATION OF NEXT GENERATION SEROLOGICAL TESTS FOR AFRICAN VL GHALIB, H.1; BHATIA, A.2; GUDERIAN, J.2; HOWARD, R.F.2; IRETON, G.2; ABDOUN, A.O.3; MUKHTAR, M.3; BEZUNEH, A.4; MEKURIA, A.H.4; REED, S.G.2 1.UNIVERSITE DE LAUSANNE, EPILANGES, SWITZERLAND; 2.INFECTIOUS DISEASE RESEARCH INSTITUTE, SEATTLE, UNITED STATES; 3.INSTITUTE OF ENDEMIC DISEASES, UNIVERSITY OF KHARTOUM, KHARTOUM, SUDAN; 4.ADDIS ABABA UNIVERSITY, ADDIS ABABA, ETHIOPIA.
Keyword:rapid diagnostic test; rk28; antibody detection
Abstract: The development of the rK39 Rapid Diagnostic Test (RDT) has addressed a critical need for the prompt confirmation of a clinical diagnosis of visceral leishmaniasis (VL; also known as kalaazar) and has been a vast improvement over invasive tissue biopsies and labor-intensive microscopy. These immunochromatography-based tests have been widely employed in South America and the Indian subcontinent, but have shown lower sensitivity in Africa. The rK28 polyprotein, composed of the individual Leishmania infantum proteins rK39, rK26, and rK9, together with new RDT platforms have increased the sensitivity and specificity of point-of-care confirmation of VL in Africa (Pattabhi et al., 2010). After further refinement of several different rK28 platforms, we report here an evaluation of four different rK28-based RDTs with panels of banked African sera. Using 100 Ethiopian sera, sensitivities of 99% (Chembio), 98% (Ease MedTrend), 98% (CTK), and 96% (InBios) were observed using the rK28 recombinant antigen. Using 100 Sudanese sera, sensitivities of 95%, 97%, 94%, and 96%, respectively, were observed on rK28-containing RDTs. DAT had a sensitivity of 95% on these same Sudanese sera. All of these values using the RDTs were clearly superior to the previously observed values that ranged as low as 70% for rK39 RDTs. The following specificities were observed for these Ethiopian sera (97.6%, 97.6%, 100%, and 97.6%, respectively) and for the Sudanese sera (96%, 91.3%, 99.3%, and 92.7%, respectively). Fresh whole blood and patientmatched Sudanese sera were compared on the same version of these RDTs. Identical sensitivities were observed for detecting antibodies in these two fluids: 95% (Chembio), 94% (CTK), 97% (Ease MedTrend), and 96% (InBios). DAT had a sensitivity of 95% on these serum samples. Any of these RDTs appear suitable for confirmation of VL diagnosis in Africa. Taking advantage of the current transmission season, a large blinded evaluation trial has been initiated
in Sudan on one of these rK28 RDTs to compare readings using blood and serum from the same patient (and in relationship to DAT) as well as consistency of results by multiple human readers. These studies were supported by the Bill and Melinda Gates Foundation (grant #49932). - CLINICAL LEISHMANIASIS [1269] P 611 - THE FIRST CASE WITH VISCERAL LEISHMANIASIS RESISTANT TO MEGLUMINE ANTIMONIATE IN ALBANIA SHKURTI, K.1; KRAJA, D.1; BINO, S.1; KOKICI, M.2; PIPERO, P.1 1.DEPT OF INFECTIOUS DISEASES, TIRANA, ALBANIA; 2.DEPT OF LABORATORY, TIRANA, ALBANIA.
Keyword:leishmaniasis; ambisome; glucantime
Abstract: Visceral leishmaniasis is known to occur in Albania since 1938, typically as a childhood disease. The highly effective drug for the treatment in 99% of cases is meglumine antimoniate. The authors aim is to present the first case, resistant to antimony compound. During the period 2006-2011 we identified four other cases in adults that were also treated with amBisome. A 38 years male from a rural zone in the south of Albania, was hospitalized in Department of Infectious Disease in Tirana with Visceral leishmaniasis. He had high fever, chronic diarrhea, weight loss, and asthenia for about six weeks. The objective examination showed pallor face, large spleen and liver. Blood microscopic examination showed: lympho-monocytosis. hypocromia, anizocytosis, normocytosis, microcytosis and poikilocytosis, RBC 2230000, Hb 7.4g%, WBC 3600, PLT 127000. Biochemical exams were: AST 117U/l, ALT 68U/l, bilirubine 1 mg%, total protein 5.6 g%, nitrogen urea 48 mg%, creatinine 0.8 mg% Electrophoresis of proteins: albumine 51.8%, alfa 1 globuline 3.4%, alfa 2 globuline 6.6%, beta globuline 8.4%, gama globuline 29.8%. Na 113 mmol/l, K 3.2 mmol/l, Chl 105 mmol/l, Ca 1.1mmol/l. The bone-marrow aspirate resulted positive for Leishmania amastigotes in the microscopic examination. The serology for leishmania was positive with ELISA. All tests for Brucellosis, Typhoid fever, HIV and malaria were negative. Cardiac sonography examiation was normal. Treatment was started with antimony (glucantime) for two weeks, but the clinical situation was the same with high fever. The blood tests after fourteen days of treatment were: RBC 1680000, Hb 5.2 g%, WBC 2300, PLT 88000. AST 92 U/l, ALT 92 U/l, bilirubine 1.1 mg%. Then stopping of antimony was decided, replaced with amphotericyne B lyposomale (AmBisome) 3 mg/kg for five days and supportive treatment with blood transfusion and vitamin therapy. After a week the clinical condition of the patient was improvement, he had no more fever and after ten days the blood examination showed only mild anemia. The bone marrow aspirate one month after treatment didn’t find more leishmania amastigote. Conclusion: Amphotericyne B lyposomale (AmBisome) it’s an alternative drug in the cases resistant to antimony, but very expensive in developing countries.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1270] P 612 - EVALUATION OF ACTIVITY OF THE ESSENTIAL OIL OF CYMBOPOGON CITRATUS (DC.) STAPF AND CITRAL AGAINST VISCERAL LEISHMANIASIS. BRITO, A.M.G.1; RODRIGUES, S.A.1; PINTO, É.G.2; TEMPONE, A.G.2; FILHO, L.X.3 1.UNIVERSIDADE TIRADENTES, ARACAJU, SE, BRAZIL; 2.INSTITUTO ADOLFO LUTZ, SÃO PAULO, SP, BRAZIL; 3.RENORBIO, FORTALEZA, CE, BRAZIL.
Keyword:natural+products; cymbopogon+citratus; visceral+leishmaniasis
Abstract: The need for new drugs more effective, safe and accessible for the treatment of leishmaniasis, visceral in particular, later on, affects liver, spleen, reticuloendothelial system, bone marrow and lymph nodes becomes relevant. Furthermore, in Brazil leishmaniasis reach 19 states, and more than 90% of human cases of the disease are concentrated in Northeast region, there is still important regions foci in the Midwest, North and Southeast regions. Studies point for the occurrence of about 20.000 new cases annually of the disease. According to the World Health Organization, the plant species are the best and major source of drugs for humanity and Brazil has 60.7% of its territory of natural and planted forests, representing the second largest forest area in the world, only behind from Russia. Thus, this study aimed investigated the activities of the essential oil of Cymbopogon citratus and Citral against Leishmania (L.) infantum. In order for this to happen, oil from fresh leaves were harvested on the Mother Earth farm, located in Santana do São Francisco/SE. The Citral was acquired from Sigma-Aldrich (Darmstadt, Germany). Gaseous Chromatography and Detections by Flame Ionization were carried to identify its chemical constituents, just like an evaluation of inhibitory concentrations 50% in promastigotes and amastigotes in Leishmania mentioned beforehand, cytotoxicity assays, nitric oxide production and fluorescence to detect possible increase of membrane permeability in the presence of Citral were developed. Pharmacological activities were found in promastigotes (CI50/48 hours 25µg/mL for oil and 30 µg/mL for Citral), however, activities were not found in amastigotes. The cytotoxicity (CI50 was 26.25 µg/mL for oil and 33.96 µg/mL for Citral). There was no increase in nitric oxide production. However, pore formation was observed in cell membranes of promastigotes in the presence of Citral. Therefore, the essential oil of Cymbopogon citratus and Citral may come to be used as preventive measure against visceral leishmaniasis. Since the promastigotes are the ways that infect vertebrates, including man. - CLINICAL LEISHMANIASIS [1272] P 613 - EVALUATION OF THE INCIDENCE OF HIV/LEISHMANIA INFANTUM COINFECTION IN TERESINA, BRAZIL, AND THE PERFORMANCE OF DIRECT PARASITOLOGICAL AND MOLECULAR DIAGNOSIS SILVA, M.R.B.1; BRANDÃO, N.A.A.2; DORTA, M.C.L.2; COSTA, D.L.3; COSTA, C.H.N.3; OLIVEIRA, M.A.P.4 1.INSTITUTO DE DOENÇAS TROPICAIS NATAN PORTELA/ IDTNP, TERESINA, PI, BRAZIL; 2.INSTITUTO DE PATOLOGIA TROPICAL E SAÚDE PÚBLICA/UFG., GOIANIA, GO, BRAZIL; 3.UNIVERSIDADE FEDERAL DO PIAUÍ/ UFPI, TERESINA, PI, BRAZIL; 4.INSTITUTO DE PATOLOGIA TROPICAL E SAÚDE PÚBLICA/UFG, GOIANIA, GO, BRAZIL.
Keyword:hiv; leishmania infantum ; co-infection
Abstract: Background: Co-infection HIV-Leishmania is a growing problem worldwide, due to the high proportion of asymptomatic infections in endemic areas. Recent observations suggest that most adults with kala-azar from the city of Teresina, Brazil, may be are infected with HIV. We decided to
evaluate the prevalence of HIV infection among hospitalized patients with kala-azar in the city’s reference hospital. Methods: We studied 93 consecutive patients older than 10 years old with typical clinical presentation of kala-azar, plus a reactive serology or the detection of parasites in direct examination or culture of bone marrow. Results: We identified 93 patients fulfilling criteria. Thirty-two patients (34.4%) had a reactive HIV1/HIV-2 test. Among the 32 HIV+, 29 had slides of bone marrow aspirate and 13 had a positive bone marrow direct examination (44,8,5%), while this proportion was 22/49 (44,9%) among those HIV negative, where in this group 49 had slides of bone marrow aspirate. A PCR-RFLP was positive in 30/32 (62,5%) among the HIV positive, while 30/59 (50,8%) were PCR-RFLP positive among the HIV negative. Conclusion: The results indicate a high prevalence of HIV infection among patients diagnosed with kalaazar. There was a very low sensitivity of bone marrow direct examination, as well as a disappointing performance of PCR-RFLP.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1283] P 614 - DIFFICULTY TO DIAGNOSE SKIN LEISHMANIASIS BY OCCUPATIONAL ACCIDENT: CASE REPORT BRITO, M.E.F.1; RODRIGUES, E.H.G.1; ARAUJO, A.I.F.1; WERKHAUSER, R.P.1; ALMEIDA, E.L.1; VIANA, J.B.M.1; ALEXANDRE, J.L.A.1; BRANDÃO-FILHO, S.P.1; ANDRADE, M.S.2; ALMEIDA, A.P.1; SILVA, C.J.3; MEDEIROS, A.C.R.2 1.CENTRO DE PESQUISAS AGGEU MAGALHÃES, RECIFE, PE, BRAZIL; 2.HOSPITAL UNIVERSITÁRIO OSWALDO CRUZ, RECIFE, PE, BRAZIL; 3.NÚCLEO DE VIGILÂNCIA EM SAÚDE E AMBIENTE, MORENO, PE, BRAZIL.
Keyword:american cutaneous leishmaniasis; diagnosis; occupational accident
Abstract: American cutaneous leishmaniasis (ACL) is a chronic infection, complicated by presenting itself in several forms, with a variety of vectors and etiologic agents. It resembles with other skin diseases, what hinder the common diagnosis. Report: Female patient, 62 years old, resident in Recife, Pernambuco, health care professional. Sought medical attention complaining of appearance of erythematous papule on the palmar aspect of the distal phalanx of the second left finger, for 4 days, with a gradual increase in volume. She underwent oral treatment with cephalexin and there were no improvement. The lesion progressed with raised edges ulceration, exudate, adjacent lymphangitis and axillary node. In the culture with antibiogram were identified Enterococcus faecalis and Acinectobacter baumannii with resistance profile. Then, she underwent the treatment with Tigecycline (50mg) and there was no improvement after the 5th day. New anamnesis was conducted which demonstrated that 4 months earlier, the patient had accidentally pierced the left finger, the re-skinning a tuberculin needle, when performed puncture of injury in patient suspected of LTA. Biological material was collected in the edge of the lesion to perform polymerase chain reaction using primers B1/B2 specific for the subgenus
Viannia, and the result was consistent with ACL. The treatment with N-methyl glucamine 15mg/kg was administrated. After 15 days the healing process could be verified. Discussion: The diagnosis of ACL has been associated with clinical, epidemiological and laboratorial aspects. There is, however, difficult to relate ACL to occupational accident, sometimes for lack of epidemiological link, by similarity with other dermatological diseases and/or the presence of co-infection. An accurate diagnosis of the disease can be slowed by inadvertently confused and self-medication. Professionals and health institutions need to be alert to the possibility of the occurrence of ACL in this context, and develop strategies aimed at the prevention of risks present in the workplace. Financial support: CNPq, FACEPE, FIOCRUZ. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1287] P 615 - NEW APPROACHES AND NEW TECHNOLOGY FOR THE PRODUCTION OF DIAGNOSTIC TESTS FOR HUMAN VISCERAL LEISHMANIASIS COLLOVATI, M.1; DUTHIE, M.2; DIAS, R.1; REED, S.2 1.ORANGELIFE, RIO DE JANEIRO, RJ, BRAZIL; 2.IDRI, SEATTLE, UNITED STATES.
Keyword:rapid tests; innovation; point of care
Abstract: Although Rapid Diagnostic Tests (RDT) are widely applied for the diagnosis of both human and canine visceral leishmaniasis (VL) local manufacturing of these tests in endemic countries has lagged behind the technology. Although VL RDT have been commercialized for over a decade, the lack of local production and of mechanisms for local regulatory approval has resulted in limited access and high prices of RDT for diagnosis of VL in Brazil. This situation has finally changed for two reasons. One is the proactive efforts of ANVISA, the Brazilian regulatory body responsible for approving new health care products, which has held important international meetings in 2012 to discuss harmonization and acceleration of the RDT approval process. Secondly, local manufacturing of RDT has begun in Brazil, by two organizations. One of these is ORANGELIFE, a Brazilian company located in Rio de Janeiro, focused on Point of Care Solutions. ORANGELIFE has worked to create a new model for Brazil involving innovative point of care solutions produced in Brazil, with the intent of reducing and eradicating targeted neglected diseases. The human VL test was our first challenge because it is one of the most devastating neglected disease, with little market potential and relatively low priority by government agencies. We started our activity registering a HVL test in OEM, from a USA company. However, our commitment to making an impact on public health over profit concerns led us to develop our own products. ANVISA, rapidly becoming one of the world’s leading regulatory agencies, has recognized and supported the efficacy of this development model. Furthermore, in order to innovate the existing point of care solutions, ORANGELIFE decided to use the intelligence of a Smart-Phone and the accuracy of our rapid test, to create a new generation of diagnostic concept: “SMART-READER CONCEPT”. Results using locally produced RDT in combination with the SMART-READER will be discussed.
- OTHER SPECIAL TOPICS [1290] P 616 - COMPARATIVE ANALYSIS OF QUICK TEST METHODOLOGIES “RK39” AND INDIRECT IMMUNOFLUORESCENCE REACTION (IIFR) IN DIAGNOSTIC LABORATORY OF HUMAN VISCERAL LEISHMANIASIS IN PERNAMBUCO A STATE OF NORTHEASTERN BRAZIL PRATA FONSÊCA, A.M.D.R.B.; CAUÁS, E.M.C.; DOWSLEY FILHO, ,.M.A.S.; DE OLIVEIRA, L.B. LACEN-PE, RECIFE, PE, BRAZIL.
Keyword:introduction: the human visceral leishmaniasis (hvl) is a disease caused by protozoa of the genus leishmania and its transmission occurs via infected sandfly vectors. it is characterized by severe clinical evolution and represents compulsory disease of great importance for public health. the rk39 and indirect immunofluorescence reaction (iifr) are important methodologies used in the laboratory diagnosis of hvl, contributing to the completion of this disease. materials and methods: we collected the epidemiological information and results of rk39 and iifr of 106 serum samples from laboratório central de saúde pública “dr. milton bezerra sobral - lacen (pernambuco-brazil), between the period may 2010 to december 2012, where it was conducted a comparative study between the methodologies. results: we observed a similar behavior between both methodologies, but 5 samples (4.72%) showed divergent results: three children under two years of age coming from the municipalities of serra talhada and petrolina, an adult resident in the municipality of caruaru with low titers in iifr (1/40) and reported classical clinical (fever and splenomegaly with or without hepatomegaly) and an elderly residing in the city of recife with interrupted treatment, presenting classical clinical and reagent iifr. discussion and conclusion: there was no significant difference between the rk39 and iifr hvl of the samples analyzed. the low maturity of the immune system of patients 1 and 10 months of age may suggest a higher sensitivity of rk39 in this age group compared to iifr and the negativity of rk39 in other divergent samples are probably due to technical limitations of the methodologies used epidemiological and clinical conditions of the patients. keywords: human visceral leishmaniasis; iifr; rk39
Abstract: Introduction: The Human Visceral Leishmaniasis (HVL) is a disease caused by protozoa of the genus Leishmania and its transmission occurs via infected sandfly vectors. It is characterized by severe clinical evolution and represents compulsory disease of great importance for Public Health. The rK39 and Indirect Immunofluorescence Reaction (IIFR) are important methodologies used in the laboratory diagnosis of HVL, contributing to the completion of this disease. Materials and Methods: We collected the epidemiological information and results of rK39 and IIFR of 106 serum samples from Laboratório Central de Saúde Pública “Dr. Milton Bezerra Sobral - LACEN (Pernambuco-Brazil), between the period May 2010 to December 2012, where it was conducted a comparative study between the methodologies. Results: We observed a similar behavior between both methodologies, but 5 samples (4.72%) showed divergent results: three children under two years of age coming from the municipalities of Serra Talhada and Petrolina, an adult resident in the municipality of Caruaru with low titers in IIFR (1/40) and reported classical clinical (fever and splenomegaly with or without hepatomegaly) and an elderly residing in the city of Recife with interrupted treatment, presenting classical clinical and reagent IIFR. Discussion and Conclusion: There was no significant difference between the rK39 and IIFR HVL of the samples analyzed. The low maturity of the immune system of patients 1 and 10 months of age may suggest a higher sensitivity of rK39 in this age group compared to IIFR and the negativity of rK39 in other divergent samples are probably due to technical limitations of the methodologies used epidemiological and clinical conditions of the patients.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1293] P 617 - STANDARDIZATION OF COLLECTION METHOD THROUGH SWAB IN HUMAN SALIVA IN PATIENTS WITH AMERICAN CUTANEOUS LEISHMANIASIS IN PERNAMBUCO, BRAZIL ALEXANDRE, J.L.A.1; ARAUJO, A.I.F.1; VIANA, J.B.M.1; BRITO, M.E.F.1; ALMEIDA, E.L.1; MEDEIROS, A.C.R.2; WERKHAUSER, R.P.1; BRANDÃO-FILHO, S.P.1; RODRIGUES, E.H.G.1 1.CENTRO DE PESQUISAS AGGEU MAGALHÃES, RECIFE, PE, BRAZIL; 2.HOSPITAL UNIVERSITÁRIO OSWALDO CRUZ, RECIFE, PE, BRAZIL.
Keyword:american cutaneous leishmaniasis; human saliva; pcr
Abstract: Leishmaniasis is characterized by a spectrum of diseases distributed endemically in tropical and subtropical regions, affecting humans and animals through different species of the genus Leishmania. In Brazil, represents a public health problem due to its wide distribution, occurrence of severe clinical forms and difficulty relating to diagnosis and treatment. The disease has a zoonotic characteristic caused by thirteen species belonging to the subgenera Leishmania and Viannia, and these subgenera are being used as a target for diagnostic tests. The control of leishmaniasis is related, among others factors, of the availability of methods with high sensitivity and specificity for detection of the parasite, which provide relevant epidemiological information for planning appropriate measures. Obtaining biological samples for diagnostic analysis provides critical care to the patient, because it is an invasive process, capable of inflammation, and there may be reactivation of the disease in some cases. The aim of this study is to improve the molecular diagnosis of American cutaneous leishmaniasis (ACL), using the technique of polymerase chain reaction (PCR) associated to saliva as a human biological sample for identification of Leishmania spp., through the standardization of the swab collection method in ACL patients. We investigated 30 patients with ACL, with occurrence of parasite DNA in 46.67 % of the samples. From the total of 14 samples in that was detected the DNA of Leishmania, nine (64.29%) match to halibut region and five of them (35.71%) to oral mucosa. This result characterizes the halibut area as the best area to collect saliva samples and detect Leishmania spp. Considering the sensitivity of the PCR test and the collection procedure through saliva swab fluids, these findings demonstrate the feasibility of the method as a new diagnostic approach of ACL, leading to patient comfort and simplicity, without any harm to health by practicality the manner and place of collection of parasites. Financial support: CNPq, FACEPE, FIOCRUZ. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1295] P 618 - TREATMENT OF DOGS EXPERIMENTALLY INFECTED WITH L. CHAGASI WITH DIETHYLDITHIOCARBAMATE. SILVA, K.R.1; NEGREIROS, A.S.2; KHOURI, R.1; WEYENBERGH, J.1; CRUZ, M.S.2; BARRAL, A.1 1.FIOCRUZ-BA, SALVADOR, BA, BRAZIL; 2.UFPI, TERESINA, PI, BRAZIL.
Keyword:detc; dog; leishmaniasis
Abstract: The visceral leishmaniasis is a systemic zoonotic disease, chronic disease mainly affecting man and dog. Canine visceral leishmaniasis (CVL) is spread by the bite of female sand fly Lutzomyia longipalpis, and the etiologic agent Leishmania infantum chagasi. The dogs are considered as the domestic reservoir of the disease, developing an important role in its cycle, mainly by intense cutaneous parasitism and live in the same environment of humans. CVL leads to a broad spectrum of clinical symptoms, which vary with the type of immune response developed by the animal. This response is controlled by T cells and cytokine-mediated. The mechanisms to destroy intracellular microorganisms of the cell are represented by oxidative responses and non-oxidative. The oxidative responses are characterized by production of reactive oxygen intermediates, mainly free radicals such as oxygen peroxide (H2O2), hydroxyl radical (OH -), superoxide anion (O2 -) and peroxynitrite (ONOO -). The production of molecules O2 - is directly linked to the action of superoxide dismutase (SOD). Several drugs have been tested in the treatment of CVL, but almost all have a factor limiting their use, either by adverse effects or failure in destroying the parasite. Testing the in vivo action of a potent inhibitor of SOD, the diethyldithiocarbamate (DETC), Khouri et al., showed a lower parasite load in the ear and lymph node of BALB / c mice with Leishmania braziliensis. The DETC has been used in other infections such as Candida albicans, HIV and Mycobacterium bovis which
showed satisfactory action. Thus, the purpose of this study was to evaluate the effects of therapy with DETC, a potent inhibitor of SOD in dogs experimentally infected with L. infantum chagasi. Methods: Animals infected with the parasite were treated with doses of 100mg/kg/day divided into two oral administrations for 15 days. We evaluated hematological, biochemical and parasite load before, during and after treatment. There was a decrease in parasite load in lymph node, accompanied by a decrease in plasma activity levels of SOD, all statistically significant (p <0.05). Furthermore, we observed a positive correlation between parasite burden and levels of SOD. Conclusions: The DETC is able to decrease parasite burden in lymph node. - CLINICAL LEISHMANIASIS [1296] P 619 - ST-SEGMENT’S ELEVATION IN THE ELECTROCARDIOGRAM OF PATIENT WITH CUTANEOUS LEISHMANIASIS IN USE OF GLUCANTIME: A CASE REPORT ARAUJO NETO, A.R.; SANTOS, E.L.; MOURA, V.A.; SÁ, V.M.H.; PARREIRA, B.L.; COUTINHO, O.M.V.C.; MILAGRES, F.A.P. UNIVERSIDADE FEDERAL DO TOCANTINS, PALMAS, TO, BRAZIL.
Keyword:electrocardiogram ; st-segment’s; glucantime
Abstract: Introduction:.Elected as first choice for the treatment of leishmaniasis by the World Health Organization and the Ministry of Health of Brazil, the pentavalent antimonials are available for clinical use since 1945.Studies report also lack of standardization in their use, as well as cases of toxicity and no response to treatment. Electrocardiographic changes are among those described in the literature. The ST-segment’s elevation is rare and is indication for discontinuation of Glucantime, because it precedes or predicts severe cardiotoxicity. Case report: M.G, 72 years old, male, brown, retired, from Xambioá- Tocantins, in empiric treatment for Cutaneous Leishmaniasis (ACL) with Glucantime for 20 days. The patient was referred to the emergency room of the Hospital Geral de Palmas due to electrocardiographic changes ST-segment’s elevation and others suggestive of myocardial ischemia according to the reporton November 5th, 2012. Patient denied any signs, symptoms or history of cardiovascular disease.The physical examination revealed an ulcerated and painful lesion with raised margin and presence of fibrin in the right leg. There was also ulcerated sores with raised and hyperemic borders, fibrinareas and necrotic spots in other body regions. There wasn’t any progression of clinical findings suggestive to cardiopathy or changes in enzyme tests, such as creatine kinase MB portion (CKMB), lactate dehydrogenase (LDH) and troponin I. It was proposed, therefore, that such alterations could have been caused by the use of Glucantime. The confirmatory diagnosis of ACL was made by scraping the lesion in the patient’s right leg, which resulted positive for Leishmania. Thus it was administered Amphotericin B Deoxycolate and suspended Glucantime, what led to an improvement of electrocardiogram changes and its normalization. After nine days, the patient presented, with an infusion of Amphotericin B, malaise, nausea, chills, vomiting and deterioration of renal function (Cr = 2.0 mg/dL). Then that drug was suspended. It was begun a treatment with Liposomal Amphotericin B (1000 mg at the end of treatment), with improvement of symptoms, renal function (Cr = 1.5 mg/dL) and the injury, onthe 4th day of the scheme. The outpatient was giveninthe next day, after 20 days of hospitalization. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1298] P 620 - DETECTION OF LEISHMANIA PARASITE IN BIOPSY SPECIMENS OF HUMAN CUTANEOUS LEISHMANIASIS BY REVERSE TRANSCRIPTION REAL TIME PCR IN ISFAHAN, IRAN HOSEINI, S.G.1; HEJAZI, S.H.2 1.INFECTIOUS DISEASE AND TROPICAL MEDICINE RESEARCH CENTER, ISFAHAN UNIVERSITY OF MEDICAL SCIENCE, ISFAHAN, IRAN, ISLAMIC REPUBLIC OF; 2.SKIN DISEASES AND LEISHMANIASIS RESEARCH CENTER, DEPARTMENT OF PARASITOLOGY & MYCOLOGY, SCHOOL OF ME, ISFAHAN, IRAN, ISLAMIC REPUBLIC OF.
Keyword:leishmaniasis; real time pcr; 18s rrna
Abstract: Real time PCR has been widely used for diagnosis of cutaneous leishmaniasis (CL) however most PCR methods are based on DNA amplification which does not provide information on viability of the pathogen and might detect persistent DNA of the Leishmania. Moreover the number of RNA copies in the cytoplasm of parasite is much more than DNA copies hence RNA amplification provides more sensitivity than DNA amplification. The 18S ribosomal RNA gene has been found to be highly efficient for the diagnosis of leishmaniasis from human clinical materials and each parasite contains a large number of copies of the 18S rRNA gene. In this study a total of 27 skin biopsies obtained from patients with skin lesions suspicious of CL were subjected to RNA extraction and real time PCR for detection of 18S rRNA gene. Patients were recruited from Isfahan province, an endemic area for CL due to Leishmania major in center of Iran. All lesions with duration of less than 6 months (n=13) had positive smears and positive PCRs but from lesions with duration of more than 6 months (n=14) two lesions with negative smears were positive for rRNA gene and three suspicious cases which were empirically under treatment with meglumine antimonate had negative PCRs but their lesions healed completely after full course of treatment. Real time reverse transcription (RT) PCR seems a promising method for detection of live parasites in skin samples particularly for chronic cases with low number of parasite in their lesions. Negative RT PCR in lesions of treated patients intends successful treatment.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1302] P 621 - EFFECT OF S-ALLYL-CYSTEINE ON VIABILITY, APOPTOSIS AND PROLIFERATION OF LEISHMANIA MAJOR IN VITRO ESLAMI, G.1; SHAMS, A.1; FASAHAT, M.1; ASHOURI, H.1; HATEFI, Z.1; NABIPOUR, Y.1; MIRZAEI, F.1; HEJAZI, S.H.2
1.SHAHID SADOUGHI UNIVERSITY OF MEDICAL SCIENCES, YAZD, IRAN, ISLAMIC REPUBLIC OF; 2.ISFAHAN UNIVERSITY OF MEDICAL SCIENCES, ISFAHAN, IRAN, ISLAMIC REPUBLIC OF.
Keyword:leishmania major ; sac, apoptosis; proliferation
Abstract: There are many researches about garlic oil extracts against Leishmania parasite but recent studies have proved that these derivatives such as Allicin are cytotoxic and therapeutic effects of garlic is related to an odorless matter ,namely, S-Ally-Cysteine. Since few studies have been conducted about effectiveness of this material on Leishmania parasites, therefore, in this study effects of SAC were investigated on viability, apoptosis and proliferation of L. major promastigotes in vitro. In this study Leishmania major promastigotes(MRHO/IR/75/ER) was used. To assess, viability, apoptosis and proliferation of promastigotes after 72 hours treatment cultured parasites were involved with SAC in end concentrations of 2.5, 5, 10, 20,40 and 50 mM. This study showed that SAC could not induce apoptosis in promastigotes. Proliferation assay showed that SAC in end concentration of 5 and 10 mM could induce promastigotes growth. On the other hand, direct observation showed cultures comprising SAC in end concentration of 20, 40 and 50 mM were produced more rosette form of promastigotes. Because antioxidant of SAC has been proved and this study has also shown that some concentrations of SAC could be an effect on the survival and growth of promstigotes in culture, it appears that this substance can be used as a stimulating factor for growth of parasites in culture media.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1306] P 622 - IMMUNOHISTOCHEMISTRY OF LEISHMANIA INFANTUM CHAGASIINFECTED BALB/C MICE TREATED WITH EUGENOL AND THYMOL DERIVATIVES VILA-NOVA, N.S.1; MORAIS, S.M.1; LOBO, C.H.2; FALCAO, M.J.C.3; MOURA, A.A.A.2; SALES, A.D.4; RODRIGUES, A.P.R.4; FIGUEREIDO, J.R.4; ANDRADE, H.F.5 1.PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS VETERINÁRIAS, UNIVERSIDADE ESTADUAL DO CEARÁ, FORTALEZA, CE, BRAZIL; 2.LABORATÓRIO DE BIOLOGIA DA REPRODUÇÃO, UNIVERSIDADE FEDERAL DO CEARÁ, FORTALEZA, CE, BRAZIL; 3.UNIVERSIDADE ESTADUAL DO CEARÁ, FORTALEZA, CE, BRAZIL; 4.LABORATÓRIO DE MANIPULAÇÃO DE OÓCITOS E FOLÍCULOS PRÉ-ANTRAIS, UNIVERSIDADE ESTADUAL DO CEARÁ, FORTALEZA, CE, BRAZIL; 5.UNIVERSIDADE DE SÃO PAULO, LABORATÓRIO DE PROTOZOOLOGIA, SÃO PAULO, SP, BRAZIL.
Keyword:immunohistochemistry; eugenol; thymol
Abstract: In Northeastern Brazil there are many places where Leishmaniasis is endemic with several lethal cases among humans and dogs. The aim of this work was to survey natural active compounds to find new molecules with more activity against Leishmania infantum chagasi and with low toxicity. The compounds thymol and eugenol were chosen to be starting compounds to synthesize acetyl and benzoyl derivatives and to test their leishmanicidal action in vivo against L. i. chagasi. This study included 30 male BALB/c mice, with the age of 21 days, maintained according to institucional guide lines and inoculated intraperitoneal with 107 infective promastigotes of L. i. chagasi. Thirty five days post infection one animal was euthanized and liver and spleen smears were used to confirm infection and verify parasite burden, and then start the treatment. Thymol and eugenol derivatives were given intra peritonially to BALB/c at 100 mg/Kg/day during 30 days. The presence or absence of Leishmania after treatment (qualitative histological events) was determined by the presence of amastigote forms, Leishmania antigens detected by immunohistochemistry. Slides containing sections of fragments from BALB/c liver and spleen obtained after the treatment of leishmaniasis were submitted to immunohistochemistry for the detection of amastigotes forms. Immunohistochemistry was performed with anti-Leishmania antibodies produced in rabbits reacted with peroxidaseconjugated goat anti-rabbit IgG (Sigma Chemical Co.). All reactions were developed in the same way using a diaminobenzidine chromogen solution (Sigma Chemical Co., MO, USAD5637) which precipitates a brown product and counterstaining was performed with Harris hematoxylin. Next, the slides were dehydrated in a growing ethanol series and mounted with Permount resin (Fisher Chemicals, NJ, USA). Immunohistochemistry demonstrated the presence of Leishmania amastigote only in the spleen of the group treated with acetyl-thymol. In conclusion, these easy-making derivatives constitute useful compounds for further studies to find new agents for Leishmaniasis treatment. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1313] P 623 - L. BRAZILIENSIS BIOCHEMICAL AND GROWTH BEHAVIOR AFTER IN VITRO PHOTODYNAMIC THERAPY USING PROTOPORPHYRIN IX SOUZA, D.M.1; ALVES, P.M.2; SILVA, M.L.F.1; RODRIGUES, D.B.2; JUNIOR, V.R.1; PAULINO, T.P.2 1.UNIVERSIDADE FEDERAL DO TRIANGULO MINEIRO / DPTO CIÊNCIAS BIOLÓGICAS / LABORATÓRIO DE IMUNOLOGIA, U FEDERAL DO TRIÂNGULO BERABA, MG, BRAZIL; 2.UNIVERSIDADE FEDERAL DO TRIÂNGULO MINEIRO / CEFORES, UBERABA, MG, BRAZIL.
Keyword:photodynamic therapy; leishmania braziliensis; protoporphyrin ix
Abstract: Leishmaniasis are parasitary infections that manifest in animals and human and are caused by many protozoan species from the leishmania genus. Photodynamic Therapy (PDT) is a
therapeutical modality in which a visible light, a dye and O2/N2 react in order to yield reactive species of oxygen and/or nitrogen (ROS, RNS). Actually exist few studies involving PDT with L. braziliensis, so, this work is important to clarify the parasites reaction when is under specific treatment, especially about the parasite biochemical behavior. A L. brasiliensis culture was divided in four sample on the same volume and cells concentration, 106 parasites. The group distribution was: Non-treated cells (NT), Cell group treated only with light (LG), Cells group treated only with dye (DG) and Cells group treated with PDT (TG). The cells from the DG and TG are previously incubated (10 minutes) with Protoporphyrin IX (10mmol/L). After incubation, the TG was light irradiated (10 joules). The same conditions of irradiation was applied to LG. The NT group do not suffer light irradiation or dye incubation. After this procedure the nitric oxide and lipidic peroxidation was determinated. A cell fraction (106 cells) of all the groups, after their respective treatment (or not) was inoculated on Schneider medium in order to evaluate a metabolic profile in a growth curve (Optical Density- OD and glucose consumption). The nitric oxide (evaluated by Griess reaction) does not show significative nitrites and nitrate yield in neither studied groups, suggesting that Leishmania does not produce RNS´s including cells PDT-PPIX mediated. The levels of lipid peroxidation were 1,75 times more in TG than a others groups. The growing curve showed a higher development on the non PDT groups and the glucose consumption was higher as well. The non production of RNS´s after PDT-PPIX mediated indicate that a alteration on biochemical parameters (glucose consumption and optical density) is due to reactive oxygen species yielded. The low growth on TG (observed after OD analysis) suggest that a cell viability was decreased and/or a cell damaged occur (like a denaturation of some Leishmania biomolecules). Supported by FAPEMIG and Cefores. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1314] P 624 - GENETIC VARIATION OF PARASITES FROM LESIONS OF PATIENTS WITH CUTANEOUS LEISHMANIASIS AND THE RELATIONSHIP WITH THE RESPONSE TO TREATMENT RODRIGUEZ, N.M.; ARMAND, A.; SOTO, P.A. UNIVERSIDAD CENTRAL DE VENEZUELA, CARACAS, VENEZUELA.
Keyword:diagnosis; inmunotherapy; genetic variability
Abstract: Leishmaniasis is one of the six most important tropical diseases worldwide, with epidemiological and clinical pleomorphism in relationship with the species. Identification of Leishmania species is very important because allowed oriented the treatment and evaluated the sensitivity of the parasite to the drugs. Amplification of DNA fragments by the Polymerase Chain Reaction (PCR), followed by the analysis of the length polymorphism of the restriction fragments (RFLP) allows the identification and characterization of species in the Leishmania genus. The objective of this study was the diagnosis of leishmaniasis, species identification and their genetic variability and the relationship with the response to the treatment, using biopsies from patients with lesions suspicious of cutaneous leishmaniasis, for this proposal we used molecular techniques such as PCR and PCR-RFLP. We carried out a prospective, descriptive and experimental study in patients that assist to the Dermatology Service in the Institute of Biomedicine of the Ministry of Health, from May to November 2009. 53 patients were included in this study, from all of them were required to sign the informed consent and the authorization to take clinical pictures of the leishmaniasis lesions. Different diagnostic tests were conducted during the study: leishmanina skin test, smears from the lesion, skin biopsy, culture, PCR and PCR-RFLP. The results confirm leishmaniasis in 98% of the patients (52/53) by one or more procedure. PCR was positive in 72% of the samples, where 76% were positive for Leishmania (V.) braziliensis and 24% positive for Leishmania (L.) mexicana; 96% of the patients were leishmanina skin test positive; smear was positive in 75% (40/53) of the cases and 66% (35/53)
had a positive culture, lesions were more frequent in the legs (45%) and in the arms (41%). Most of the patient were treated with immunotherapy (66%); and a combination of both (glucantime and immunotherapy(33%) ; the promedy of time for clinical healing of the lesions was 18 weeks for patients with L(L.) mexicana and 14 weeks for patients with L(V) braziliensis. We also confirm the existence of genetic variability in some samples with L(V) braziliensis parasites which are coincident with the patients more resistant to treatment, genetic variation was not found for L(L.)mexicana. We conclude that the combination of molecular techniques is not only useful for diagnosis of the disease, also allows observing the epidemiological and clinical behavior, and can suggest the relationship with the response to the treatment. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1316] P 625 - ANTILEISHMANIAL ACTIVITY OF GERANYLGERANIOL BY APOPTOSIS LIKE DEATH MECHANISM LOPES, M.V.; DESOTI, V.C.; CALEARE, Â.O.; UEDA-NAKAMURA, T.; SILVA, S.O.; NAKAMURA, C.V. UNIVERSIDADE ESTADUAL DE MARINGÁ, MARINGÁ, PR, BRAZIL.
Keyword:geranylgeraniol; apoptosis; mitochondria superoxide anion production
Abstract: Localized cutaneous leishmaniasis treatments usually involves daily injections for 20-30 days of pentavalent antimonials Pentostam® or Glucantime®, however, a high rate of adverse effects and limited efficacy are related. Previous studies demonstrated that seed crude extract of Bixa orellana, known as “the annatto plant”, was found to be active against Leishmania amazonesis in vitro. In this work, we investigated the potential effect of geranylgeraniol (GGOH), a compound isolated from annatto seeds, in L. amazonensis and its possible targets in the parasite. GGOH was isolated from annatto seeds and identified through 1H and 13C nuclear magnetic resonance imaging and DEPT. The compound was evaluated against promastigotes and intracellular amastigotes. For this, promastigote forms of the parasite in the logarithmic-phase were cultured in the absence or in the presence of geranylgeraniol different concentrations by 24 to 96 h of incubation. For the antiamastigote activity, peritoneal resident cells from BALB/c mice were infected with 10 promastigotes per host cell and incubated for 6 h at 37 C in 5% CO2 atmosphere. GGOH was added to infected macrophages and after 24 h the parasites were counted in a Neubauer chamber. The cytotoxicity assay was evaluated in J774G8 macrophage cells after 24 h by sulforhodamine assay and the percentage of hemolysis by hemoglobin released in the supernatant of bloods cells treated with the compound. GGOH mechanism in Leishmania was evaluated by electronic microscopy and mitochondria membrane potential and cell membrane integrity alterations by Rh123 assay using flow citometry. Additionally, mitochondria superoxide production was analysed by MitoSOX detection in a fluorescence reader and DNA fragmentation by TUNEL assay. The compound inhibited the promastigote and intracellular amastigote forms with IC50 of 11 ± 1.0 and 17.5 ± 0.7 g/mL, respectively. This compound was also more toxic to parasites than to macrophages and did not cause lysis in human blood cells. The alterations induced by geranylgeraniol were observed in the protozoan by electronic microscopy, Rh 123 and TUNEL assays and included mainly mitochondria alterations and an abnormal chromatin condensation in the nucleus. Moreover, geranylgeraniol induces an increase in superoxide anion production. Collectively, our in vitro studies indicate geranylgeraniol as a selective antileishmanial that appears to be mediated by apoptosis-like cell death.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1323] P 626 - HISTOLOGICAL AND EXTRACELLULAR MATRIX ANALYSES OF SKIN, SPLEEN, LIVER AND LYMPH NODES OF BALB/C MICE EXPERIMENTALLY INFECTED WITH LEISHMANIA AMAZONENSIS AND TREATED WITH EXTRACT OF MORINDA CITRIFOLIA (NONI). OLIVEIRA, R.M.1; SOUZA, F.A.2; DE SÁ, J.C.1; KALABRESE, K.S.2; SILVA, D.C.M.1; SILVA, A.L.A.1
1.UNIVERSIDADE ESTADUAL DO MARANHÃO, SÃO LUÍS, MA, BRAZIL; 2.FUNDAÇÃO OSWALDO CRUZ - FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:morinda citrifolia; infecção experimental; balb/c
Abstract: Leishmaniasis is a zoonosis of major public health importance, which occurs in several Brazilian states. In the state of Maranhão, the city of São Luís is an endemic area. Currently, the chosen treatment for Leishmaniasis in humans remains the pentavalent antimony; however, the large number of side effects leads to its interruption in many cases. This way, we seek an alternative for the treatment of this disease. The aim of this study was to evaluate the effect of the plant Morinda citrifolia (noni) in the treatment of Leishmaniasis in mice. Noni fruit were collected and stored in flasks to obtain the extract. For testing, we used 4-6 weeks BALB/c mice from the Breeding Center of Laboratory Animals, of the Oswaldo Cruz Foundation, Rio de Janeiro, Brazil. The animals were divided into six groups, corresponding to the treated, untreated and control groups. The treatment period ranged from thirty to sixty days. Every thirty days, three animals from each group were euthanized for collection of the footpad, spleen, liver and lymph nodes. The organs were fixed in buffered formalin and proceeded routine histopathological processing. The slides were examined and photographed under a light microscope. As the results, it was found in the not treated group severe extracellular matrix destruction, hyperplasia on the white pulp of spleen and inflammatory cell infiltration on the liver. In the treated animals, we observed an intense production of extracellular matrix, characterized by the predominance of mature collagen, and no injury to the other organs analyzed. The results about the evaluation of the extracellular matrix of the skin showed that in groups treated with the noni extract were regressions of the lesions caused by infection with Leishmania amazonensis, which demonstrates that Morinda citrifolia induces an effective control of the infection. - OTHER SPECIAL TOPICS [1335] P 627 - FROM PHASE III CLINICAL TRIALS TO IMPLEMENTATION: AN EXAMPLE OF SSG AND PM COMBINATION THERAPY IN EASTERN AFRICA WASUNNA, M.1; MUS, A.2; ASRAT, H.3; KHALIL, E.2; OLOBO, J.4; ELLIS, S.5; STRUBWOURGAFT, N.5; BALASEGARAM, M.6 1.KEMRI/DNDI, NAIROBI, KENYA; 2.INSTITUTE OF ENDEMIC DISEASES, KHARTOUM, SUDAN; 3.ADDIS ABABA UNIVERSITY, ADDIS ABABA, ETHIOPIA; 4.MAKERERE UNIVERSITY, KAMPALA, UGANDA; 5.DNDI, GENEVA, SWITZERLAND; 6.MSF ACCESS CAMPAIGN, GENEVA, SWITZERLAND.
Keyword:access; registration; harmonization
Abstract: The Leishmaniasis East Africa Platform (LEAP), founded by the Drugs for Neglected Diseases initiative (DNDi) in 2003, has three main objectives: to evaluate, validate and facilitate registration of new treatments for visceral leishmaniasis (VL) in the region; to strengthen clinical research capacity; and to serve as a base for on-going educational cooperation among Eastern African countries (Ethiopia, Kenya, Sudan and Uganda), facilitating standardization of procedures and practices in the region, within the confines of local regulations.
A phase 3 clinical trial to evaluate the efficacy and safety of paromomycin (PM) alone versus sodium stibogluconate (SSG), as well as a short course combination of SSG&PM was completed by LEAP in January 2009. Consequently, in March 2010, the World Health Organization expert working group on Leishmaniases recommended the use of SSG&PM as the first line treatment for VL in East Africa. Shortly afterwards, the Federal Government of Sudan approved this recommendation in selected Ministry of Health sites, in collaboration with DNDi and Médecins Sans Frontières. The process of inclusion in national treatment guidelines and registration of PM is on-going in the other LEAP member countries. After the phase 3 clinical trials, the following steps were taken before the treatment was made available to patients: • Review by the expert advisory body of national drug regulatory authorities • Review by the national expert groups towards inclusion of the short course SSG&PM combination in national VL guidelines • Revision of national VL guidelines and inclusion of SSG&PM on the essential medicines list in LEAP countries • Registration of PM • Pharmacovigilance studies were initiated • Community sensitization • Training of health workers Despite these measures and political will from the LEAP country governments, challenges remain and hinder comprehensive evaluation and registration of new medicines. Harmonization by Eastern African countries of the steps that follow successful completion of phase 3 studies is a priority to make sure effective medicines are actually available to patients.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1340] P 628 - LABORATORY PARAMETERS OBSERVED IN PATIENTS WITH AMERICAN VISCERAL LEISHMANIASIS DURING TREATMENT WITH PENTAVALENT ANTIMONIALS. ESTECHE, M.V.1; GODOY, E.1; ANDRES, C.2; MIRTHA, M.3
1.UNIVERSIDAD DEL NORTE, ASUNCIÓN, PARAGUAY; 2.PROGRAMA NACIONAL DE CONTROL DE LAS LEISHMANIOSIS., ASUNCIÓN, PARAGUAY; 3.HOSPITAL GENERAL PEDIÁTRICO NIÑOS DE ACOSTA ÑU, SAN LORENZO, PARAGUAY.
Keyword:visceral leishmaniosis; rk39; pentavalent antimonials
Abstract: Currently in Paraguay, the number of registered cases of american visceral leishmaniasis (AVL) has increased dramatically, with small children the most important population group affected. The aim of this study was to describe the changes in laboratory parameters observed in patients in the pediatric general hospital " Niños de Acosta Ñu ", with confirmed diagnosis of AVL and treated with pentavalent antimonials.
We included 32 children of 0-16 years old, admitted to the pediatric general hospital " NiĂąos de Acosta Ă‘u ", in the city of San Lorenzo, Paraguay, located 15 km from the capital Asuncion, between 1 January 2007 and 1 January 2012, who were diagnosed with AVL by the clinic, rK39 serology and direct visualization of the parasite in bone marrow smears stained with Giemsa. Laboratory tests were performed to the patients to measure hematological parameters and functions of liver and kidney. The age mean was 2 years old and 78% of patients came from Central department. The most prominent symptoms present in 100% of patients were: prolonged fever, esplenomegaly and anemia. The eutrophic state prevailed in 87% of patients, leukopenia was present in 56% of cases; neutropenia in 100% of cases, increased erythrocyte sedimentation rate in 100% of cases, 37% of patients had altered prothrombin time, thrombocytopenia was present in 28% of the patients and hypoalbuminemia in 28% of cases. The successful development of all cases was evidenced by the recovery of hematologic values and increased albumin. The absence of nephrotoxicity and hepatotoxicity in all of the patients, confirmed the effectiveness of N-methylglucamine antimonate as a good choice for therapy in children. - CELLULAR AND MOLECULAR BIOLOGY [1347] P 629 - AMERICAN TEGUMENTARY LEISHMANIASIS: EFFECTIVENESS OF AN IMMUNOHISTOCHEMICAL PROTOCOL FOR THE DETECTION OF LEISHMANIA IN SKIN TAFURI, W.1; ALVES, C.1; ALVES, C.1; FIGUEIREDO, M.1; COELHO, G.2; MELO, M.1; TAFURI, W.1; RASO, P.1; SOARES, R.3 1.UFMG, BELO HORIZONTE, MG, BRAZIL; 2.UFOP, BELO HORIZONTE, MG, BRAZIL; 3.FIOCRUZ-BTHE, BELO HORIZONTE, MG, BRAZIL.
Keyword:american tegumetar leishmanasis ; diagnosis ; immunohistochemistry
Abstract: American tegumentary leishmaniasis (ATL) is endemic in Latin America, where Brazil has over 27 thousand cases per year. The aim of the present study was to develop an immunohistochemical method (IHC) for ATL diagnosis. For this purpose, we used serum from a dog naturally infected with Leishmania (Leishmania) infantum chagasi (canine hyperimmune serum) as the primary antibody, followed by a detection system with a secondary biotinylated antibody. Skin samples were obtained from 73 patients in an endemic area of Caratinga, Minas Gerais (MG) State, Brazil all testing positive for ATL with the Montenegro skin test, microscopy, and PCR. Canine hyperimmune serum was employed as a primary antibody in an immunohistochemical diagnostic method using streptavidin-biotin peroxidase. To assess the specificity of this reaction, IHC assays employing two monoclonal antibodies were carried out. As the polymer-based technology is less time-consuming and labor intensive than the IHC labeled streptavidin-biotin peroxidase method, we compared the two methods for all samples. The IHC method detected ATL in 67 of the 73 cases (91.8%). Immunolabeled parasites were primarily detected inside macrophages either in the superficial or the deep dermis. Detection was facilitated by the high contrast staining of amastigotes (dark brown) against the light blue background. A lower detection rate (71.2%) was observed with the both of the monoclonal Leishmania antibodies compared to the canine hyperimmune serum. This may have been due to a non-specific background staining observed in all histological samples rendering positive detection more difficult.
The higher efficacy of the canine hyperimmune serum in the IHC method was confirmed by the method using streptavidin-biotin peroxidase as well as that with the polymer-based technology (biotin-avidin-free system). The data are encouraging with regard to validating IHC as a standard alternative method for ATL diagnosis. - RESERVOIRS [1350] P 630 - EVALUATION OF IMMUNOTHERAPY ASSESSMENT LEISHTEC® ASSOCIATED WITH ALLOPURINOL IN DOGS NATURALLY INFECTED BY LEISHMANIA INFANTUM - PRELIMINARY RESULTS. RIBEIRO, V.M.1; BAHIA, E.M.2; TELES, P.P.A.1 1.ESCOLA DE MEDICINA VETERINARIA PUC MINAS, BETIM, MG, BRAZIL; 2.CLINICA VETERINARIA SANTO AGOSTINHO, BELO HORIZONTE, MG, BRAZIL.
Keyword:immunotherapy; canine leismaniosis; canine leishmaniosis treatment
Abstract: Sixteen dogs of different breeds and ages naturally infected by Leishmania infantum were selected. The infection in these animals was confirmed by indirect immunofluorescence (IFI), PCR and parasitological examination. Nine animals were symptomatic and seven asymptomatic; five of them showed up anemia and 11 had normal hematocrit; fraction Albumin / Globulin (A/G) was below 0.6% dL in five dogs and normal in 11; the search of parasites on the skin of the apex of the inner ear via the method of immunohistochemistry (IHC) was positive in 6 animals and negative in 10. In full dilution of serum antibodies by IFI, 5 animals had titers of 1:40 to 1:320, 10 of 1:640 to 1:5120 and one showed the title of 1:10240. The animals were treated with Leish-Tec® using double dose, in three applications, with intervals of 21 days reapplying every six months. They were also treated with oral allopurinol, 10 to 20 mg/kg twice a day during the entire period. The evaluation of the animals after 6 months of treatment revealed that 13 animals were asymptomatic with normal hematocrit, and the fraction A/G in 12 was normal. Out of the 16 evaluated animals by IHC, amastigotes were found only in one. Serological evaluation showed seroconversion in 8 animals and 7 had titles of 1:320. Conclusion: We can conclude that immunotherapy with Leish-Tech® associated with allopurinol can induce clinical and laboratorial improvements in dogs with visceral leishmaniasis, as long as the reduction of cutaneous parasitism, and the reduction of the anti-L. infantum titers. Becoming, therefore, an option of treatment in the treatment protocol of the of the disease in dogs.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1354] P 631 - EVALUATION OF EHRLICHIA CANIS ANTIBODIES IN DOGS EXPOSED TO A LEISHMANIA INFANTUM NATURAL CHALLENGE IN AN ENDEMIC AREA FOR VISCERAL LERISHMANIASIS ARAUJO, M.F.L.1; CUTOLO, A.A.2; TANIGUCHI, H.H.1; HIRAMOTO, R.I.M.1; KASTELLÃO, K.G.1; GAVA, R.1; PEREIRA-CHIOCCOLA, V.L.1; MEIRA, C.S.1; BENETI, S.A.A.3; TOLEZANO, J.E.1; MOLLA, L.M.4; MENZ, I.5 1.INSTITUTO ADOLFO LUTZ, SÃO PAULO, SP, BRAZIL; 2.PREFEITURA DE MONTE MOR, MONTE MOR, SP, BRAZIL; 3.CLINICA QUATRO PATAS-DRACENA, DRACENA, SP, BRAZIL; 4.VIRBAC DO BRASIL, SÃO PAULO, SP, BRAZIL; 5.UNIVERSIDADE ESTADUAL DE CAMPINAS-UNICAMP, CAMPINAS, SP, BRAZIL.
Keyword:cross-reaction; diagnostic; serology
Abstract: Diagnostics for Canine Visceral Leishmaniasis (CVL) are complex and the use of different immunological and direct methods are important to confirm Leishmania (L.) infantum infection. The use of serological diagnostic methods for CVL are questioned by veterinarians suggesting cross reaction with Ehrlichia canis, considering the possibility of false positive VL diagnostic results. In a longitudinal study 111 healthy dogs, originally brought from a non VL transmission area, were evaluated after 6 months exposure period to a natural L. infantum challenge in a VL endemic area. Confirmed parasitological and/or molecular methods were evaluated through serology by ELISA and DPP (Dual Path Platform®) for detection of K28 antibodies and compared to Ehrlichia canis dog sera tested by a chromatographic method (Speed Duo Leish/Ehrli®). Blood and bone marrow were collected from CVL exposed dogs and serology was performed by the following methods: ELISA with crude parasite L. major soluble antigen and two different immunochromatographic tests, DPP for detection of K28 antibodies and Speed Duo Leish/Ehrli® (L. infantum and Ehrlichia canis antigens). Parasitological diagnosis was performed on bone marrow smears, and DNA detection by real time PCR (qPCR) for L. infantum. Comparisons were made between serological and parasitological techniques. Leishmania infection was demonstrated in 26.1% (29/111) dogs, where 79.3% (23/29) were qPCR positive, some of them positive for amastigotes in smear samples; 20.7% (6/29) were only amastigote smear positive. From the Leishmania infected dogs 34.5% (10/29) were positive on E. canis antibody test. From the 82 non-infected dogs 37.8% (31/82) were E. canis positive. From these dogs 16.1% (5/31) were positive in ELISA and DPP; 6.4% (2/31) negative in ELISA and positive in DPP; 9.7% (3/31) were negative in ELISA and positive in DPP; 67.7% (21/31) were negative in both methods, ELISA and DPP. Contact to E. canis antigen was demonstrated only by serological chromatographic test. From the positive E. canis sera 32.3% (10/31) were positive also at one or both Leishmania sp. serological methods, but it is possible that dogs with solely E. canis infection may present non-specific antibodies that cross react with Leishmania sp., mainly at the ELISA method. In this study it was also observed 34.5% of dogs with a possible co-infection Leishmania/Ehrlichia confirming a high prevalence and a high transmission rate for both protozoa in the area. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1357] P 632 - ISOLATION OF LEISHMANICIDAL DITERPENE PRODUCED BY ONE SPECIES OF ASTERACEAE MOREIRA, C.P.S.; SANTOS, C.N.; ROCHA, M.N.; BERTOLLO, C.M.; ALMEIDA ALVES, T.M. FIOCRUZ, BELO HORIOZNTE, MG, BRAZIL.
Keyword:asteraceae; diterpene; natural product
Abstract: The few therapeutic alternatives available to treat leishmaniasis present limitations due to occurrence of serious side effects, among other facts. In this regarding, it is urgent to discover new drugs. The natural products are important sources of drug leaders. Therefore, the objective of this work is to isolate and identify the compounds responsible for the leishmanicidal activity of one species of Asteraceae plant. Ethanolic leaves extract was fractionated by high speed counter-current chromatography (HSCCC) using the biphasic system hexane/ethyl acetate/methanol/water (2:3:2.5:2.5). The similar fractions according to thin layer chromatography analysis were grouped (F1 to F21). The 21groups were tested in murine macrophages infected with L. amazonensis at a concentration of 50 µg/mL (except 1, 10 and 12, that were 20 µg/mL). The percentage of inhibition was determined by counting under an optical microscope. The active groups are being refractionated by high performance liquid chromatography (HPLC) in order to isolate bioactive compounds. Five groups displayed leishmanicidal activity without damages to macrophages cells. They showed 54-100% reduction
of parasitemia. Compound C1 was isolated as active compound (parasitemia reduction of 73%) from one of the active group. Mass spectrometry and nuclear magnetic resonance data analysis indicate C1 as a diterpene. The chemical fractionations of other active groups are underway. Financial Support: CNPq/Fiocruz and Fapemig - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1358] P 633 - AMERICAN CUTANEOUS LEISHMANIASIS OUTBREAK IN AN INDIGENOUS COMMUNITY IN THE AGRESTE REGION OF PERNAMBUCO, BRAZIL. DA SILVEIRA NETO, M.M.1; DE BRITO, M.E.F.2; DE ALMEIDA, É.L.2; FILHO, S.P.2; SALES, R.H.F.1; DE LUCENA, N.C.1; RODRIGUES, M.M.1 1.FPS-IMIP, RECIFE, PE, BRAZIL; 2.CENTRO DE PESQUISAS AGGEU MAGALHÃES/FIOCRUZ, RECIFE, PE, BRAZIL.
Keyword:american cutaneous leishmaniasis.; leishmania (viannia) braziliensis; ecoepidemiology
Abstract: Introduction: American Cutaneous Leishmaniasis (ACL) has clinical and epidemiological characteristics that vary between the regions of Brazil. The distribution of the disease occurs throughout the country but 62% of cases occur in the state of Pernambuco. Objective: To evaluate the clinical, epidemiological and laboratory aspects of cases of ACL in an indigenous community in the Agreste (sub-humid hill) region of Pernambuco. Material and Methods: In 2012, 30 suspected cases of ACL were detected in Ororubá, a Xucuru village in Pesqueira, in the Agreste region of Pernambuco. The coordinator of the Pernamubuco Indigenous Health District (DSEI) reported the cases to the State Health Department (DSEI), which in turn reported the situation to the Federal Ministry of Health. The suspected cases were verified by: parasitological diagnosis, histopathological analysis, cultures and Polymerase chain reaction (PCR) tests, utilizing B1 and B2 primers for the Viannia subgenus, at the Instituto de Medicina Professor Fernando Figueira (IMIP) and the Fiocruz Leishmaniasis Reference Service (PE). The majority of cases were of indigenous children and adolescents, male and female, between 2- 12 years-old, with lesions on their faces, arms and legs. Results: Of 30 patients, 27 underwent parasitological diagnosis with 16 positive results. Of the 18 selected samples, 16 were confirmed with PCR, and of these, 2 cultures were obtained and identified as Viannia subgenus via PCR. Fine needle aspiration (FNA) of cervical lymph nodes of one subject was preformed and the presence of the parasite was detected. The cutaneous lesions were predominantly 1 to 5 cm in diameter; nevertheless, There was a case in the mucosal detected in a 10 year-old child. Discussion: There have been several outbreaks of the disease in Pernambuco, such as the 26 cases reported in 1996 at a military training center located in the Zona da Mata (coastal forest) region, and 71 cases reported in 2007 at the same location. Other outbreaks occurred in the Imbu district, in Vicenza, (72 cases) and Igarassu (25 cases), both in the Zona da Mata Norte (northern coastal forest) region. Conclusions: The occurrence of these outbreaks demonstrates the persistence endemic of the disease, probably due to exposure to environmental factors and the deficiencies in preventative measures.
Keywords: American Ecoepidemiology.
cutaneous
leishmaniasis.
Leishmania
(Viannia)
braziliensis,
Financial Support: FACEPE (Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco), IMIP (Instituto de Medicina Integral Professor Fernando Figueira)
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1363] P 634 - COMPARISON OF PCR WITH STAINED SLIDES OF BONE MARROW AND LYMPH NODES ASPIRATES WITH SUSPECT DIAGNOSIS FOR LEISHMANIASIS SANTOS, T.R.1; CARREIRA, V.S.2; FERRARI, H.F.2; MOREIRA, M.A.3; LUVIZOTTO, M.C.R.2
1.DEPARTMENT OF ANIMAL PATHOLOGY, CPPAR, SÃO PAULO STATE UNIVERSITY (UNESP),, JABOTICABAL, SP, BRAZIL; 2.LABORATORY OF PATHOLOGY, DEPARTMENT OF ANIMAL CLINIC, SURGERY AND REPRODUCTION, SCHOOL OF VETERINARY, ARAÇATUBA, SP, BRAZIL; 3.CLINICAL PATHOLOGY LABORATORY, ANHEMBI-MORUMBI UNIVERSITY, SÃO PAULO, SP, BRAZIL.
Keyword:canine leishmaniasis; pcr; cytology
Abstract: Visceral leishmaniasis (VL), also known as kala-azar, is a disseminated protozoan infection caused by Leishmania donovani complex. Traditionally the definite diagnosis is made by amastigote detection in the tissue. The aim of this study was to evaluate the PCR technique in stained slides of bone marrow and lymph nodes aspirates with suspect diagnosis for leishmaniasis. Slides were selected totaling 62 suspect cases (33 bone marrow samples and 29 lymph node samples) and 17 positive cases (8 bone marrow and 9 lymph node). From 62 suspect cases, 39 (62.90%) were confirmed to be positive being 17 (n=29) lymph node aspirates and 22 (n=33) bone marrow. This finding is in agreement with the higher sensitivity of the PCR assay compared to direct microscopic observation. In conclusion, the findings of this study supports the use of PCR on archive cytological preparation stained slides for the diagnosis of canine visceral leishmaniasis, emphasizing the higher sensitivity of this technique when compared to direct microscopic examination and mostly the use of the suspect status for the cytology samples that presents the previously mentioned particularities with focus on detecting the oligosymptomatic or assymptomatic dogs in endemic areas functioning as potential reservoirs for this disease. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1369] P 635 - NON USUAL CLINICOPATHOLOGICAL PRESENTATION OF CANINE VISCERAL LEISHMANIASIS LUVIZOTTO, M.C.R.; DA COSTA, L.D. FACULDADE DE MEDICINA VETERINÁRIA, ARAÇATUBA, SP, BRAZIL.
Keyword:leishmaniasis; dog; tongue
Abstract: The Canine Visceral Leishmaniasis (CVL) is a chronic progressive disease caused by protozoa of the genus Leishmania sp., whose transmission involves phlebotomus and is a zoonosis of worldwide importance. The most common clinical signs are lymphadenopathy, hepatosplenomegaly, skin changes, hyporexia, cachexy and onychogryphosis. Other less common clinical presentations are emesis, diarrhea, conjunctivitis and uveitis. The aim of this report is to describe a non usual clinical-pathological presentation of Visceral Leishmaniasis in a dog, Rottweiler, female, adult. The animal was euthanized and sent to the Department of
Pathology of UNESP/Araçatuba due to present neoformations in the tongue. During the necropsy, on external examination, there were multiple neoformations in the dorsal and lateral tongue, measuring 1.0 x 1.0 x 1.5 cm, polypoid, smooth and not ulcerated. In histological sections stained with hematoxylin and eosin, such lesions were covered with intact epithelium; in the submucosa, sharp and diffuse lymphoplasmacytic inflammatory infiltrate was observed, and rare typical amastigotes of Leishmania sp. within the cytoplasm of macrophages. The cross sections submitted to immunohistochemistry method, using an anti-Leishmania polyclonal antibody produced in mice showed positive staining, confirming the diagnosis. This finding suggests that the CVL can manifest itself in various ways, requiring, therefore, a differential diagnosis for many other diseases, such as proliferative diseases. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1377] P 636 - DEVELOPMENT OF NEW DRUGS FOR LEISHMANIASIS : WHAT CAN WE LEARN FROM THE IN VIVO PROFILING OF SEVERAL PROMISING CHEMICAL SERIES BRAILLARD, S.1; CHATELAIN, E.1; MOWBRAY, C.E.1; LAUNAY, D.1; MAES, L.2; THOMPSON, A.3 1.DNDI, GENEVA, SWITZERLAND; 2.LABORATORY OF MICROBIOLOGY, PARASITOLOGY AND HYGIENE, UNIVERSITY OF ANTWERP, ANTWERP, BELGIUM; 3.AUCKLAND CANCER SOCIETY RESEARCH CENTRE, UNIVERSITY OF AUCKLAND, AUCKLAND, NEW ZEALAND.
Keyword:drug development; in vivo efficacy; pk/pd
Abstract: Drugs for Neglected Diseases initiative (DNDi) is a collaborative, patients’ needs-driven, notfor-profit organization whose mission is to develop and make available new drugs for the most neglected tropical diseases, such as visceral leishmaniasis (VL). DNDi’s Lead Optimization programs aim to develop and improve chemical entities that have to fulfill the Target Product Profile (TPP) in terms of efficacy, safety and cost. To this end, building a robust and accurate screening cascade for identifying potential candidates from discovery screening to preclinical development is crucial. This is a dynamic, two-way process that depends on the results obtained through in vivo profiling and, retrospectively, from clinical outcomes. Partnership between DNDi, academic laboratories with leishmaniasis or series specific medicinal chemistry expertise and committed Contract Research Organizations led to the generation of a reasonable number of in vivo data both on pharmacokinetics and efficacy. Efficacy data was obtained through in vivo assessment in the Balb/c mouse model, and more recently in the Syrian hamster model developed at LMPH (Laboratory of Microbiology, Parasitology and Hygiene). This hamster model, which aims to predict results in humans as much as possible, showed robust and highly reproducible results. Supplemented by pharmacokinetic data in different species, this efficacy model allowed us to start building a preliminary assessment of the Pharmacokinetic/Pharmacodynamic (PK/PD) relationships for different new VL chemical series, such as oxaboroles and nitroimidazoles. Moreover, evaluation of existing drugs or new formulated drugs will allow us to benchmark this model, and, more importantly, will reinforce the accuracy of DNDi’s screening cascade through a better understanding of the in vitro / in vivo profiling as a function of the different mechanisms of action.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1381] P 637 - ISOLATION OF LEISHMANIA (LEISHMANIA) MAJOR AND LEISHMANIA (LEISHMANIA) AMAZONENSIS PROMASTIGOTES RESISTANT TO TUBERCIDIN AOKI, J.I.; SILVA, M.G.; YAMASHIRO-KANASHIRO, E.H.; RAMOS-SANCHES, E.M.; OZAKI, C.Y.; ROCHA, M.C.; COTRIM, P.C. INSTITUTO DE MEDICINA TROPICAL DE SÃO PAULO, SÃO PAULO, SP, BRAZIL.
Keyword:leishmania; tubercidin; drug resistance
Abstract: Purine metabolism in Leishmania is not well understood. Purine is essential to DNA and RNA synthesis, and it is also an essential difference between the parasite and its host, suggesting an important target to new anti Leishmania drugs. Tubercidin (TUB), a purine toxic analogue is a potential antiparasite by DNA synthesis inhibition. A protein with 606 amino acids, about 70kDa, one transmembrane domain, unknown function to date is being characterized by our laboratory as involved in TUB resistance when transfected and overexpressed. In order to verify if this protein is actually involved in resistance pathway, we generated L. (L.) major and L. (L.) amazonensis mutants resistant to TUB, by drug pressure. L. (L.) major and L. (L.) amazonensis mutants showed a 30 and 40 fold resistance, respectively, when compared with wild type cells. These results showed both mutants were well adapted to high TUB concentrations (5µM). An interesting result showed that this resistance was reversed in the absence of TUB, suggesting that this resistance is due to DNA amplification. Gene amplification has been frequently observed in Leishmania promastigotes mainly when the resistance is induced in vitro. To confirm, a real time PCR with genomic DNA was performed. Real time PCR data analysis showed an increase about three times higher in mutants than in wild type, suggesting that TUB resistance is the result of an overexpression caused by DNA amplification. Real time RT-PCR assay of this protein, using total RNA, showed a significant increase in transcripts levels between resistant and wild type parasites (about 60-fold resistance). Finally, no cross resistance was observed with other drugs used in leishmaniasis treatment, as glucantime, allopurinol and pentamidine, indicating a TUB specific mechanism. Supported by: CAPES, FAPESP, CNPq and LIM48-FMUSP. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1383] P 638 - EVALUATION OF THE LEISHMANICIDAL AND CYTOTOXIC ACTIVITIES OF SOME SEAWEED FROM NORTHEAST SEA COAST, BRAZIL DOS ANJOS, K.F.L.1; ALIANÇA, A.S.S.1; BORGES, A.1; BIANCO, E.M.2; FIGUEIREDO, R.C.B.Q.1
1.CENTRO DE PESQUISAS AGGEU MAGALHÃES, RECIFE, PE, BRAZIL; 2.UNIVERSIDADE FEDERAL DE SANTA CATARINA, FLORIANÓPOLIS, SC, BRAZIL.
Keyword:leishmania amazonensis; seaweed; leishmanicidal
Abstract: Leishmaniasis, an infectious disease caused by parasites of the genus Leishmania, is one of the major neglected tropical disease in the world, mainly in Latin America with a rapid spread of this illness in Brazil. For over half a century, the clinical forms of disease have been treated almost exclusively with pentavalent antimonial compounds. Besides their cytoxicity, this treatment requires long courses of parenteral administration and generalized resistance to these agents have been also reported. In this sense, the development of new medicines that can replace the presently available therapeutic alternatives is an urgent need. Seaweeds are considered as source of bioactive metabolites characterized by a broad spectrum of biological activities
including antiprotozoal activity. In this work we evaluated the in vitro leishmanicidal and cytotoxic activities of 10 extracts of seaweed macroalgae from Brazilian Northeast coast, on Leishmania amazonensis, the etiologic agent of cutaneous leishmaniasis. The concentration of seaweed extract necessary to inhibit 50% of the promastigote growth was determined by cell counting after 72 hours of incubation extracts. The cytotoxicity was estimated by MTT colorimetric assay and the 50% cytotoxic concentration (CC50) was determined by regression analysis. The selective index was determined as the rate of CC50 to IC50 values. All the extracts tested are able to inhibit the parasite growth in a time-dependent manner with IC50 values varying from 49 to 160 g/mL. The extracts from Canistrocarpus cervicornis, Dictyota mertensii and Laurencia dendroidea, were the most effective with IC50 values of about 50, 71 and 51 g/mL respectively. Our results also showed that all seaweed extracts presented no singificant toxicity to mammalian cells (CC50 >100 g/mL) and were more selective against the parasite with IS value varying from 1.2 to 5.59. The low cytotoxicity of Canistrocarpus cervicornis, Dictyota mertensii e Laurencia dendroidea extracts allied to their significant leishmanicidal activities points these seaweeds as promissory candidates for chemotherapeutic agent against Leishmania amazonensis. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1384] P 639 - CYTOKINE EXPRESSION PATTERN IN THE SPLEEN OF A PATIENT WITH A CLINICAL TREATMENT-RESISTANT FORM OF VISCERAL LEISHMANIASIS DE FREITAS, L.A.1; PAGLIARI, C.2; SANTOS, L.G.3; ALMEIDA, V.A.1; SILVA, T.L.V.E.3; DUARTE, M.I.S.2; DOS-SANTOS, W.L.C.1; COSTA, C.H.N.3 1.FUNDAÇÃO OSWALDO CRUZ, CENTRO DE PESQUISAS GONÇALO MONIZ, SALVADOR, BA, BRAZIL; 2.FACULDADE DE MEDICINA, UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL; 3.UNIVERSIDADE FEDERAL DO PIAUÍ, INSTITUTO DE DOENÇAS TROPICAIS NATAN PORTELA, TERESINA, PI, BRAZIL.
Keyword:spleen; cytokine; histology
Abstract: The spleen is a secondary lymphoid organ interposed in the blood stream that monitors blood for circulating antigens and pathogens and hemocateresis of old or damaged red blood cells, limiting the availability of free iron, which is necessary for bacterial metabolism. Spleen is affected in practically all cases of visceral leishmaniasis, and a spleen regression index is used as indicative of response during the treatment against human visceral leishmaniasis. Furthermore, splenectomy is used as supplementary treatment of non-responsive patients to the usual drug therapy schedules. We present herein the case of a 25 yr. old male patient, with recurrent hospitalizations in the past 3 years due to fever and asthenia, mucocutaneous pallor and abdominal swelling, always diagnosed as visceral leishmaniasis, confirmed by tests. On clinical examination, the abdomen was distended with mild pain on deep palpation and splenomegaly (8 cm from the left costal margin). Laboratory tests showed pancytopenia, positive rapid test for leishmaniasis and presence of Leishmania in the bone marrow smears. The patient had received all treatment in use for the disease: glucantime, amphotericin deoxycholate, amphotericin lipid complex, liposomal amphotericin and pentamidine. There was transient symptomatic improvement. However, splenomegaly persisted and the symptoms relapsed a few months afterwards. Faced with this resistance framework, splenectomy was performed on May 2, 2012. The spleen weighed 4.800g. Spleen sections were stained with hematoxylin-eosin and with antibodies against CD3, CD4, CD8, CD20, CD79alpha, CD138, CD68, IL4, IL10, IL17, IL-6, IFN-gamma, TNF-alpha, Foxp-3. Spleen showed the general aspect of the red and white pulp architecture preserved, with large lymphoid follicles, with large germinal centers and welldefined mantle areas, marginal zone had a usual aspect. Plasmacytosis and lymphoid depletion were observed in the PALS. The red pulp showed increased capillarization, congestion, and erythrophagocytosis. Amastigote-containing macrophages were observed in non-granulomatous nodular aggregates and in the reticular sinuses. Preliminary analysis of cytokine expression
pattern showed an increase of IL-17 and to a lesser extent of IL-6, IL-10 and Foxp-3 producing cells. Such pattern is suggestive of a possible transition pattern of TH2 to TH17 immune response and a failure of progression towards a TH1 immune response. Now we are confirming this analysis by morphometry. The patient is now asymptomatic, rapid test for leishmaniasis is negative and parasites ware not observed in bone marrow. Sensitivity test of Leishmania infantum isolated from the patient to the used drugs (antimony, amphotericin B, and pentamidine) showed no resistance pattern. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1386] P 640 - EVALUATION OF LEISHMANICIDAL ACTIVITY OF NAPHTOQUINONES ADUTS ON PROMASTIGOTE FORMS OF LEISHMANIA AMAZONENSIS. MAIA, F.C.1; FIGUEIREDO, R.C.B.Q.2; DAVID, C.C.3; CÂMARA, C.A.3; SILVA, T.M.S.3; DA SILVA JUNIOR, V.A.4; PORTO, A.L.F.4
1.DEPARTAMENTO DE MEDICINA VETERINÁRIA/PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIA VETERINARIA/UFRPE, RECIFE, PE, BRAZIL; 2.CENTRO DE PESQUISAS AGGEU MAGALHÃES/FIOCRUZ, RECIFE, PE, BRAZIL; 3.DEPARTAMENTO DE CIÊNCIAS MOLECULARES/UFRPE, RECIFE, PE, BRAZIL; 4.DEPARTAMENTO DE MORFOLOGIA E FISIOLOGIA ANIMAL/UFRPE, RECIFE, PE, BRAZIL.
Keyword:naphtoquinones; leishmania amazonensis; lapachol derived
Abstract: Leishmaniasis, a infectious disease caused by protozoa belong to the genus Leishmania, is considered by World Health Organization (WHO) an increasing public health concern, particularly in Africa, Asia and Latin America. The classical treatment for this illness is still based on pentavalent antimony in the form of sodium stibobluconate (Pentostan) or Nmethylglucamine antimoniate. These drugs are in general toxic, expensive and require long-term treatment. The clinical resistance to these compounds has been reported. Therefore, there is an urgent need for development of new effective and safe drugs for the treatment of Leishmaniasis. Quinones are naturally occurring compounds which have antibacterial, antifungal and larvicidal activities. In this work, we evaluated the effect of four lapachol derived naphthoquinone aduts on the growth of promastigote forms of Leishmania amazonensis, the etiologic agent of cutaneous leishmaniasis in Brazil. The concentration of naphtoquinones necessary to inhibit 50% of the promastigote growth was determined by regression analysis, after 48 hours of drug incubation. Parasites incubated in free-drug medium were used as a control. All naphtoquinones presented an inhibitory effect on the parasite growth in a dose-dependent way with IC50/48h values of about 11; 19; 25 and 26 for compounds NAF31 (C34H35N2O2); NAF16 (C33H34N2O3); NAF15 (C32H32N2O3) and NAF17 (C33H34N2O) respectively. The higher activity of compound NAF 31 could be due to the presence of one n-buthyl group. The comparison of IC50 values for isomers NAF16 and NAF 17 showed the importance of naphtoquinones functional groups. NAF16 a methyl ester, showed to be significantly more effective than NAF17, an alcohol, which presented the lower IC50 value among the compounds tested. Although the preliminary results obtained herein point towards the use of these naphtoquinones as potential chemotherapeutic agent against L. amazonensis, further studies to evaluate the effects of these compounds on mammalian cells and amastigote forms of the parasite are still needed. Financial support: CNPq, PRONEM/FACEPE
- OTHER SPECIAL TOPICS [1387] P 641 - BIOGUIDED FRACTIONATION OF PIPER ADUNCUM L. (PIPERACEAE) EXTRACTS TO ISOLATION OF ANDULCHALCONE A, A NEW ANTILEISHMANIAL
METABOLITE LAGO, J.H.G.1; PASSERO, L.F.D.2; TEMPONE, A.G.3
1.UNIFESP, SAO PAULO, SP, BRAZIL; 2.FM-USP, SAO PAULO, SP, BRAZIL; 3.INSTITUTO ADOLFO LUTZ, SAO PAULO, SP, BRAZIL.
Keyword:piper aduncum; natural products; leishmanicidal
Abstract: Parasitic protozoan diseases affect the poorest population in developing countries. Leishmaniasis has been included among the most important threats for public health in Central and South American continent, with few therapeutic alternatives and highly toxic drugs. In the course of selection of novel drug candidates from Brazilian flora, we undertook the bioguided fractionation of MeOH extract from leaves of Piper aduncum L. (Piperaceae). After extraction using MeOH and complete evaporation of the solvent under reduced pressure, the obtained extract displayed leishmanicidal activity against promastigote forms of L. (L.) amazonensis (100% of inhibition at 200 microg/mL). Thus, this extract was subjected to silica gel column chromatography eluted with hexane containing increasing amounts of EtOAc (up to 100%), to give six fractions (A – F). After antiprotozoal evaluation (100% of inhibition at 200 microg/mL), the activity was concentered at fraction D, which was subjected to silica gel column chromatography eluted with increasing amounts of hexane in EtOAc (up to 100%) to afford nine fractions (D1 – D9). Since the leishmanicidal activity was detected in fraction D3 (100% of inhibition at 100 microg/mL), this material was purified using preparative TLC to afford 14 mg of adunchalcone A, a new natural product. The structure of this compound was established on the basis of spectroscopic data analysis, mainly nuclear magnetic resonance (including bimensional analysis) and high resolution mass spectrometry. This compound showed activity against promastigotes of Leishmania (L.) amazonensis, Leishmania (V.) shawi, Leishmania (L.) braziliensis, and Leishmania (V.) guyanensis showing IC50 of 3.1, 5.7, 13.5, and 61.7 microg/mL, respectively, while IC50 to amphotericin B was determined as 0.1 microg/mL to all tested Leishmania species. However, despite of the potential against promatigotes forms, this compound was not effective against amastigote forms of Leishmania. The cytotoxicity study using peritoneal macrophages, demonstrated that andunchlacone showed CC50 of 39 microg/mL. The obtained results showed that bioguided fractionation was a rational approach to detection and isolation of adunchalcone A, a new antileishmanial metabolite from P. aduncum L. Additionally, these data suggested that this new compound, as well as other similar secondary metabolites or synthetic analogs, might be useful tolls for drug design for in vivo studies against protozoan diseases.
This abstract was submitted to presentation in scientific section "New anti-leishmanial drug discovery from natural products resources". - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1389] P 642 - TOWARDS THE IDENTIFICATION OF A BACK-UP CANDIDATE FOR DNDI-VL2098 TO TREAT VISCERAL LEISHMANIASIS LAUNAY, D.1; CHATELAIN, E.1; BRAILLARD, S.1; VISHWAKARMA, P.2; GUPTA, S.2; PURI, S.2; YARDLEY, V.3; MAES, L.4; THOMPSON, A.5 1.DRUGS FOR NEGLECTED DISEASES INITIATIVE, GENEVA, SWITZERLAND; 2.CENTRAL DRUG RESEARCH INSTITUTE, LUCKNOW, INDIA; 3.LONDON SCHOOL OF HYGIENE AND TROPICAL MEDICINE, LONDON, UNITED KINGDOM; 4.LABORATORY OF MICROBIOLOGY, PARASITOLOGY AND HYGIENE, UNIVERSITY OF ANTWERP, ANTWERP, BELGIUM; 5.AUCKLAND CANCER SOCIETY RESEARCH CENTRE, UNIVERSITY OF AUCKLAND, AUCKLAND, NEW ZEALAND.
Keyword:screening; lead optimization; in vivo efficacy
Abstract: The Drugs for Neglected Diseases initiative (DNDi) is a collaborative, patients’ needs-driven, not-for-profit organization whose mission is to develop new drugs for the most neglected tropical diseases, including Visceral Leishmaniasis (VL). Through collaboration with the TB Alliance/Auckland University, DNDi identified DNDI-VL2098 as a potential drug candidate for VL, and is currently completing a set of preclinical studies. Since nitroimidazoles still hold great potential as treatment for neglected tropical diseases (including VL), the project team decided to focus its efforts on a back-up program. The main objectives were as follows: a) modify the core structure of DNDI-VL-2098 (nitroimidazooxazole) to encompass potential safety liabilities; b) improve pharmacological properties; c) decrease hERG inhibition potential and d) show efficacy in in vivo models. To address these objectives, it was decided to concentrate on substituted nitroimidazooxazine analogues. High-throughput screening of a library of nitroimidazooxazines at IPK (Institut Pasteur Korea) led to a number of hits from two subseries that were progressed into lead optimization. Medicinal chemistry work on those hit series was mainly concentrated on side chain variations, and particularly on incorporating new heteroaryl systems designed to improve the overall pharmacological profile of the current lead. To date, around 230 compounds were synthesized and screened in vitro at CSIR-CDRI against the intramacrophagic amastigote stage of the parasite (VL) expressing luciférase firefly reporter gene. From these, 190 compounds showed IC50 < 1 µM and more than 60 compounds had IC50 < 100 nM. Selected compounds with excellent in vitro potential (potency as well as microsomal stability) were evaluated in vivo. Those compounds with the most encouraging efficacies in both mouse and hamster models of VL are currently being progressed as back-up candidates. Additional studies are in progress to further characterize these and to select a back-up compound with the best chance of becoming a successful clinical candidate.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1392] P 643 - DEVELOPMENT OF FUNCTIONAL AND MORE COMPLEX ASSAY IN VITRO BY FLOW CYTOMETRY THAT ENABLES SIMULTANEOUS ASSESSMENT OF POTENTIAL ANTI-LEISHMANIA COMPOUNDS AND THEIR CYTOTOXICITY BANDEIRA, C.1; PERUHYPE-MAGALHÃES, V.2; SOUZA-FAGUNDES, E.M.1; COSTA-SILVA, M.F.2; TEIXEIRA-CARVALHO, A.2; MARTINS-FILHO, O.A.3; PIETRA, R.S.4; SCIO, E.5; ALVES, R.J.4 1.INSTITUTO DE CIÊNCIAS BIOLÓGICAS UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL; 2.CENTRO DE PESQUISAS RENÉ RACHOU, FUNDAÇÃO OSWALDO CRUZ, BELO HORIZONTE, MG, BRAZIL; 3.B. CENTRO DE PESQUISAS RENÉ RACHOU, FUNDAÇÃO OSWALDO CRUZ, BELO HORIZONTE, MG, BRAZIL; 4.FACULDADE DE FARMÁCIA UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL; 5.INSTITUTO DE CIÊNCIAS BIOLÓGICAS, UNIVERSIDADE FEDERAL DE JUIZ DE FORA, BELO HORIZONTE, MG, BRAZIL.
Keyword:l. (v.) braziliensis; phagocytosis; flow cytometry
Abstract: Therapeutic options for the treatment of leishmaniasis are insufficient and have problems such as low efficiency, high toxicity and the emergence of resistant strains. This scenario is compounded by the limited number and lack of innovation in development programs in the area of new drugs for neglected diseases. In this context, the process of discovery and development of pharmaceuticals, biological assays are essential for the identification of bioactive compounds. The tests used for the screening of drugs with cytotoxic activity for protozoa of the genus Leishmania, include different processes as the use of intact parasite (free or intracellular) or
specific enzymes of metabolism as a target cell. These assays allows the screening of large numbers of samples, and those that present activity, are then subjected to secondary confirmatory assays to certify the observed activity and its toxicity, by methodologies with more complexes parameters. In the present work, we describe the development of a new functional and more complex assay that enables simultaneous assessment of potential anti-Leishmania compounds and their cytotoxicity. This method comprises the evaluation of the internalization of L. (V.) braziliensis promastigote form labeled with FITC by human peripheral blood monocytes and cytoxicity by simultaneous analysis by flow cytometry. Was established the conditions for labeling of parasites with the fluorochrome to achieve greater efficiency of phagocytosis by setting the ratio of number of parasites per cell as 1 to 2, at incubation time of 6 hours. The cytotoxicity assessment was performed by the quantification of cells undergoing apoptosis early, late and necrosis. The reagent 7AAD was used for analysis of late apoptosis and necrosis. The evaluation of early apoptosis by staining with Annexin-V. Hemolysis was additional parameter to test cytotoxicity. Two drugs used on clinic (Amphotericin-B and Glucantime®) were evaluated in this new methodology and their leishmanicidal activity as well as immunotoxicity could be detected. This methodological innovation has proven to be a test more complex, more sensitive and quantitative. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1402] P 644 - EVALUATION OF BIOLOGIC ACTIVITY OF DICTYOTA MERTENSII AGAINST LEISHMANIA AMAZONENSIS DOS ANJOS, K.F.L.1; ALIANÇA, A.S.S.1; BORGES, A.1; HIGINO, T.M.M.1; BIANCO, E.M.2; FIGUEIREDO, R.C.B.Q.1 1.CENTRO DE PESQUISAS AGGEU MAGALHÃES, RECIFE, PE, BRAZIL; 2.UNIVERSIDADE FEDERAL DE SANTA CATARINA, FLORIANÓPOLIS, SC, BRAZIL.
Keyword:leishmania amazonensis; dyctiota mertensii; seaweed
Abstract: Seaweeds are considered as source of bioactive metabolites characterized by a broad spectrum of biological activities including antiprotozoal activity. In this work we evaluated the biologic activity of organic extract from Dictyota mertensii (DME) on Leishmania amazonensis as well as its cytotoxic potential against mammalian cells. The concentration of DME extract necessary to inhibit 50% of the promastigote and amastigote growth was determined by cell counting after 72 and 18 hours of incubation with DME respectively. The cytotoxicity was estimated by MTT colorimetric assay and the 50% cytotoxic concentration (CC50) was determined by regression analysis. The selective index was determined as the rate of CC50 to IC50 values. Our results demonstrated that DME strongly inhibited the growth of promastigote and presented low cytotoxicity to mammalian cells as determined by the selective index, which was higher than 3.0. The extract also inhibited the survival of amastigote forms inside the macrophages with IC50 of about 35.8 g/mL. At the higher concentrations tested DME completely abolished the macrophage infection. To elucidate whether the inhibition of macrophage infection could be related to an increased nitric oxide (NO) production by these cells, control and treatedmacrophages were evaluated for nitrite by Griess reagent. The increasing of NO production found in cells treated with DME suggests an immunomodulatory effect of this extract on macrophages. In order to investigate the putative intracellular target of DME, treated or untreated promastigote forms were submitted to propidium iodide and Rhodamin 123 staining and evaluated by confocal microscopy and cytometry. The slight increase of PI+ parasites treated with DME suggested that necrosis was not the main mechanism of cell death induced by DME treatment. On the other hand, DME caused strong decrease of mitochondrial membrane potential as demonstrated by confocal microscopy and cytometry. Taken together our results suggested the DME as promissory agent against Leishmaniasis.
Supported by CPqAM/FIOCRUZ/FACEPE
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1407] P 645 - SEROREACTIVITY TO LEISHMANIA RK39 RAPID TEST IN HIV PATIENTS COSTA, C.H.N.1; LIMA, S.S.1; SANTOS, C.S.2; PEREIRA, T.J.C.F.2; COSTA, D.L.1 1.FEDERAL UNIVERSITY OF PIAUÍ, TERESINA, PI, BRAZIL; 2.INSTITUTO DE DOENÇAS TROPICAIS NATAN PORTELA, TERESINA, PI, BRAZIL.
Keyword:rapid test; hiv; reactivity
Abstract: Introduction: The coinfection HIV / Leishmania sp is a new event in several regions of the world with prospects of progressive impact in the coming years due to geographic overlap of the two infections. Morbidity for each disease is increased in coinfected patients, the chance of relapse of leishmaniasis is higher in patients with HIV and the cure rate of Kala-azar is significantly lower in this population. This scenario is even worse because the diagnosis of Leishmania infection remains a challenge Patients and Methods: A case-control study was carried in Teresina, Piauí, Brazil. Cases were HIV infected patients and controls were HIV-free patients admitted in the same hospital in the same period. The rK39 rapid test for leishmaniasis (Kala- azar Detect Rapid Test®) was applied to all patients. Results were confronted with parasitological diagnosis of leishmaniasis. Statistical analysis was performed with Stata/SE10.0. A significance level of 95% was accepted Results: A total of 28 cases and 13 controls were included. 3/13 HIV-free patients tested reagent for rK39; 2/28 HIV patients had a parasitological diagnosis of leishmaniasis (two visceral leishmaniasis and one cutaneous leishmaniasis) but no HIV patient tested reagent for leishmaniasis (p = 0.008) Conclusion: The rK39 reactivity is lower in immunocompromised patients than in immunocompetent ones. The diagnosis of Leishmania infection in HIV positive patients may not rely solely on serological tests.
- RESERVOIRS [29] P 646 - TOLL LIKE RECEPTORS, NOVEL CYTOKINES AND FOXP3 EXPRESSION IN A SUSCEPTIBLE EXPERIMENTAL MODEL OF CANINE LEISHMANIA INFANTUM INFECTION HOSEIN, S.1; RODRIGUEZ-CORTES, A.2; BLAKE, D.1; ALLENSPACH, K.1; ALBEROLA, J.2; SOLANO-GALLEGO, L.2 1.THE ROYAL VETERINARY COLLEGE, LONDON, UNITED KINGDOM; 2.UNIVERSITAT AUTONOMA DE BARCELONA, BARCELONA, SPAIN.
Keyword:tlr; leishmania; canine
Abstract: A wide spectrum of clinical manifestations occurs in canine Leishmania infantum infection as a result of complex interactions between the parasite and host immune responses. Toll-like receptors (TLRs) are essential components of the innate immune system and facilitate the early
detection of infection. The TLR signalling pathway is thought to be one of the first systems to respond against Leishmania infection, although information about TLR expression during canine L. infantum infection is scarce.
The objective of this study was to investigate transcription of TLRs, cytokines and Foxp3 in a susceptible model of canine L. infantum infection. Popliteal lymph node, liver, skin and spleen samples were taken post-mortem from experimentally infected beagles at different stages of infection [Group 1 (n=6); non-infected clinically healthy control dogs; Group 2 (n=24); six months post-infection and Group 3 (n=7); 15 months post-infection]. Transcription of TLR 2, 3, 4 and 9, cytokines (IL17 and IL22) and Foxp3 and a panel of tissue-specific housekeeping genes were assessed by quantitative real-time PCR.
Absolute quantification of lymph node TLR transcription revealed down-regulation in both infected groups when compared to controls for almost all TLRs except for TLR2 which was upregulated when Group 3 was compared with Group 2. Down-regulation was also noted for spleen and liver TLR3 transcripts in infected groups compared to controls. Liver TLR4 mRNA levels were also down-regulated in both infected groups compared to controls. In contrast, TLR4 and TLR3 up-regulation was only encountered in skin in Group 2 when compared to controls. No statistical differences were found between TLR9, IL-17, IL-22, Foxp3 and dog groups in spleen, liver or skin. FoxP3 levels were consistently down regulated in the lymph node in infected groups compared with controls while no statistical differences were found between IL17 and IL-22. These results demonstrate progressive TLR down-regulation in the majority of tissues with L. infantum infection leading to parasite dissemination and disease development. Overall, there appears to be little impact on cytokine and Foxp3 transcription in the majority of tissues studied in agreement with a silent phase needed for the establishment of L. infantum infection in dogs.
- RESERVOIRS [31] P 647 - CLONING AND SEQUENCING OF TH1, TH2 AND TH17 PEROMYSCUS YUCATANICUS CYTOKINES. SOSA-BIBIANO, E.I.1; LORÍA-CERVERA, E.N.1; VILLANUEVA-LIZAMA, L.E.1; VAN WYNSBEGHE, N.1; SCHOUNTZ, T.2; ANDRADE-NARVÁEZ, F.J.1
1.UNIVERSITY OF YUCATAN, MERIDA, MEXICO; 2.UNIVERSITY OF NORTHERN COLORADO, GREELEY, UNITED STATES.
Keyword:peromyscus yucatanicus; leishmania (leishmania) mexicana; cytokines
Abstract: Yucatan deer mouse (Peromyscus yucatanicus) is the main reservoir of Leishmania (Leishmania) mexicana, the etiologic agent of cutaneous leishmaniasis (CL) in the Yucatan peninsula of Mexico. We have demonstrated that P. yucatanicus reproduces both clinical and subclinical infections similar to those of humans as well as production of nitric oxide in response to L. (L.) mexicana infection. Therefore, we proposed the possibility to employ this wild rodent as a novel experimental model to study CL caused by this parasite. Nevertheless, the specific reagents to study the immune response in P. yucatanicus infected by L. (L.) mexicana are lacking. To address this issue, we used P. maniculatus primers to identify and analyze the sequences of Th1 (IL-12, IFN- , TNF- ), Th2 (IL-4, IL-10, TGF- ) and Th17 (IL-
17) cytokine partial genes of P. yucatanicus. These cytokines were amplified by RT-PCR; partial cDNAs were cloned into pCR 2.1 vector and sequenced. The resulting nucleotide sequences and predicted polypeptides were identified by BLAST against their orthologous genes from other mammal species. All of P. yucatanicus genes with the expected size were amplified using P. maniculatus primers. Most of these cytokine genes were highly conserved with orthologous sequences and functional domains were identified in each amino acid predicted sequences. These data would enable to quantify and analyze the expression of cytokines involved in the P. yucatanicus immune response to L. (L.) mexicana infection. It will also permit us to study the immune pathways leading to the clinical and subclinical infection in Yucatan deer mice and compare with the immune response observed in humans. - EPIDEMIOLOGY [37] P 648 - INFLUENCE OF TREATMENT ON THE DEVELOPMENT OF LEISHMANIASIS RECIDIVANS CUTIS: A CASE-CONTROL STUDY. GOMES, C.M.1; CESETTI, M.V.1; DE MORAIS, O.O.1; MENDES, M.S.T.1; ROSELINO, A.M.F.2; RIBEIRO SAMPAIO, R.N.1 1.UNIVERSIDADE DE BRASÍLIA, BRASÍLIA, DF, BRAZIL; 2.UNIVERSIDADE DE SÃO PAULO, RIBEIRÃO PRETO, SP, BRAZIL.
Keyword:leishmaniasis, cutaneous; case-control studies; risk factors
Abstract: In the Americas, the recurrence of American cutaneous leishmaniasis (ACL) after long-term cure is often called leishmaniasis recidivans cutis (LRC) and is considered an unusual form of ACL. This case-control study aimed to estimate the risk factors related to the clinical presentation and treatment of primary ACL and the development of LRC. The analysis included 105 patients, 82 (78%) of whom had been cured for more than 5 years, and 23 (22%) of whom were in the LRC case group. The odds ratio (OR) for the cured patients who underwent Nmethylglucamine treatment, the treatment of choice, was 2.94 compared with patients who did not receive such treatment (95% confidence interval [CI] = 1.10-7.77). Patients who underwent what was considered adequate treatment with N-methylglucamine and second-choice drugs had a 2.12-fold greater chance of cure compared with patients who underwent treatment with inadequate doses and duration (95% CI = 0.74-6.04). Multivariate analysis that included only patients who used N-methylglucamine showed an OR of 6.02 for the chance of cure compared with patients who received the treatment of choice (90% CI 1.50-27.16, p = 0.03). We concluded that the use of pentavalent antimony (PA) remains the primary technique for treating ACL and avoiding long-term recurrence. Although other drugs have shown promising results in some studies, more scientific evidence is needed to ascertain their true equivalence to PA. - VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [38] P 649 - CATALOGUE OF TYPE MATERIAL OF PHLEBOTOMINAE (DIPTERA: PSYCHODIDAE) IN THE COLLECTION OF INSTITUTO EVANDRO CHAGAS, BRAZIL PINHEIRO, M.S.B.1; DOS SANTOS, T.V.1; DE ANDRADE, A.J.2 1.INSTITUTO EVANDRO CHAGAS, ANANINDEUA, PA, BRAZIL; 2.UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL.
Keyword:type material; phlebotominae; collection
Abstract: Biologic collections are important font of information for researchers of all area of knowledge, such as medical and veterinary entomology. Phlebotomine sand flies play a important tool in the transmission of leishmaniasis and a total of 54 valid species were described since 1941 to 1986 for researchers directly related to Instituto Evandro Chagas, Brazil. Unfortunately the type
material of most of these described species seems to be lost. In ways to facilitate the search of types for entomologists, the present study aims to catalog the type material of Phlebotominae in the collection of Instituto Evandro Chagas. Active search for glass slides stored in boxes were taken after to check original descriptions and additional bibliographic material. After screening and comparison with original drawings, the types were identified and reorganized in separated boxes with red labels to holoypes/ alotypes; and green labels to paratypes/ syntypes. It was catalogued a total of 103 type specimens belonging 13 species as follow: Psychodopygus lainsoni (2 males syntypes), Psychodopygus leonidasdeanei (male holotype, female alotype and 45 females paratypes), Psychodopygus llanosmartinsi (2 females paratypes), Psychodopygus wellcomei (1 male and 4 females syntypes), Pintomyia gruta (1 male and 2 females paratypes), Nyssomyia richardwardi (6 females paratypes), Nyssomyia shawi (9 males and 25 females paratypes), Nyssomyia umbratilis (female alotype and 1 female paratype), Nyssomyia yuilli yuilli (1 male and 1 female paratypes), Trichophoromyia brachipyga (1 male paratype), Trichophoromyia readyi (male holotype, female alotype and 1 male paratype). An image database and digitalization of information was performed, and this last is available at http://splink.cria.org.br. Financial Support: IEC, FAPESP.
- VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [47] P 650 - LEISHMANIA (VIANNIA) BRAZILIENSIS DETECTION IN ANIMALS AND THEIR ECTOPARASITES BY PCR AND REAL-TIME PCR DE MORAIS, R.C.S.; GONÇALVES, S.C.; E SILVA, R.P.; COSTA, P.L.; SILVA, K.G.; SILVA, J.F.; BRANDÃO-FILHO, S.P.; DANTAS-TORRES, F.; DE PAIVA-CAVALCANTI, M. AGGEU MAGALHÃES RESEARCH CENTER, RECIFE, PE, BRAZIL.
Keyword:leishmania (viannia) braziliensis ; ectoparasites; domestic animals
Abstract: American cutaneous leishmaniasis (ACL) is a parasitic disease caused by different species of protozoa of the genus Leishmania (Kinetoplastida: Trypanosomatidae). This disease occurs in the Americas, and it is transmitted through the bites of phlebotomine sand flies of the genus Lutzomyia, with vector species varying from region to region. In Brazil, the ACL has been increasing since 1987 and it is considered a serious public health problem, with autochthonous cases in all Brazilian regions, being Leishmania (Viannia) braziliensis the main species detected in humans and found in all endemic regions of the country. In this study, two rural areas in Pernambuco, northeastern Brazil, were selected. Genomic DNA was extracted from blood samples of dogs and cats and ectoparasites (ticks, fleas, and lice) using commercially available DNA extraction kits. The eluted DNA was tested using a conventional PCR and a quantitative real time PCR. A total of 280 blood samples were collected from animals (five cats and 275 dogs) and 117 ectoparasites only from dogs. In total, 255 (91.07%) animals were positive for detection of L. (V.) braziliensis, being four (80%) of the cats and 251 (91.27%) of the dogs, with mean parasite loads of 1.08 fg/µL and 17,384.56 fg/µL, respectively. In relation to ectoparasites, 51 (43.60%) of them were positive for L. (V.) braziliensis, with a mean parasite loads 14.14 fg/µL. The high mean parasite load found in dogs assessed in this study (17,384.56 fg/µL) was remarkable. Further studies to better understand the role of dogs in the epidemiology of ACL, particularly in situations of domestic transmission, are necessary.
- VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [53] P 651 - BLOOD MEAL IDENTIFICATION IN WILD CAUGHT BLOOD FED PHLEBOTOMUS ARGENTIPES (DIPTERA: PSYCHODIDAE) USING CYTOCHROME B PCR AND REVERSE LINE BLOTTING IN BIHAR, INDIA. GARLAPATI, R.B.; POCHE, D.; POCHE, R. GENESIS LABORATORIES, WELLINGTON, UNITED STATES.
Keyword:blood meal of sand flies; cyt b ; reverse line blot
Abstract: Blood meal identification in vectors plays an important role in epidemiological studies by determining the host preferences of wild sand flies in natural habitat. The anthropophilic index is a crucial component in vector borne diseases like visceral leishmaniasis. Blood meal analysis can identify the reservoir hosts of vector borne diseases. The amplification of the mitochondrial cytochrome b gene followed by reverse line blot analysis helps to identify the blood meal ingested by the wild caught sand flies. A clear understanding of the host preferences and their vectorial capacity for disease agents plays an important role in developing efficient control strategies for vector borne diseases. In the current study, blood fed sand flies were collected from four different villages in Bihar, India by using CDC traps with incandescent light. Traps were placed in five different sites in the villages. In one village traps ta were set on top of the palm trees. Whole DNA was extracted from the blood fed sand flies and was amplified for the cytochrome b region, followed by reverse line blot analysis. Human, cow, buffalo and goat were the major blood meals identified followed by chicken. In some of the blood fed sand flies multiple blood meals were identified. - VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [61] P 652 - ECOLOGICAL ASPECTS OF THE SANDFLY FAUNA (DIPTERA, PSYCHODIDAE) IN AN AMERICAN CUTANEOUS LEISHMANIASIS ENDEMIC AREA UNDER THE INFLUENCE OF HYDROELECTRIC PLANTS IN PARANAPANEMA RIVER, STATE OF PARANÁ, BRAZIL ROSA CRUZ, M.F.1; GALATI, E.B.2; ROSA CRUZ, C.F.1; MARQUEZ, E.S.3 1.UNIVERSIDADE ESTADUAL DO NORTE DO PR, BANDEIRANTES, PR, BRAZIL; 2.FACULDADE SAUDE PUBLICA USP, SAO PAULO, SP, BRAZIL; 3.UNIVERSIDADE ESTADUAL NO NORTE DO PR, BANDEIRANTES, PR, BRAZIL.
Keyword:ecological aspects. ; flebotomine sandflies; vectors
Abstract: INTRODUCTION: An epidemiological study was undertaken to identify determinant factors in the occurrence of American cutaneous leishmaniasis in areas under the influence of hydroelectric plants in Paranapanema river, State of Paraná, Brazil. The ecological aspects of the phlebotomine fauna were investigated. METHODS: Sandflies were sampled with automatic light traps from February 2004 to June 2006 at 25 sites in the urban and rural areas of Itambaracá, and in Porto Almeida and São Joaquim do Pontal. RESULTS: A total of 3,187 sandflies of 15 species were captured. Nyssomyia neivai predominated (34.4%), followed by Pintomyia pessoai (32.6%), Migonemyia migonei (11.6%), Nyssomyia whitmani (8.8%), and Pintomyia fischeri (2.7%), all implicated in the transmission of Leishmania. Males predominated for Ny. neivai, and females for the other vector species, with significant statistical differences (p < 0.001). Nyssomyia neivai, Pi. pessoai, Ny. whitmani, Brumptomyia brumpti, Mg. migonei, and Pi. fischeri presented the highest values for the
Standardized Species Abundance Index (SSAI). The highest frequencies and diversities were found in the preserved forest in Porto Almeida, followed by forests with degradation in São Joaquim do Pontal and Vila Rural. CONCLUSIONS: Sandflies were captured in all localities, with the five vectors predominating. Ny. neivai had its highest frequencies in nearby peridomestic environments and Pi. pessoai in areas of preserved forests. The highest SSAI values of Ny. neivai and Pi. pessoai reflect their wider dispersion and higher frequencies compared with other species, which seems to indicate that these two species may be transmitting leishmaniasis in the area.
- EPIDEMIOLOGY [70] P 653 - AMERICAN TEGUMENTARY LEISHMANIASIS (LTA) IN THE MUNICIPALITY OF BANDEIRANTES-PARANÁ BETWEEN 2000 AND 2009 ROSA CRUZ, M.F.1; ROSA CRUZ, C.F.1; GALATI, E.B.2; MARQUEZ, E.S.1 1.UNIVERSIDADE ESTADUAL DO NORTE DO PR, BANDEIRANTES, PR, BRAZIL; 2.FACULDADE SAUDE PUBLICA USP SP, SAO PAULO, SP, BRAZIL.
Keyword:american cutaneous leishmaniasis ; flebotomine sandflies; epidemiology
Abstract: Introduction: in the State of Paraná the American cutaneous leishmaniasis (ACL) is endemic, occurring in 276 of its 399 municipalities. Objectives: to describe the occurrence of the LTA in the municipality of Bandeirantes between 2000 and 2009, to report aspects of clinical and therapeutic forms, identify the species of Leishmania rolling in the human population and the species of sandflies envolved in disease transmission in the municipality. Method: retrospective and descriptive study of the secondary backing ACL ecological group of insects and vectors of Leishmania. We used data of epidemiological investigation of LTA. For the identification of Leishmania sp, tissue samples were used from the patients with suspicion of ACL and subjected to polymerase chain reaction (PCR). For Entomological analysis were performed with automatic light traps, during one year, every two weeks in 11 urban zones, peridomiciliary points and monthly in 12 rural points, Shannon trap with modified pickups at one point of the countryside, in three seasons. Results: you can highlight a moment epidemic in 2007, when the incidence was 1.038 cases/1,000 inhabitants/year. The distribution of cases in space occurred unevenly, with higher concentrations in the urban area. Of the 123 ACL notifications, 61.79% were female. The predominant age group was 60 years, in both sexes. There was a predominance of domestic activity of sandflies (40.65%), skin clinic (95.94%), the drug (Pentavalent Antimonial was the most widely used drug (98.37%). Leishmania (Viannia) braziliensis was identified by PCR in three patients of LTA. In the research of vectors in 24 points sampled with the CDC and Shannon traps, captured a total of 5.652 sandfly (51.66% male) belonging to 17 species. Nyssomia neivai predominated (47.36%), followed by Nyssomia whitmani (35.77%). Conclusion: Socio-economic data show the ACL affecting predominantly women, suggesting that transmission of Leishmania major likely vectors, Nyssomia neivai and Ny. whitmani, is occurring in domiciliary and peridomiciliary areas. These areas, whose attraction to the sandfly can increase the risk of acquiring the disease by residents. - VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [71] P 654 - LABORATORY AND FIELD EVALUATION OF AN IMIDACLOPRID TREATED RODENT BAIT FOR FEED THROUGH AND SYSTEMIC CONTROL OF PHLEBOTOMUS PAPATASI SCOPOLI (DIPETRA: PSYCHODIDAE) ZHIOUA, E.1; DERBALI, M.1; CHELBI, I.1; CHERNI, S.1; BARHOUMI, W.1; BOUJAAMA, A.2; RABAN, R.3; POCHÉ, R.4
1.PASTEUR INSTITUTE OF TUNIS, TUNIS, TUNISIA; 2.NATIONAL INSTITUTE OF STATISTICS, TUNIS, TUNISIA; 3.GENESIS LABORATORIES INC., WELLIGNTON, UNITED STATES; 4.GENESIS LABORATORIES INC;, WELLIGNTON, TUNISIA.
Keyword:zoonotic cutaneous leishmaniasis; phlebotomus papatasi; systemic and feed through insecticide
Abstract: The objective of this study was to evaluate an imidacloprid-treated rodent oral bait for its systemic and feed through insecticidal activity against Phlebotomus papatasi Scopoli, 1786 vector of Leishmania major Yakimoff & Schokhor, 1914 (Kinetoplastida: Trypanosomatidae), etiologic agent of zoonotic cutaneous leishmaniasis (ZCL). Meriones shawi Duvernoy, 1842 (Rodentia, Gerbillidae) were treated with imidacloprid-treated bait (0.05%). In the laboratory, effects on adult and larval of Phlebotomus papatasi fed on treated M. shawi and on its faeces were studied. The effectiveness of this approach was tested under field conditions. In the laboratory, 100.0% of P. papatasi were killed within 24 hours after blood feeding on the gerbil, M. shawi treated up to 4 weeks prior with a single application of imidacloprid (0.05 %) bait. In addition, none of the P. papatasi larvae that consumed faeces from M. shawi treated with the imidacloprid bait survived to pupation. In the field, application of the imidacloprid bait resulted in a 90.0% reduction in the P. papatasi population up to 4 weeks prior with a single application of imidacloprid (0.05 %) bait. This is the first study to demonstrate field efficacy of insecticidetreated rodent baits for P. papatasi control and the first study to evaluate this approach on M. shawi, a principal ZCL reservoir host. These results suggest that insecticide-treated rodent baits could be used to effectively reduce the populations of P. papatasi associated with M. shawi in ZCL endemic areas. - EPIDEMIOLOGY [78] P 655 - DIAGNOSIS OF CANINE LEISHMANIASIS IN SOUTH OF BAHIA CARVALHO, F.S.; GROSS, E.; WENCESLAU, A.A.; OLIVEIRA, H.C.; SANTOS, I.A. UNIVERSIDADE ESTADUAL DE SANTA CRUZ, ILHÉUS, BA, BRAZIL.
Keyword:tegumentary leishmaniasis; zoonosis; ilhéus
Abstract: Leishmaniasis is a zoonotic disease known worldwide, and affects million people each year. The main etiologic agents of canine leishimaniasis in Brazil are Leishmania infantum (visceral form), and Leishmania braziliensis (cutaneous form), which are transmitted by the bite of the diptera vector belonging to species Phlebotomus and Lutzomyia. In the country there are records about the appearing news sources of disease beyond of the expansion of cases in urban areas. In Northeast gathers almost half of cases occurred in the country and the State of Bahia is an important endemic region for disease. The epidemiologic information obtained from blood sample of dogs revealed prevalence average of 24% of positive animals. Although this information, the south of Bahia not presents epidemiologic information of disease in dogs, although there are many cases of tegumentary leishmaniasis in human. Therefore, the objective was to research occurrences of canine leishmaniasis in animals of south of Bahia utilizing serological and molecular techniques. To accomplish this research were utilized 25 blood samples, the skin of animals of Zoonosis Control Center (CCZ) were scraped in cities Ilhéus and Itabuna. After to realize clinical tests was collected 10 mL of blood of jugular vein, to hematologic evaluation, to extract of DNA, and obtaining of serum. The genomic DNA was stracted with phenol:chloroform:isoamyl alcohol. The serologic diagnosis was realized through of reaction of indirect immunofluorescence (kit IFI-LVC Bio-Manguinhos®). The molecular diagnosis was realized with the primers (forward) 5’CTTTTCTGGTCCCGCGGGTAGG3’ e (reverse) 5’CCACCTGGCCTATTTTACACCA3’ e (forward)
5’GGGGTTGGTGTAATATAGTGG3’ e (reverse) 5’CTAATTGTGCACGGGGAGG3’, to L. infantum e L. braziliensis respectively. Clinicaly 13 (52%) of the animals presents cutaneous alteration that can be related to American tegumantary leishmaniasis (LTA), although the skin scraped and direct tests of the hair revealed the presence of Demodex canis scabies and fungics spores in 4 (16%) and 5 (20%) animals, respectively. 16 (64%) of the animals presented anemia and 14 (56%) thrombocytopenia. In serodiagnosis 2 (8%) were seropositive. The molecular diagnosis revealed one 1 (4%) and 10 (40%) of animals positive for L. infantum and L. braziliensis respectively. The characteristic geo-climatic regions associated vector presence, may justify the results. Positive dogs in urban environment presents risk to regional population. - OTHER SPECIAL TOPICS [86] P 656 - EMERGENCE OF VISCERAL AND CUTANEOUS LEISHMANIASIS: OUTCOME OF URBANIZATION AND AGRICULTURAL DEVELOPMENT AOUN, K.; BOURATBINE, A. RESEARCH LAB LR 11-IPT-06 “MEDICAL PARASITOLOGY, BIOTECHNOLOGY & BIOMOLECULES”, INSTITUT PASTEUR, TUNIS, TUNISIA.
Keyword:leishmaniasis emergence; urbanization; agricultural development
Abstract: Visceral and Cutaneous Leishmaniasis (VL & CL) have emerged in these last two decades with an increase of the incidence of cases and a spread of their geographical distribution. Such epidemiological situation is largely related to environmental changes auspicious to Leishmania transmission. Unstructured urbanization and agricultural development seem to be the most important factors. The extension of the cities’ size, as well as the establishment of new villages, sometimes occur at the expense of zoonotic biotopes, such as chenopods areas and forests infested with wild reservoirs of L. major and L. braziliensis respectively. This introduces man in active foci, increasing his exposure to parasites. Moreover, such anarchic urbanization usually offers household designs and habitat conditions suitable for sandflies vectors and reservoir hosts (dog, rodents). Urbanization also promotes the migration of non immune susceptible populations with dramatic burden as observed in Kabul (Afghanistan) counting for thousands of CL cases in 2003. The huge mobilization of water resources started in the 80’s in North African and Middle Eastern countries has permitted the extension of irrigated areas and thereby the development of agriculture in arid regions. As a consequence, Meriones reservoir has swarmed, inducing many epidemics of Zoonotic CL. On the other hand, new rural settlements with animals breeding and dogs also favored L. infantum life cycle leading to the emergence of zoonotic VL. Much data is still missing concerning the complex Leishmania life cycles, their risk factors and their interactions. The knowledge of such parameters, including those environmental, using multidisciplinary and integrated approaches is crucial for a better management and control of Leishmaniasis. - EPIDEMIOLOGY [89] P 657 - : CLINICAL COMPLICATIONS OF VISCERAL LEISHMANIASIS IN CHILDREN ADMITTED TO A PEDIATRIC HOSPITAL DOS SANTOS, N.F.B.; TORRES, O.D.S. UCSAL, SALVADOR, BA, BRAZIL.
Keyword:viceral leishmaniasis; parasitology; pediatrics
Abstract: Leishmaniasis is a parasitic disease caused by the flagellate protozoan Leishmania. It is a systemic infectious disease characterized by a chronic irregular fever, hepatomegaly and splenomegaly. It is most common in children, especially children under ten years of age. The clinical picture may include bone marrow, liver and spleen disease. In order to assess the clinical complications of leishmaniasis in children admitted to a referral hospital in Salvador, Bahia, we conducted a retrospective study. This study was based on data collected from medical records of hospitalized patients with visceral leishmaniasis in 2011. Of the twelve charts analyzed, 41.6% belonged to preschool children, aged from two to seven years. Of these, 58.3% were male and 41.6% female. Most cases came from the Salvador microregion. The most common clinical complications in this study were: anemia, opportunistic infections, hemorrhage, renal dysfunction, edema and malnutrition. It was found that the complications of the disease in our cases were similar to those found in the literature. The origin of the patients in this study showed the endemicity of schistosomiasis in areas of the state of Bahia. It also shows how neglected the disease is in the state.
- EPIDEMIOLOGY [98] P 658 - ECOEPIDEMIOLOGY OF LEISHMANIA SPP IN THE ENZOOTIC FORESTED ENVIRONMENT IN TWO REGIONS OF THE STATE OF SÃO PAULO, BRAZIL. STUDY OF A COHORT OF WILD SMALL MAMMALS TANIGUCHI, H.H.1; TANIGUCHI, H.H.1; GALATI, E.A.B.2; ELIAS, C.R.1; LAROSA, R.1; PEREIRA-CHIOCCOLA, V.L.1; SHAW, J.J.3; TOLEZANO, J.E.1
1.INSTITUTO ADOLFO LUTZ, SÃO PAULO, SP, BRAZIL; 2.FACULDADE DE SAÚDE PÚBLICA/USP, SÃO PAULO, SP, BRAZIL; 3.INSTITUTO CIÊNCIAS BIOMÉDICAS/USP, SÃO PAULO, SP, BRAZIL.
Keyword:ecoepidemiology of leishmaniasis; reservoir; vector
Abstract: The ecoepidemiology of Leishmania spp. was investigated in natural forested areas in São Paulo State, Brazil by the capture-release-recapture of small wild mammals, dogs and phlebotomines between 2000-2009. Leishmania infection were detected by clinical observations, direct examination of smears or imprints of aspirates and biopsies, rk39 antibody in whole blood, skin test with antigen of L. braziliensis, inoculation in vivo and in vitro and DNA extraction for molecular tests. After the tissue samples were taken the animals were marked on their ears and released at their capture site. At each recapture all the tests were repeated. Phlebotomines were collected in the same places and in chicken hen by Shannon trap, Disney trap, Falcão modified light trap, Castro aspirator. Of 2,714 small mammals collected 2,048 were recaptures. Trinomys iheringi and Philander opossum were recaptured up to 35 times in Ilhabela and one specimen of Ph. opossum was recaptured 79 months after the first capture. In Itupeva one specimem of Didelphis albiventris was recaptured 15 times and another specimem of D. albiventris was recaptured 35 months after the capture. L. (V.) braziliensis were identified in dogs from Ilhabela and Itupeva, in Rodentia (Trinomys iheringi from Ilhabela, Nectomys squamipes and Lutreolina crassicaudata from Itupeva) and Marsupialia (Di. aurita from Itupeva). L. (L.) amazonensis was isolated in Ilhabela from Tr. iheringi. L. (L.) infantum chagasi, was observed in areas of LTA in Itupeva (Akodon spp, D. marsupialis). In certain areas it was possible to determine concurrent cycles of 2-3 Leishmania species. The results of recapture and the long residence time in the same locations indicate that the circulation and dissemination of Leishmania is closely linked to the existence of these animals territories. Regarding vectors Nyssomyia intermedia and Migonemyia migonei predominated (93.81%) in Ilhabela, as many in forested areas as in
chicken. On the other side, in Itupeva, predominated Ny. neivai (33.06%), Ny. whitmani (24.06%) and Lutzomyia longipalpis (20.62%) in both environments. The presence of Lu. longipalpis requires concern due its role as Leishmania infantum chagasi vector. In this region was described a enzootic cycle of this parasite. The natural Leishmania infection in wild and domestic animals associated with the presence of sandfly vectors indicates the potential risk for human infection and perpetuation of cycles of the parasite.
- VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [101] P 659 - ECOLOGY OF LUTZOMYIA LONGIPALPIS IN AN AREA OF VISCERAL LEISHMANIASIS TRANSMISSION IN NORTH-EASTERN BRAZIL COSTA, P.L.1; TORRES, F.D.1; DA SILVA, F.J.1; GUIMARAES, V.F.2; GAUDÊNCIO, K.3; BRANDAO FILHO, S.P.4
1.CENTRO DE PESQUISAS AGGEU MAGALHAES, RECIFE, PE, BRAZIL; 2.CENTRO DE PESQUISAS AGGEU MAGALHÃES, RECIFE, PE, BRAZIL; 3.CENTRO DE PESQUISAS AGGEU MAGAÇHAES, RECIFE, PE, BRAZIL; 4.CENTRO DE PEQUISAS AGGEU MAGALHAES, RECIFE, PE, BRAZIL.
Keyword:sand flies; phlebotominae; lutzomyia longipalpis
Abstract: Visceral leishmaniasis is a major public health issue in South America, where the disease is rapidly spreading. Changes in ecology and distribution of the principal vector, Lutzomyia longipalpis are among the factors accounting for the increasing incidence of the disease in this region. However, information about the ecology of L. longipalpis is still incipient, which may directly impair the implementation of effective control programs. Herein, the ecology of L. longipalpis was studied in a focus of visceral leishmaniasis in north-eastern Brazil. From August 2009 to August 2010, phlebotomine sand flies were monthly collected in four localities using CDC light traps (~37 per month) and lantern-baited Shannon trap with mouth aspirators. A total of 24,226 phlebotomine sand flies were collected with light traps and 375 with mouth aspirators. The most abundant species was L. longipalpis, representing 97.9% of the specimens collected with light traps and 91.5% with the mouth aspirator. Other species (Lutzomyia evandroi, Lutzomyia lenti and Lutzomyia sallesi) were found in low numbers. Most phlebotomine sand flies (94.6%) were collected at chicken coops and corrals. No significant correlation was found between the monthly abundance of phlebotomine sand flies and the monthly averages of temperature, relative humidity or rainfall. However, interestingly enough, 82.4% of L. longipalpis specimens were collected in months when relative humidity surpassed 75%. This study points out that this vector is well adapted to live in different habitats and to different climate conditions. It also suggests that some north-eastern populations of L. longipalpis may be more xerotolerant than southern populations. Further studies to assess the relationship between microclimate and L. longipalpis density in different Brazilian regions are advised. - VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [109] P 660 - COMPOSITION OF COMMUNITIES OF PHLEBOTOMINES AS AN INDICATOR OF ENVIRONMENTAL CHANGES: INFLUENCE ON THE EPIDEMIOLOGY OF LEISHMANIASES IN PARÁ, BRAZIL GONÇALVES, R.1; SOUSA, G.R.1; COSTA, M.D.2; GARCEZ, L.M.1 1.INSTITUTO EVANDRO CHAGAS, BELÉM, PA, BRAZIL; 2.SECRETARIA DE ESTADO DE SAÚDE PÚBLICA DO PARÁ, SANTARÉM, PA, BRAZIL.
Keyword:communities; ecology; epidemiology
Abstract: Variations in the composition of communities of phlebotomine sand flies in different areas result from environmental changes, which influence the population dynamics of the insects and the epidemiology of both visceral (VL) and tegumentary (TL) leishmaniases. The municipality of Santarém, in the Brazilian state of Pará, lies within the Tapajós river basin. It presented high incidences of both TL and VL (48 and 6 cases per 100 000 habitants. respectively) from 2007 to 2010. We describe the composition of phlebotomine communities in Jatobá (-2,569 -54,861), a locality impacted by ecotourism in the municipality of Santarém. Six CDC light traps were placed in the peri- and intra-domiciliary areas of three houses from 18:00 – 06:00 for three nights and a Shannon trap hung in the surrounding forest from 19:00 – 21:00 for a single night. This sampling regime was carried out on two occasions, once each in the rainy (March) and dry (September) seasons of 2012. Indices of abundance and species richness were calculated (Margalef: DMg; 0-10), as well as of diversity (Shannon: H; 0-10) and evenness (Pielou: J; 0-1) (PAst v.2.03). In the rainy season nine species were captured in the peridomicile, including vectors of both VL (Lutzomyia longipalpis: 63.0%) and TL (Bichromomyia flaviscutellata: 1.2%), and the index values were as follows: DMg = 1.820; H = 1.153 and J = 0.525. Three species were collected indoors, including Lu. longipalpis (48.0%) and the index values were DMg = 0.621; H = 0.833 and J = 0.759. Three species were captured in the forest, two of them being vectors of TL (Psychodopygus complexus = 85.7%; Ps. davisi = 9.5%) and the following index values calculated: DMg = 0.657; H = 0.501; J = 0.456. In the dry season the predominant species among the eight captured in the peridomicile were Nyssomyia antunesi (44.0%,), a probable vector of TL, and Lu. longipalpis (4.0%). The indices were as follows: DMg = 2.427; H = 1.72; and J = 0.782. The predominant species among the four captured indoors was Ny. antunesi (40.0%). This was also captured in the forest, where it comprised 5.0% of a sample containing only two species. The vector species (but not their numbers) varied between the two seasons. Thus Lu. longipalpis (both within and around human habitations), and Ps. complexus (in the surrounding secondary forest), only became predominant during the rainy season, with correspondingly low indices of species richness and diversity reflecting profound changes in the environment. Nyssomyia antunesi tended to predominate in the peridomicile during the dry season, exceeding the numbers of Lu. longipalpis. The consequences of environmental changes on phlebotomine communities should be monitored using ecological indices in longitudinal studies.
- EPIDEMIOLOGY [118] P 661 - RISK FACTOR ANALYSIS IN INDIAN VISCERAL LEISHMANIASIS MULTICASE FAMILIES MISHRA, A. CCMB, HYDERABAD, INDIA, HYDERABAD, INDIA.
Keyword:vl-visceral leishmaniasis; (ugc-srf-fellowship); acknowledgement-bihar state population
Abstract: VL is multifactorial disease where environmental and socio-economic risk factors play their role in outcome of VL. we have conducted a cross-sectional case-control study to identify risk factor
in Indian VL in endemic region of Bihar. This study was conducted in Multicase familes (two or more than two were affected in family), total 136 Case families and 136 matched control families were enrolled (unrelated) for study. Cases selected were VL diagnosed between 2000 to 2009 were eligible. For each cases, we had selected one control (individually matched), with no history of previous VL and matched on caste, sex, age group and same geographical region (same village). All families were subjected to a structured interview for micro environmental factors using a predesigned program. SPSS 16.0 software was used for the analysis. We found no significant associations between Education (Literate/Illiterate), Consumer Item (Radio/Watch/TV/Other), Communication (Telephone/Mobile), Transportation (Cycle / Riksha / Troley / Motorcycle / Car), Source of Water reservoir (Well / Pound/ River/Canal), TreeNumber, Tree-Variety and Landed property. Many micro-environmental factors show significant association in analysis were Cattle number (10 meter Radius from House-hold) in control family (63.2%) case family (86.7%) (p < 0.0001; Fisher’s exact test), cattle variety (Poultry, Goats, Cow/Buffalow and mixed (any two or three variety). (p< 0.0001; Fisher’s exact test), The complex structure of House-hold (Mud, Foos, Mix (brick or brick and bamboo but not mud) and Pakka (Cemented or Plasterd walls) (p < 0.0001; Pearson Chi-Square test), Plantation (Bamboo, Banana, Litchi, Mango etc) near 50 meter from House-Hold for cases (30.9%) and controls (11.8%) ( p < 0.0001; Pearson Chi-Square test), sleeping on floor ( p < 0.0001; Fisher’s exact test), then for Hand-pump present near house-hold (10 meter distance) for cases (63.2%) and controls (50.7%).; p = 0.037; Pearson chi-square test) Further, Monthly income and Landed property reflect economic status of families and is related to poverty and ultimately malnutrition. The groups (cases and controls) were shown to have no association in landed property while there was significant association between the groups for monthly income ( p= 0.018; Pearson Chi-Square test). Preventive measure analysis on bed net show that there was 56.6% case family not using bednet in comparison to 40.4% control families in this study ( p= 0.015;Fisher’s exact test). This is the first study which shows risk factor study in multicase families among the population of endemic region of VL in India. Our data suggest that combination of certain risk factors will lead to maximum cases in a family, while neighbour population always face risk to get the disease. These results will be useful in developing VL control programs for reducing transmission and incidence of this disease.
- VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [119] P 662 - FIRST DETECTION OF LEISHMANIA SIAMENSIS AMONG SAND FLIES AND ANIMAL RESERVOIRS IN THAILAND CHUSRI, S.1; THAMMAPALO, S.2; SILPAPOJAKUL, K.1; SIRIYASATIEN, P.3 1.PRINCE OF SONGKLA UNIVERSITY, SONGKHLA, THAILAND; 2.THE OFFICE OF DISEASE PREVENTION AND CONTROL 12 SONGKHLA, SONGKHLA, THAILAND; 3.CHULALONGKORN UNIVERSITY, BANGKOK, THAILAND.
Keyword:lishmania ; siamensis; vector
Abstract: Background: Leishmania siamensis was documented as the main causative pathogen of an emerging autochthonous leishmaniasis in Thailand. The data of potential vector and reservoir
was still unknown. After confirmed leishmaniasis had been reported, this study was objective to identify the vectors and reservoirs of Leishmania siamensis in affected area. Methodology/Principal Findings: An environmental survey was carried out in Songkhla, the Pacific coast province in southern Thailand. Forty villagers, seventy seven sand flies, and potential animal reservoirs were examined for the presence of L. siamensis by conventional culture and identification PCR product of the ITS1gene. Leishmania DNA amplicon was identified in Sergentomyia sand flies (S.gammea and S. barraudi) and black rats (Rattus rattus). A Phylogenetic tree of confirmed patient, sand flies and black rat fell into single clade and separate from other Leishmania species. Conclusions/Significance: This study demonstrated the potential involvement of R.rattus and Sergentomyia sand flies in transmission of L. siamensis - OTHER SPECIAL TOPICS [124] P 663 - MULTIFACETED LEISHMANIA NETWORKING; THE LEISHRIIP EXPERIENCE VICTOIR, K.1; INSTITUT PASTEUR INTERNATIONAL NETWORK, L.2 1.INSTITUT PASTEUR, PARIS, FRANCE; 2.RIIP, PARIS, FRANCE.
Keyword:networking; multidisciplinar; leishmania
Abstract: The Institut Pasteur International Network consists of 32 Institutes spread over 5 continents. In more than 10 of these institutes, Leishmania research is being performed. Studies range from fundamental research e.g. approaching drug discovery and design by protein crystallography and structure-based hit optimization (IP Montevideo) to more applied research such as the study of the reservoirs in North African countries (IP Algeria, Tunis and Morocco). The strength of the network is its geographical broadness, overarching of the widest variety of Leishmania species, in combination with a high diversity of in-house expertise including epidemiology, entomology, immunology, vaccinology, drug discovery and diagnostics. It is meant to be a core network which is open to collaborating partners in order to become a pool of scientific expertise for Leishmania research. During this session some of the main investigation axes will be presented.
- EPIDEMIOLOGY [126] P 664 - OUTBREAK OF CUTANEOUS LEISHMANIASIS IN INDIGENOUS VILLAGE AT PESQUEIRA, PERNAMBUCO, BRAZIL. SIVINI, M.A.1; SILVA, A.P.2; LEAL-NETO, O.B.1; LIMA, P.M.1; MOREIRA, I.M.1; ASSUNÇÃO, R.S.1 1.SECRETARY OF HEALTH OF PERNAMBUCO, RECIFE, PE, BRAZIL; 2.SPECIAL SECRETARIAT FOR INDIGENOUS HEALTH, MINISTRY OF HEALTH, RECIFE, PE, BRAZIL.
Keyword:epidemiology; indigenous; leishmaniasis
Abstract: The American Cutaneous Leishmaniasis (ACL) today begins a transition characterized by the model of urban occupation of wild spaces and poor housing conditions. On indigenous peoples, despite the culture and tradition cultivated by the tribals, the habits of modern man mingle with
the living habits of the indians bringing inadequate when dealing with sanitary conditions favoring the occurrence of infectious and parasitic diseases such as ACL. The objective was to characterize the outbreak of ACL occurred in the Guarda village, the indigenous territory Xukuru OrorubĂĄ, Pesqueira, Pernambuco, Brazil, in October 2012. This is a descriptive exploratory spatial approach. The population of the village of 311 people was investigated. From a GPS device were constructed computerized templates of georeferenced locality spatialising all elements of interest. A structured questionnaire was applied to cases of ACL to knowledge of environmental and household characteristics of the subjects. The spatial data was in ArcGIS 10.0 environment and the data analyzed using Epi Info version 3.5.1. To identify the phlebotomine fauna light traps were used. We identified 11 cases of ACL, 82% male, showing a 3.5% incidence in the population of the village. The average age was 11.54 (SD Âą 3.20), with concentration of cases in the age group 10-14 years (64%). The Kernel and thematic map showing the distribution of households and cases in the territory. Detected in 100% of the homes the presence of household animals, and 90.9% intended garbage in the open. After entomological classification, we identified the presence of Lutzomyia migonei and Lu. intermedia at home and peridomicile. Held practice of health education, focused on environmental management, cleaning and disposal of trash. Since this is an area where one death has been verified by Visceral Leishmaniasis, human blood samples for serology were conducted in order to diagnose Canine Visceral Leishmaniasis in 100% of dogs and those with reagent results, serum samples were collected to be confirmed with ELISA. Given the above, there is a need to change some habits of the indigenous population in order to decrease the risk of occurrence and spread of the ACL. - CLINICAL LEISHMANIASIS [141] P 665 - GLOBALLY FIRST TIME INTRODUCTION OF LLIN (LONG LASTING INSECTICIDE TREATED NET) FOR ELIMINATION OF KALA AZAR (BOTH VISCERAL & POST KALA-AZAR DERMAL LEISHMANIASIS ) IN HYPER- ENDEMIC SUBDISTRICTS OF BANGLADESH. CHOUDHURY, T.A.
VISCERAL LEISHMANIASIS (KALA-AZAR) ELIMINATION PROGRAM, DIRECTORATE GENERAL OF HEALTH SERVICES, MINI, DHAKA, BANGLADESH.
Keyword:long lasting insecticide treated net for, ; visceral leishmaniasis elimination program; in eight hyper endemic sub-districts of bangladesh
Abstract: Background: Bangladesh along with Nepal and India is committed to Eliminate Kala-azar (Visceral Leishmaniasis) by 2015. Effective Integrated Vector Management (IVM) is one of the main strategies for Kala azar elimination and LLIN (Long Lasting Insecticide treated Net) is one of the tools for this IVM for preventing human- vector contact Objectives: Globally for the first time to implement the Integrated Vector Management strategy through LLINs distribution for Elimination of Visceral Leishmaniasis in Hyper Endemic sub-districts of Bangladesh and thus pave the way for elimination status by 2015 and to examine the effectiveness of LLIN tool in reducing the human- vector (Sand fly) contact, and improving Awareness building in community for helping in Identification of new KA /PKDL cases through Campaign distribution approach. Target groups:
Total 9494 patient both kala-azar and PKDL, registered since 2008 in 8 hyper endemic sub district of Bangladesh had received the nets. Activities: Village-unit approaches were followed: 1. Baseline data collection and selection of sub-district on endemicity criteria. 2. Case identification from 2008 -- August 2012. 3. Development of LLIN distribution strategy and Micro plan. 4. Development of BCC material (pre and post distribution) 5. Active case search and selection of patient through field visit. 6. Advocacy meeting at Sub-district level to create mass awareness on LLINs. 7. Village wise campaign for distribution of LLINs among new and old cases. ----One LLIN for each patient and extra one for his/her family members. 8. Monitoring, Evaluation, Follow- up program- by field managers. Deliverables: • Globally practiced for first time, as a tool for Integrated Vector Management (IVM) for Visceral Leishmaniasis Elimination Program, LLIN distribution was successfully implemented and can be practiced in other countries. • To reduce the transmission of Kala azar from reservoir to Vector, LLIN distribution can play a vital role for PKDL patients. • Lessons learned that LLIN distribution among KA/KDL cases, with prior campaign for community participation can act as Catch-up strategy for new case identification.
- EPIDEMIOLOGY [153] P 666 - NATURAL INFECTION OF LUTZOMYIA LONGIPALPIS BY LEISHMANIA AMAZONENSIS IN THE CITY OF CORRIENTES (ARGENTINA) BORDA, C.E.; STEINDEL, M.; REA, M.J.; CAMPAGNARO, G.; MIÉREZ, M.L. CENTRO NACIONAL DE PARASITOLOGÍA Y ENFERMEDADES TROPICALES (CENPETROP), FACULTAD MEDICINA, UNNE, CORRIENTES, ARGENTINA.
Keyword: natural infection ; leishmania amazonensis; corrientes (argentina)
Abstract: In the province of Corrientes, located in the Northeast region of Argentina, important foci of American tegumentary leishmaniasis (ATL) have been reported. The Municipality of Corrientes is an endemic zone of human ATL (27°28 00 S, 58°50 00 W).
Information on phlebotomine species distribution is limited in the province and their role as Leishmania vectors has not been elucidated yet. Entomological surveys were performed in different districts of the Province of Corrientes, based on the reported cases of ATL in humans. Six sand flies species are present in the district including Nyssomyia neivai, Migonemyia migonei, Psathyromyia shannoni, Evandromyia cortelezzi, Pintomyia pessoai, Micropygomyia quinquefer and Lutzomyia longipalpis. Considering all the study sites and the number of sand flies, the predominant species in Corrientes city was Lutzomyia longipalpis, followed by Nyssomyia neivai. A total of 2,780 L. longipalpis were collected in one housing in peridomiciliary environment from April to December 2010. The aim of this study was to investigate the natural infection and the molecular identification of Leishmania in this city. Groups of 10 sandflies were subjected to DNA extraction by standard phenol-chloroform method. For parasite detection, a 120 bp fragment of Leishmania kDNA mini-circle was PCR amplified using primers 150 and 152. DNA amplification was performed in an Eppendorf Mastercycler Gradient apparatus using the following thermal conditions: initial denaturation at 94oC for 4 min, 30 cycles of 94oC for 1 min, annealing at 57oC for 1 min, extension at 72oC for 1 min followed by an final extension step at 72oC/5 min. Five µl of the amplified DNA fragment was digested with endonucleases AvaI and HaeIII and subjected to electrophoresis in a 10% polyacrylamide gel, stained with ethidium bromide, visualized under UV light and digitally recorded. For southern blot assays, the amplified DNA fragment was transferred to charged nylon membrane and hybridized overnight at 42oC with L. braziliensis - M2904 and L. amazonensis -LTB0016 k-DNA horseradish peroxidase labelled probes and revealed using an ECL kit. The expected 120 bp kDNA band was detected in two sandfly samples and PCR-RFLP and southern blot analysis confirms the presence of L. amazonensis. This is the first report of L. amazonensis in northeastern of Argentina and the found of naturally infected Lu. longipalpis by L. amazonensis suggest its possible role in the parasite transmission. Considering the high density of phlebotomine in the urban area, there is a trait to transmission of both cutaneous and visceral leishmaniasis in the municipality of Corrientes. Financial support by CNPq - VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [156] P 667 - DISTRIBUTION OF PHLEBOTOMINE FAUNA (DIPTERA: PSYCHODIDAE) ACROSS AN URBAN-RURAL GRADIENT IN AN AREA OF ENDEMIC VISCERAL LEISHMANIASIS IN NORTH INDIA, SPECIAL EMPHASIS ON UTTARANCHAL REGION SINGH, N.S.1; -SINGH, D.P.1; IPE, I.M.2; IPE, A.2; LAL, D.2 1.SANDFLY RESEARCH LABORATORY,LUCK NOW CHRISTIAN COLLEGELUCK NOW CHRISTIAN COLLEGE,, LUCKNOW, INDIA; 2.SCHOOL OF ENTOMOLOGY, ST. JOHN’S COLLEGE, AGRA-282002, INDIA.
Keyword:phlebotomus argentipes; leishmaniasis,; uttaranchal region, india
Abstract: Visceral Leishmaniasis in the Indian sub-continent, also known as Kala-azar, is caused by Leishmania donovani transmitted by Phlebotomus argentipes. This study endeavours to delineate the suitable locations of Phlebotomus argentipes with relation to environmental characteristics between endemic and non-endemic districts in India. The aim of this study was to evaluate the urbanization of VL vectors in North India, special emphasis on Uttaranchal Region where VL is endemic. Sandflies were captured using, light traps along an urban-rural gradient. The traps were installed outside and inside mud houses at a height of 200 cm. A total of 2709 sandflies were collected and 9 species were identified. The predominant species was Sergentomyia punjabaensis (rate of 67.12%) with P. argentipes (0.5%). A total of 1357 females were dissected and no Leishmania infections were detected. Most of the sandflies were captured at the border of a forest (76.37%) and no flies were captured in the urban area, which suggests that transmission is still restricted to rural sites. However, the fact that a specimen was collected in an intermediate area indicates that urbanization is a real possibility and that vector monitoring is important. The present study should be utilized to make critical decisions for vector surveillance and controlling Kala-azar disease vectors. At present in India, Bihar (31 out of 38 districts), West Bengal (10 out of 24 districts), Uttar Pradesh (4 districts), that border Bihar, and a small portion of the Jharkhand district are endemic at varying levels. This study provides the report evaluating the prevalence of Leishmania infection in sand flies in a region endemic for VL Uttaranchal region in India. Sergentomyia species are the most common species of sand fly. Knowledge of the infection rate in female P. argentipes may help in predicting severity of disease and in vector elimination programs. - CLINICAL LEISHMANIASIS [177] P 668 - VISCERALIZATION POTENTIAL OF CUTANEOUS LEISHMANIASIS INFECTIONS IN SRI LANKA: EARLY SEROLOGICAL EVIDENCE SIRIWARDANA, Y.1; RANASINGHE, S.2; SOYZA, P.3; KARUNAWEERA, N.1 1.DEPARTMENT OF PARASITOLOGY, FACULTY OF MEDICINE, UNIVERSITY OF COLOMBO, COLOMBO, SRI-LANKA; 2.MINISTRY OF HEALTH, COLOMBO, SRI-LANKA; 3.DEPARTMENT OF PARASITOLOGY, FACULTY OF MEDICINE, COLOMBO, SRI-LANKA.
Keyword:l.donovani; immunology; cutaneous
Abstract: Sri Lanka is affected by cutaneous leishmaniasis (CL) caused by a genetically distinct variant of L. donovani, the agent of visceral leishmaniasis (VL). However, sero-prevalence of antiLeishmania antibodies among CL patients in Sri Lanka has not been studied. Study performed with the aim of investigating the sero-prevalence of anti-Leishmania antibodies in CL, using an in-house serological assay. An ELISA was developed using local Leishmania antigen and optimized under local conditions. Serum samples from a total of 101 CL patients (presented between years 2001-2008) were tested on the ELISA for the presence of anti-Leishmania antibodies. Sero-positive patients were also tested by rK39 assay. Clinical and socio-demographical parameters were compared with the serological findings. Sero-prevalence of anti-Leishmania antibodies was 14.85% (n=15/101) in the study group and 2/15 were also found to be rK39 positive. CL cases reported from multiple regions demonstrated anti-Leishmania antibodies (Southern: 53.3%, n=8/15, Northern: 13.3%, n=2/15 and other provinces: 33.3% n=5/15). A higher proportion of females (22.7%, n=5/22) as compared to males (13.5%, n=10/74) and patients < 20 years, (23.5%, n=4/17) as compared to older age groups (13.0% n=11/84) showed a higher sero-conversion rate (SCR). Also, a higher proportion of single lesions (16.9%, n=11/65) as
compared to multiple lesions ( 8.3%, n=1/12), lesions within 7-9 months duration (30%, n=3/10) as compared to other groups were associated with higher SCRs. In spite of small lesions (<1cm) (35.0%, n=7/20) having a higher SCR, ulcerating nodules had the highest SCR (22.7% n=10/44). This study provides early evidence for the ability of local parasite L.donovani to evoke a systemic immune response in some patients. A study is ongoing for careful follow-up of patients to detect time of onset of sero-conversion and possible evidence of visceral involvement. Grants from the National Science Foundation of Sri Lanka (RG/2011/HS/13), WHO/SEARO (55760) and NIAID, NIH, USA(1 R01 AI099602-01) are acknowledged.
- EPIDEMIOLOGY [180] P 669 - TRENDS IN CLINICAL AND EPIDEMIOLOGICAL PATTERNS OF CUTANEOUS LEISHMANIASIS SRI LANKA SIRIWARDANA, Y.1; SIRIMANNA, G.2; KARUNAWEERA, N.1 1.DEPARTMENT OF PARASITOLOGY, FACULTY OF MEDICINE, UNIVERSITY OF COLOMBO, COLOMBO, SRI-LANKA; 2.NATIONAL HOSPITAL, COLOMBO, SRI-LANKA.
Keyword:l.donovani; trends; sri lanka
Abstract: An epidemic of autochthonous cutaneous leishmaniasis (CL) started in Sri Lanka in year 2001 with over 2000 cases passively detected up to date. CL in this focus is caused by an atypical parasite variant belonging to L.donovani complex. However, main clinical picture remains as CL up to date. Trends in clinico-epidemiological characteristics of CL in Sri Lanka were studied. A total of 1422 clinically-suspected, referred cases (1400 CL, 2 mucosal and 20 visceral) were investigated. Microscopy, culture/ PCR on lesion material in CL/ MCL, on bone marrow in VL and rK39 on serum enabled confirmation of 1006 CL, 4VL and 2 MCL patients. (VL and ML discussed in different abstracts). During the early part of the outbreak (2001-3 period) majority of CL cases were males (81%) in 21-40 year group (69%), and were soldiers. In recent years, more children, elderly individuals, females and more civilians have been reported (only 55% males, 38% young adults and 0.7% soldiers in 2010-12). At the time of presentation, more single and early (<3 months) lesions were observed during the recent years (2010-12). Surface squamation (if non-ulcerative) irregular shape with squamation and inflammation in surrounding skin was observed. Case numbers referred from North declined with time while an increasing numbers were detected from South. Proportion of cases from other parts of the country was minimal throughout and also decreased with time. However, biannual seasonal variation of case presentation with 2 peaks in April-June and August-October was observed throughout. Involvement of more females and children indicate possible peridomestication of the transmission cycle. Rapid progression of lesions and associated features might be due to changing levels of host immunity. Better awareness among clinicians and general public might explain the early reporting of cases.Grants from the National Science Foundation of Sri Lanka (RG/2011/HS/13), WHO/SEARO (55760) and NIAID, NIH, USA (1 R01 AI099602-01) are acknowledged.
- EPIDEMIOLOGY [196] P 670 - EARLY PATTERN OF RK39 SERO-PREVALENCE IN A CUTANEOUS LEISHMANIASIS ENDEMIC AREA IN SRI LANKA RANASINGHE, P.S.1; WICKREMASINGHE, R.; MUNASINGHE, A.2; HULANGAMUWA, S.3; SENEVIRATNE, K.4; BANDARA, S.4; ATHAUDA, I.1; NAVARATNE, C.1; SILVA, O.1; WACKWELLA, H.1; MATLASHEWSKI, G.5; WICKREMASINGHE, R.1 1.UNIVERSITY OF SRI JAYEWARDENEPURA, NUGEGODA, SRI-LANKA; 2.REGIONAL EPIDEMIOLOGY UNIT, ANURADHAPURA, SRI-LANKA; 3.DERMATOLOGY UNIT, ANURADHAPURA, SRI-LANKA; 4.OFFICE OF THE MEDICAL OFFICER OF HEALTH, THALAWA, SRI-LANKA; 5.MCGILL UNIVERSITY, MONTREAL, CANADA.
Keyword:leishmania; rk39; seroprevalence
Abstract: Leishmaniasis is a newly emerged and established disease in Sri Lanka. At present more cutaneous leishmaniasis (CL) cases are reported than mucosal (ML) and visceral (VL). The strain isolated from endogenous CL and VL was Leishmania donovani Mon-37. This study was the first to report a cross sectional survey to assess the sero-prevalence of rK39 antibodies in an endemic region for leishmaniasis in Sri Lanka. The study was conducted in Thalawa Medical Officer of Health (MOH) area which reported the first VL case, 3 ML cases and 2nd highest passive CL case detection incidence at present. Participants (n=955) were randomly selected out of an estimated population of 61674. rK39 immunochromatographic test was used to assess the sero-prevalence. Giemsa stained slit skin smear was used to detect amastigotes positive CL lesion. A total of 31 cases of CL were identified in this study of which 21 were previously identified and 10 were new cases giving a prevalence of 3.2% of CL in this area. None of the CL patients were rK39 positive. This indicates the absence of sero-conversion among CL patients. One person was sero positive but did not have clinical features of CL or VL indicating a low VL transmission in this area. The sero-prevalence of rK39 antibodies was 0.1%. Note: The data describe here are under review for a publication.
- EPIDEMIOLOGY [202] P 671 - MULTILOCUS MICROSATELLITE TYPING REVEALS GENETICALLY DISTINCT POPULATIONS WITHIN THE SPECIES LEISHMANIA TROPICA GENERALLY CORRELATING WITH GEOGRAPHICAL ORIGINS KRAYTER, L.1; MALIK, M.D.1; WUTTKE, J.1; AZMI, K.2; SCHNUR, L.F.3; SALOTRA, P.4; SCHÖNIAN, G.1 1.INSTITUTE OF MICROBIOLOGY, CHARITÉ UNIVERSITY MEDICINE BERLIN, BERLIN, GERMANY; 2.AL-QUDS NUTRITION AND HEALTH RESEARCH INSTITUTE, FACULTY OF MEDICINE, AL-QUDS UNIVERSITY, ABU-DEIS, ISRAEL; 3.KUVIN CENTRE FOR THE STUDY OF INFECTIOUS AND TROPICAL DISEASES, HEBREW UNIVERSITY, JERUSALEM, ISRAEL; 4.NATIONAL INSTITUTE OF PATHOLOGY (ICMR), SAFDARJUNG HOSPITAL CAMPUS, NEW DELHI, INDIA.
Keyword:mlmt, population genetics; leishmania tropica; leishmania aethiopica
Abstract: Leishmania tropica is a highly diverse species as shown by biochemical, serological, and molecular techniques, manifestations of disease and geographical distribution. This population genetic study examined variation at 12 hyper-variable microsatellite loci of 178 strains, 173 of L. tropica from different endemic areas and 5 of L. aethiopica. Eighty-eight strains exhibited unique microsatellite profiles that were analysed by different types of population genetic analyses that included Bayesian inference, distance-based and
factorial correspondence analysis, and F statistics. This revealed three populations that generally correlated with strain geographical origins. The first population consisted only of strains from Israel and Palestine and subdivided into at least two subpopulations, showing no geographic correlation. The second population was genetically quite heterogeneous and was comprised of strains from north, east and southern Africa (Morocco, Tunisia, Kenya and Namibia) and a very specific focus in Israel above the northern coast of the Sea of Galilee where these variants of L. tropica are transmitted by Phlebotomus arabicus in contrast to other Israeli and Palestinian foci where P. sergenti transmits L. tropica in the former and assumed to in the latter. The 5 strains of L. aethiopica also belonged to this population, suggesting that, within the context of this study, L. aethiopica might not be a distinctly separate species but a subgroup of L. tropica. The third population comprised strains from many different countries (Iraq, Azerbaijan, Turkey, India, Jordan, Israel, Kenya, Palestine) that divided in two subpopulations, one of which consisted only of strains isolated during an epidemic outbreak of cutaneous leishmaniasis (CL) in Turkey in 1995. Distance-based data analysis also revealed that Indian strains isolated from cases of CL clustered together and were distinct from those from cases of visceral leishmaniasis (VL). - EPIDEMIOLOGY [209] P 672 - IDENTIFICATION OF DIVERGENT LEISHMANIA DONOVANI STRAINS FROM NEPAL MANNAERT, A.1; VANAERSCHOT, M.1; KHANAL, B.2; BHATTARAI, N.2; IMAMURA, H.1; DOWNING, T.3; COTTON, J.4; BERRIMAN, M.4; DUJARDIN, J.1; RIJAL, S.2 1.INSTITUTE OF TROPICAL MEDICINE, ANTWERPEN, BELGIUM; 2.B.P. KOIRALA INSTITUTE OF HEALTH SCIENCES, DHARAN, NEPAL; 3.NATIONAL UNIVERSITY OF IRELAND, GALWAY, IRELAND; 4.WELLCOME TRUST SANGER INSTITUTE, HINXTON, UNITED KINGDOM.
Keyword:genome diversity; epidemiology; evolution
Abstract: The population of Leishmania donovani in the Indian subcontinent is highly homogeneous, to such an extent that only minimal intraspecific variation can be detected with classical genotyping methods. This degree of homogeneity is likely the result of insecticide spraying campaigns under the malaria control program in the 1950s and 1960s, which also affected sandflies and thus the parasites. The parasites underwent a population bottleneck, and the current Indian and Nepalese L. donovani population likely originated by clonal expansion of a few survivors, resulting in nearly identical strains at the sequence level. This is evident from the genome sequences of 196 strains isolated from visceral leishmaniasis (VL) patients from India and Nepal. However, several strains originating from the Hill region of East Nepal, called ‘Yeti strains’, show high sequence divergence and differ at the structural level, as they do not possess the episomes that are present in the other strains. Phylogenetic analyses reveal distinct clades within the group of Yeti strains, indicating that they may represent a relict population that managed to escape the eradication and subsequent bottleneck, possibly due to isolation. We will also present epidemiological and clinical data of these strains, which were collected in the antimonial and miltefosine drug eras. Since this unexpected conservation of genetic diversity in L. donovani can have implications for the treatment of VL patients and the emergence of drug resistance in the Indian subcontinent, we examined the existence of any correlation between genotype of the strains, epidemiology and clinical factors. - EPIDEMIOLOGY
[212] P 673 - THE KNOWLEDGE ABOUT THE ECO-EPIDEMIOLOGY OF CUTANEOUS LEISHMANIASIS (CL) IN THE COMMUNITIES MANOELZINHO AND BAIXÃO DOS JÚLIOS, BARREIRINHAS, MARANHÃO, BRAZIL. FILHO, A.A.P.1; BANDEIRA, M.C.A.2; FONTELES, R.S.2; MORAES, J.L.P.2; DE SOUSA, A.C.2; SILVA, S.O.1; REBÊLO, J.M.M.2; MELO, M.N.1 1.UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL; 2.UNIVERSIDADE FEDERAL DO MARANHÃO, SÃO LUÍS, MA, BRAZIL.
Keyword:communities; eco-epidemiology; knowledge about leishmaniasis
Abstract: The CL is a parasitic disease caused by protozoa of the genus Leishmania. Barreirinhas, located in Maranhão, have the most of the cases of leishmaniasis in the State, about 40%. In this sense, the knowledge of communities affected by this disease provides a base of information that may assist in the eventual control of this disease. This study aimed to perform a diagnosis of knowledge of the people about the CL. Two villages of Barreirinhas were visited, a focusing of active transmission as found in Manoelzinho (MN), and no other case was reported in Baixão dos Julios (BJ), which served as control. Questionnaires were applied on these locations. They contained questions the knowledge in the ecology of sandflies and aspects of the epidemiology, treatment and prevention of the disease. We interviewed the heads of families who agreed and signed a consent form. When asked about the existence of sandflies, the population of the village MN (100%) proved to have greater knowledge than BJ (68.2%). All respondents said they had heard of insect and that it frequents the yard, including livestock shelters, with high occurrence indoors. But also knowing that insects live in the forest, by the river, in their natural environment, being very common during the rainy season. However, it is noteworthy that the population of MN showed greater knowledge about the issues on the vector, most likely, because of the fact that the village is a source of transmission of leishmaniasis, which has been maintained over time. The opposite is observed in BJ where there is no record of this case. On matters of the epidemiological issues of the disease, the population of the MN’s village had higher knowledge (97%) than the population of BJ (54.5%), since the majority of interviewed people said they had heard of leishmaniasis, their symptoms and treatment, as well as the occurrence of the disease in relatives or acquaintances. Although the population have a reasonable level of knowledge about some of the epidemiological aspects of the disease, it appears that the county’s staff of the education on health should develop appropriate strategies and make frequent visits to the communities teaching about the preventive measures and the transmission of the disease. - VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [217] P 674 - COMPOSITION AND ABUNDANCE OF SAND FLIES (PSYCHODIDAE, DIPTERA) IN FOCUS AREAS OF LEISHMANIASIS, BARREIRINHAS, MARANHÃO, BRAZIL. FILHO, A.A.P.1; FONTELES, R.S.2; MORAES, J.L.P.2; DE SOUSA, A.C.2; SILVA, S.O.1; REBÊLO, J.M.M.2; MELO, M.N.1 1.UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL; 2.UNIVERSIDADE FEDERAL DO MARANHÃO, SÃO LUÍS, MA, BRAZIL.
Keyword:sand flies; leishmaniasis; abundance
Abstract: Sand flies belong to the family Psychodidae, subfamily Phlebotominae and are responsible for the transmission of Leishmaniasis. Two aspects contributes to the Maranhão as a endemic area for Cutaneous Leishmaniasis (CL) and Visceral Leishmaniasis (VL): the geographical position (between the Amazon and arid Northeast) and the fact that the State that it’s territory of all
species of sandfly that are considered effective vectors of leishmaniasis, (L. longipalpis, L. flaviscutellata, L. migonei, L. umbratilis, L. wellcomei, L. whitmani). Although several studies were conducted in the areas of transmission of leishmaniasis, works in villages in Barreirinhas are scarce. Therefore this study aimed to know the composition and abundance of sand flies on the villages of Barreirinhas. We used CDC light traps from June to August 2011 in five villages: Olha D’Agua, Palmeira dos Eduardos, Andiroba, Morro Alto, and Mirinzal considered emergency outbreaks of Leishmaniasis. 458 sand flies were captured, 276 males (60.2%) and 182 females (39.7%), belonging to nine species: L. whitmani (65.9%), L. longipalpis (26.2%), L. flaviscutellata (2.4%), L. antunesi (0.8%), L. lenti (0.8%), L. wellcomei (0.4%), L. anduzei (0.4%), L. aragaoi (0.2%), and Lutzomyia sp. (2.2%). The latter corresponds to morphospecies with lost characters, disallowing identification at species level. L. whitmani was the most abundant, present in all locations. This represents an important aspect of this species that is recognized as a vector of LT. L. longipalpis was second in abundance and corresponds to one of the main vectors in the region of VL. The presence of others vectors species (L. flaviscutellata, L. wellcomei, L. antunesi) reveals the need for constant epidemiological and entomological surveillance in the area. Thus, this study demonstrated the existence of diverse vectors species of Leishmania, which indicates the need for vector control measures in the area in order to decrease the incidence of CL and VL in these localities. - VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [221] P 675 - SAND FLIES (DIPTERA, PSYCHODIDAE): DISTRIBUTION IN DIFFERENT PHYTOGEOGRAPHIC REGIONS OF THE STATE OF MARANHÃO (MA), BRAZIL. FILHO, A.A.P.1; BASTOS, V.J.C.2; FONTELES, R.S.2; SILVA, S.O.1; MELO, M.N.1; REBÊLO, J.M.M.2 1.UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL; 2.UNIVERSIDADE FEDERAL DO MARANHÃO, SÃO LUÍS, MA, BRAZIL.
Keyword:sand flies; phytogeographic; phlebotominae
Abstract: Maranhão is one of the Brazilian states with the highest rates of cases of visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). One factor is the geographical position, between the Amazon and the arid Northeast, besides that, this state holds in its territory all species of sandfly considered effective leishmaniasis vectors, among them are: L. longipalpis, L. complexa, L. flaviscutellata, L. migonei, L. umbratilis, L. wellcomei, L. whitmani and others. Among the tools for vector control, studies on the distribution of sand flies in relation to vegetation in the state of MA, comes to constitute a source of information revealing the factors that contribuites for their presence. In this sense intend to analyze the distribution of sand flies in different phytogeographic regions the state of Maranhão. For this we carried out a study with a quantitative approach from studies involving the collection of sandflies from 1982 to 2005, which used CDC light traps. Altogether 91 species were found, including four belonging to the genus Brumptomyia and 87 to the genus Lutzomyia, distributed in the following subgeneres: Evandromyia (6), Lutzomyia (5), Micropygomyia (2), Nyssomyia (9), Pintomyia (2), Pressatia (3) Psathyromyia (6), Psychodopygus (14), Sciopemyia (4), Trychophoromyia (2), Viannamyia (2); group of species: Aragaoi (2), Baityi (1), Dreisbachi (1), Migonei ( 12), Oswaldoi (8), Hairy (1), Saulensis (2), Verrucarum (4) and ungrouped (1). Species diversity was higher in areas of dense evergreen seasonal forest (52 species), rainforest (31) and southern savannas (23), and lower in areas of mixed forest (forest with babaçu palms, cerrado and caatinga). It can be observed that the fauna of Maranhão has a big similarity with the others amazonic states (80.2%) corresponding to 73 species, such as found are found in the neighbor state of Pará. The greatest similarity index was observed between for restinga and open evergreen seasonal forest (J = 0.48). The dense evergreen seasonal forest showed greater similarity to the rain forest (P = 0.38). The sand fly fauna proved to be rich and heterogeneously distributed in Maranhão,
agreeing with the complexity phytogeographic state, which manifests itself in the wealth of ecosystems and climate zones. - OTHER SPECIAL TOPICS [225] P 676 - ON LINE HEALTH INFORMATION SYSTEM FOR MONITORING AND EVALUATION OF MEASURES FOR SURVEILLANCE AND CONTROL OF VISCERAL LEISHMANIASIS BIGELI, J.G.; DE OLIVEIRA, E.F.; DA SILVA, L.C. SECRETARIA DE SAÚDE DO ESTADO DO TOCANTINS – DIRETORIA GERAL DE VIGILÂNCIA E PROTEÇÃO À SAÚDE, PALMAS, TO, BRAZIL.
Keyword:chemical vector control; field work; tocantins
Abstract: In health, the information should be seen as a reducer of doubts, a tool for detecting priority foci, leading to responsible planning and execution of actions that determine the transformations necessary to reality. The World Health Organization defines Health Information System as a mechanism for the collection, processing, analysis and transmission of information needed to plan, organize, operate and evaluate health services. After approximately one year of study, the Secretaria de Saúde do Estado do Tocantins, through the Gerência de Núcleo das Leishmanioses implemented in early 2012, the Health Information System for Monitoring and Evaluation of Measures for Surveillance and Control of Visceral Leishmaniasis (VL-HIS, available after registration on www.saude.to.gov.br/svps). In the initial module of VL-HIS, only the chemical vector control is monitored, however, the next modules should include monitoring of reservoir hosts control and environmental management. Through VL-HIS, the chemical vector control pacts are registered for all municipalities in the state with this action recommended, according to epidemiological criteria, detailing the number of properties to be sprayed with insecticide in each block of each location selected, following the recognition geographic code established by the Superintendência de Campanhas de Saúde Pública (SUCAM), currently disabled. In service routine, Weekly Briefing (VL.2), which summarize all the activity of indoor residual spraying by community health agents throughout the week, are entered into the system and can be monitored in real time, since the data transmission is on line. VL-HIS also provides evaluation reports of the goals and performance of municipalities, used in official technical reports. Of the 18 municipalities in Tocantins with indication of chemical vector control, 17 (94.4%) used the system successfully along 2012. Therefore, the assessment which was previously conducted on a monthly periodicity, given the frequency of input data, can now be made weekly, which allows the timely resolution of problems, improving the monitoring of activities. - EPIDEMIOLOGY [231] P 677 - INFLUENCE OF ABIOTIC ENVIRONMENTAL FACTORS ON THE INCIDENCE OF HUMAN VISCERAL LEISHMANIASIS. CARNEIRO, J.C.; LIMA, A.L.; SANTOS, G.J.; SAMPAIO-FILHO, A.P.; NUNES-PINHEIRO, D.C. UNIVERSIDADE ESTADUAL DO CEARÁ, FORTALEZA, CE, BRAZIL.
Keyword:visceral leishmaniasis; abiotic environmental factors; correlation
Abstract: Leishmaniasis are a complex of diseases with great relevance in the world, being the city of Fortaleza an endemic area, with human deaths recorded in recent years. It has been reported, in other regions, the increase in incidence of canine and human visceral leishmaniasis (CVL and HVL), during rainy season, mainly due to the increased of sandflies density. However, the
influence of these variables, on HVL and CVL, has not been evaluated in endemic areas of the Ceará state. Therefore, the objective of this study was to evaluate the correlation and influence of precipitation index (mm), the air relative humidity (%) and mean temperature (°C) on the incidence coefficient of HVL in the city of Fortaleza, Ceará, Brazil. The experimental protocol was approved by the Ethics Committee for Animal Use of the State University of Ceará (CEUA/UECE), protocol nº 08622833-1. As the data source for analysis of HVL was used the quantitative of cases reported and registered at the Health Secretary of the Ceará State, between 2006 and 2011. The population estimate, the monthly mean of precipitation index, air relative humidity and temperature of the Fortaleza city, in the same period, were provided by the Brazilian Institute of Geography and Statistics (IBGE). The data were analyzed by Spearman correlation and linear regression, using the statistical program GraphPad Prism 5.0, in a significance level of 5%. The monthly mean of precipitation index, air relative humidity and temperature demonstrate correlation ( = -0.119, = -0.459, = -0.476, respectively) and negative influence on the incidence coefficient of HVL (y = 262.4 – 6.54x, r2 = 0.02; y = 93.47 – 0.89x, r2 = 0.97; y = 29.92 – 0.15x, r2 = 0.22, respectively). Therefore, from the foregoing, we can conclude that HVL presents a relation of direct dependency between human cases and abiotic environmental factors. This work creates perspectives for the identification of other factors that may influence in the incidence of human and canine cases, as the vector density and the participation of other possible reservoirs in the domestic cycle of the disease. Moreover, it is suggested further evaluation of changes in vegetation, employed by human action. - VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [253] P 678 - FIRST REPORT OF EVANDROMYIA EDWARDSI (DIPTERA: PSYCHODIDAE: PHLEBOTOMINAE) IN RISK AREA OF THE STATE OF RIO GRANDE DO NORTE, BRAZIL BEZERRA, A.C.D.S.1; DA SILVA, G.P.1; OLIVEIRA FILHO, R.D.1; SILVA, H.C.A.1; AMORIM, C.F.1; FERNANDES, A.M.M.M.2; SILVA, R.A.3; AMÓRA, S.S.A.1
1.UNIVERSIDADE FEDERAL RURAL DO SEMI-ÁRIDO, MOSSORÓ, RN, BRAZIL; 2.PREFEITURA MUNICIPAL (VIGILÂNCIA A SAÚDE), MOSSORÓ, RN, BRAZIL; 3.MINISTÉRIO DA SAÚDE, MOSSORÓ, DF, BRAZIL.
Keyword: phlebotomine; evandromyia; leishmaniasis
Abstract: Leishmaniasis is endemic in several continents, with high prevalence in tropical regions such as Brazil, having as etiologic agents, several species of the gender Leishmania presenting features pathogenic for humans. Is transmitted by sandflies, since some species are capable vector may transmit the etiological agent of diseases caused by protozoa. In Rio Grande do Norte (RN), 85% of the sand flies Lutzomyia longipalpis captured corresponds to a density associated with poor hygiene conditions, found in peridomestic environments associated with breeding of animals. In this same environment, has been highlighting the gender Evandromyia, with reports in different areas of Brazil, associated with the transmission of trypanosomatids. Evandromyia edwardsi has been found in several regions of Brazil, such as São Paulo, Espírito Santos and Rio de Janeiro, being related to the presence of these sand flies mainly in caves. Furthermore, E. edwardsi was found naturally infected by Leishmania (Viannia) braziliensis, but presenting an array of features not anthropophilic. This study aimed to report the capture of phlebotomine E. edwardsi in risk areas in the Mossoró, RN. Samples were collected with CDC traps installed and removed at 18:00 h to 06:00 h in the peridomestic residential area endemic for leishmaniasis, a female specimen vector E. edwardsi, identified by morphological characteristics and spermatheca. The identification of this specimen highlights of paramount importance, since it may be related to the transmission of the parasite between the tanks, leaving the man in a hazardous situation.
- RESERVOIRS [254] P 679 - DO ASYMPTOMATICS CIRCULATE ANTIMONIAL RESISTANT LEISHMANIA DONOVANI DURING THE MILTEFOSINE TREATMENT ERA IN NEPAL? BHATTARAI, N.R.1; DUJARDIN, J.C.2; VANAERSCHOT, M.2; BOELAERT, M.2; RIJAL, S.1 1.B. P. KOIRALA INSTITUTE OF HEALTH SCIENCES, DHARAN, NEPAL; 2.INSTITUTE OF TROPICAL MEDICINE, ANTWERP, BELGIUM.
Keyword:asymptomatics; antimony resistant; nepal
Abstract: Asymptomatic infections are much more frequent than clinical cases of kala-azar (KA) in the Indian sub-continent (ISC). However, their precise role in the transmission cycle is not fully elucidated so far and adequate treatment strategies are not presently available. About five years ago, disease control programme in the region has replaced the antimonial drugs, which were used to treat KA in the past, with an oral drug, miltefosine, because of the high rate of treatment failure with antimonials that was linked to drug resistance in parasites and the inherent toxicity of the drug. The question now arises what the fate is of any antimony-resistant parasite strains after the introduction of miltefosine in the ISC. Do these parasites persist and continue to circulate or have they been wiped out and replaced by new strains which are evolving under the miltefosine pressure? To monitor the fate of antimony resistant parasites in the community, a panel of recently developed and validated molecular markers for antimony resistant strains was tested in a sample of purposefully selected asymptomatically infected individuals in Nepal. Altogether 231 blood samples were collected from healthy households contacts (HHC) of miltefosine-treated KA cases and 68 samples from fully recovered past Kala-azar (HPK) cases who had been treated with miltefosine. All samples were analyzed by Leishmania-specific PCR to ascertain any Leishmania donovani infection. The overall analysis of PCR results demonstrated 33.4% of L. donovani infection which were distributed as follows: 35% positivity in HHC and 29% positivity in HPK respectively. These PCR positive samples will be further evaluated with the panel of three molecular markers for sodium stibogluconate resistance. These results will be discussed in frame of a better understanding of the long term circulation of antimony resistant parasites in this region. - VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [265] P 680 - NATURAL LEISHMANIA INFECTION IN SANDFLIES FROM THE SOUTHERN PERUVIAN AMAZON BASIN VALDIVIA, H.O.1; ZORRILLA, V.O.; PEREZ, J.G.1; RAZURI, H.R.2; VERA, H.3; FERNANDEZ, R.4; GHERSI, B.M.2; VASQUEZ, G.M.4; EDGEL, K.A.1; STELL, F.4; BURRUS, R.G.4; LESCANO, A.G.1; MONTGOMERY, J.M.2 1.DEPARTMENT OF PARASITOLOGY, U.S. NAVAL MEDICAL RESEARCH UNIT N째. 6, LIMA, PERU; 2.EMERGING INFECTIONS DEPARTMENT, U.S. NAVAL MEDICAL RESEARCH UNIT N째. 6, LIMA, PERU; 3.MINISTERIO DE SALUD, DIRECCION REGIONAL DE SALUD, MADRE DE DIOS, PERU; 4.DEPARTMENT OF ENTOMOLOGY, U.S. NAVAL MEDICAL RESEARCH UNIT N째. 6, LIMA, PERU.
Keyword:leishmania; sandflies; peruvian amazon basin
Abstract: We recently reported Leishmania infection in Lutzomyia auraensis in the Southern Peruvian Amazon Basin. However, information regarding leishmaniasis vector species in this region is still very limited. In this study we aimed to assess natural Leishmania infection in sandflies collected along a section of the Interoceanic Highway in the Southern Peruvian Amazon Basin. Six study sites were selected along this highway (Iberia, La Novia, Alto Libertad, El Carmen, Florida Baja and Mazuko). An additional site (Malinowsky) located in the Tambopata Reserve (Madre de Dios) was selected as an undisturbed habitat control. In each site, transects were
established at a distance of 200, 600 and 1000m from both sides of the highway. Sandflies were collected between October 2009 and October 2010. Six CDC light traps with CO2 (1800: 0600h) were placed 50m apart along each transect, a Shannon trap (1800:0600h) and protected human bait (1100:1330h) were placed at one of the ends of the transect. Sandfly species were identified based on keys by Young and Duncan (1994) modified for PCR analysis. Non-blood fed female sand flies were separated into 528 pools with 1-10 individuals/pool according to study site, transect and trapping method. DNA was extracted using the Gentra Puregene kit (QIAGEN). Kinetoplast based PCR was used to detect Leishmania spp. in each pool and Fluorescence Resonance Energy Transfer based Real Time PCR approach was used to identify Leishmania species. A total of 288 pools have been tested to date, of which five were positive for Leishmania spp., including Lu. (Helcocyrtomyia) spp. (n=2), Lu. hirsuta (n=2) and Lu. carrerai (n=1). All positive pools were collected from the 1000m transect at El Carmen (10% of all pools) using Shannon trap (4 pools) and CDC light trap (1 pool). No positive pools were detected to date at the other study sites. Interestingly, El Carmen is at a higher elevation and has a lower anthropogenic impact than the other sites. This is the first report of natural Leishmania infection in these sand fly species in Peru although there are reports of positivity from other countries. Based on our preliminary results it appears that multiple Lutzomyia species may be involved in leishmaniasis transmission in the Peruvian jungle, but natural infection appears to be focal and heterogeneous. - EPIDEMIOLOGY [273] P 681 - EPIDEMIOLOGY OF VISCERAL LEISHMANIASIS IN TOCANTINS FROM 2001 TO 2010 BANDEIRA, D.S.1; DE OLIVEIRA, J.H.2; PETERKA, C.R.L.1
1.FUNDAÇÃO DE MEDICINA TROPICAL DO TOCANTINS, ARAGUAÍNA, TO, BRAZIL; 2.INSTITUTO DE PATOLOGIA TROPICAL E SAÚDE PÚBLICA, GOIÂNIA, GO, BRAZIL.
Keyword:visceral leishmaniasis; epidemiology; tocantins
Abstract: Tocantins is one of the most important states that contribute to increase number of visceral leishmaniasis (VL) in the north region of Brazil. Despite several factors could be responsible for the expansion and urbanization of this neglected disease, the climatic and geographic characteristics of the Tocantins are important aspects for development of the leishmaniasis vector. To evaluate the epidemiological aspects os VL in Tocantins, a descriptive study was carried out using secondary data from SINAN (Information System for Reportable Diseases) from 2001 to 2010. In addition, a review of the vectors species in the state was done. In the period were registered 2842 cases of VL. From these cases, 2688 was new and 152 deaths were recorded. The prevalence range from 1,24/ 10000 habitants, in 2001, to 3,48/10000 habitants, in 2008. The higher number of cases was in male gender, race/skin pardo (multiracial). Although it was registered cases in all ages, the category 1 to 4 years old was the highest, followed by the category 20 to 39 years old. In 2008 was registered the highest number of cases (445) and the highest lethality of the disease (12,8%). According to the bibliographic review, in the state was recorded 46 leishmaniasis vector species. The disease was registered in 111 of the 139 municipalities of the state and the most important are Araguaína, Palmas, Porto Nacional and Paraíso do Tocantins. - EPIDEMIOLOGY [281] P 682 - OBJECT-ORIENTED REMOTE SENSING IMAGE CLASSIFICATION TO CHARACTERIZE LAND COVER FEATURES AND LAND SURFACE TEMPERATURE TO STUDY THE EPIDEMIOLOGY OF VISCERAL LEISHMANIASIS IN THE CITY OF
TERESINA, BRAZIL ALMEIDA, A.S.; WERNECK, G.L.
INSTITUTO DE MEDICINA SOCIAL/UNIVERSIDADE DO ESTADO DO RIO DE JANEIRO, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:leishmaniasis ; remote sensing; thermal band
Abstract: The use of remote sensing technologies (RS) brings new perspectives and also methodological challenges for the analysis of vector-borne diseases in particular those closely related to environmental conditions, such as visceral leishmaniasis (VL). The objective of this study is to evaluate the applicability of object-oriented classification of remote sensing imagery to characterize land cover features and land surface temperature correlated to the incidence of urban VL in the city of Teresina, Brazil. Average annual incidence rates of VL for the periods of 1993-1996 and 2001-2006 were calculated at the level of census tracts (CT). Landsat 5 Thematic Mapper (TM) scenes from 1990 and 2003 were used to capture environmental information and temperature for each period. An object-oriented classification approach was used to extract land cover features such as water, densely occupied areas, green areas, bare soil, dense vegetation and grass/pasture. We used the Spearman correlation coefficient to evaluate the association between the incidence of VL and environmental features and mean temperature for both periods of analysis (1993-1996 e 2001-2006). For the period of 1993-1996, the incidence of VL was positively correlated with the census tract’s area covered by dense vegetation, grass/pasture and bare soil (all p-values <0.01) and negatively associated with the area covered by water and densely occupied areas (p-value <0.05 for both). In the subsequent period (20012006), positive correlations were found for dense vegetation, grass/pasture, bare soil and densely occupied areas (all p-values <0.01); and negative correlation was detected for green vegetation (p<0.01). The average land surface temperature was negatively associated with the incidence of VL in both periods (p<0.05). Understanding the complexity of urban space and how it may affect the occurrence of VL requires a multiplicity of tools. In this setting, object-oriented classification of remote sensing images showed to be useful to identify landscape features associated to VL that might be helpful in defining risk areas for prioritizing the delivery of health interventions against the disease. - RESERVOIRS [288] P 683 - FACTORS ASSOCIATED WITH THE INFECTION BY LEISHMANIA SPP. IN A POPULATION OF DOGS FROM A BRAZILIAN MUNICIPALITY SILVA, E.S.1; PENAFORTE, K.M.1; BELO, V.S.2; TEIXEIRA-NETO, R.G.3; RIBEIRO, R.A.N.1; OLIVEIRA, R.B.D.2; SCHETTINI, D.1 1.UNIVERSIDADE FEDERAL DE SÃO JOÃO DEL REI, DIVINÓPOLIS, MG, BRAZIL; 2.ESCOLA NACIONAL DE SAÚDE PÚBLICA SERGIO AROUCA, FUNDAÇÃO OSWALDO CRUZ,, RIO DE JANEIRO, RJ, BRAZIL; 3.CENTRO DE PESQUISAS RENÉ RACHOU, FUNDAÇÃO OSWALDO CRUZ, BELO HORIZONTE, MG, BRAZIL.
Keyword:canine visceral leishmaniasis; clinical-epidemiological; diagnoses
Abstract: The identification and quantification of the factors associated with canine visceral leishmaniasis is important not only to the design of strategies aimed at the prevention of infection in the canine population but also to the better targeting of control activities. In the recommendations of the Brazilian Ministry of Health there are no activities directly related to the investigation of such factors. The present cross-sectional study was conducted during the period March to October 2011 and analyzed factors associated with infection by Leishmania spp. in a population of dogs attended at the Center of Reference in Health Vigilance (CREVISA), located in the municipality of Divinópolis, in the central western region of Minas Gerais state – Brazil. All the samples of animal blood, were subjected to the Enzyme-Linked Immunosorbent Assay technique. Those
that tested positive or indeterminate were then tested using the Indirect Fluorescent Antibody Test. A clinical-epidemiological data sheet was developed by the study team, together with the CREVISA professionals, that contained the exposure variables analyzed in this study. The most frequent clinical signs in the positive dogs were described and the association between the exposure variables and the occurrence of infection was analyzed using Prevalence Ratios (PR) and logistic regression models. Of the 755 animals examined, 103 (13.6%) were positive (in the both techniques). Lymphadenopathy, alopecia, weight loss, dermatitis and onychogryphosis were the most frequent signs of the disease in positive animals while 24 of them (23.3%) were asymptomatic. There was a higher prevalence of infections in dogs: a) of pure breeds (PR= 0.87, Confidence interval (CI)= 0.59 - 1.28), b) with short hair (PR=1.13, CI= 0.70 - 1.84) c) over five years of age, (PR=1.27: CI= 0.84 - 1.92) d) males (Odds Ratio (OR)=1.47; CI= 0.98 - 2.06; p=0.06) and e) with medium and large size (OR= 1.71; CI= 1.14 - 2.59). The last two variables were statistically significant in multivariate analyzes. The epidemiological associations described are plausible and consistent with what has been identified in the literature. This pattern demonstrates that although there is a certain complexity in the factors associated with CVL, consistent patterns can be identified when analyzing not only statistical significance but also the strength and direction of the associations. These results might be considered in the disease control strategies in Brazil. - EPIDEMIOLOGY [290] P 684 - SEROLOGICAL AND MOLECULAR SURVEY OF LEISHMANIA PARASITES IN APPARENTLY HEALTHY DOGS IN THE WEST BANK, PALESTINE HAMARSHEH, O.1; NASEREDDIN, A.2; DAMAJ, S.2; SAWALHA, S.2; AL-JAWABREH, H.3; AZMI, K.2; AMRO, A.4; ABDEEN, Z.2; EREQAT, S.2; AL-JAWABREH, A.3 1.DEPARTMENT OF BIOLOGICAL SCIENCES, FACULTY OF SCIENCE AND TECHNOLOGY, AL-QUDS UNIVERSITY, JERUSALEM, PALESTINA; 2.AL-QUDS NUTRITION AND HEALTH RESEARCH INSTITUTE (ANAHRI), AL-QUDS UNIVERSITY, JERUSALEM, PALESTINA; 3.LEISHMANIASES RESEARCH UNIT, JERICHO, PALESTINA; 4.FACULTY OF PHARMACY, AL-QUDS UNIVERSITY, JERUSALEM, PALESTINA.
Keyword:serological and molecular diagnosis; leishmania infantum; palestine
Abstract: Canine visceral leishmaniasis (CVL) is caused by Leishmania infantum in all Mediterranean countries. The Leishmania parasite is transmitted by the bite of a corresponding sand fly vector and primarily maintained in nature by wild and domestic reservoirs, including dogs, foxes and jackals. Infected dogs are the primary reservoir host in endemic regions and are the most significant risk disposing humans to infection. The present study aimed at assessing the prevalence of infection with Leishmania and identification of Leishmania infantum in domestic dogs in the West Bank, Palestine. The infection rate among domestic dogs collected from seven districts in the Palestinian West Bank was investigated by examination of parasites in culture from the buffy coat using serological and molecular methods; based on ELISA, internal transcribed spacer 1 (ITS1) and cysteine protease (CPB) PCR. Out of 215 dogs examined for Leishmania, 36 (16.7%) were positive in at least one method. Twenty three animals (11.5%) were positive for Leishmania DNA, whereas, ELISA and culture revealed 16 (7.5%), and 4 (1.5%) respectively. CPB-PCR on one of three culture-positive isolates revealed Leishmania infantum as the causative agent for Leishmania infection in dogs. Our study showed that canine leishmania infection is prevalent with varying degrees in all the seven studied districts in Palestine despite the absence of human VL cases in 4 of these districts. The causative agent was confirmed to be Leishmania infantum.
- EPIDEMIOLOGY [295] P 685 - MOLECULAR AND SEROLOGICAL DETECTION OF LEISHMANIA INFANTUM CHAGASI FROM MARANHÃO STATE, BRAZIL, PRELIMINARY RESULTS COSTA, A.P.1; MARCILI, A.1; COSTA, F.B.1; SOARES, H.S.1; CANDANEDO GUERRA, R.M.S.N.2; GENNARI, S.M.1
1.UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL; 2.UNIVERSIDADE ESTADUAL DO MARANHÃO, SÃO LUIS, MA, BRAZIL.
Keyword:leishmania; dog; maranhão
Abstract: Visceral leishmaniasis is an anthropozoonosis caused by a protozoan Leishmania infantum chagasi. In Brazil, it is considered reemergent and in expansion. Dogs are important reservoir and source of L. infantum chagasi infection to humans due to their high prevalence of infection, the presence of amastigote forms in the skin and because they are live in domestic and peridomestic environment. The present study aimed to determine the frequency of L.chagasi infection in dogs from 3 municipalities (São Bento, Cururupu and Caxias), in Maranhão, Brazil. Serum samples from 480 domiciliated dogs, collected from August to September 2012, were analyzed by the presence of antibodies by indirect fluorescent antibody assay (IFAT 40). All the dogs were subjected to popliteus lymphonode punsion and the aspirated material was placed in test tubes containing biphasic medium consisting of solid phase BAB (blood agar base with 15% rabbit blood) and liquid phase of LIT medium and sent for isolation of the parasite. A PCR was used for the amplification region V7V8 SSU rDNA of the isolates. The products were sequenced and the results compared with the sequences of the Leishmania previously deposited in the GenBank. A total of 9.16% (44/480) of the dogs reacted to L.chagasi antigen. In São Bento the occurrence was 1.25% (2/160), in Cururupu 7.5% (12/160) and in Caxias 18.75% (30/160) and this difference (Chi square test) was statistically significant (P <0.05), with higher occurrence in Caxias, followed by Cururupu and São Bento. Two samples were positives in culture, but only one was isolated and cryopreserved (from Caxias). After the isolation, the strain was identified as L. infantum chagasi with 100% similarity with L. infantum (GQ332357). This difference in the occurrence values indicates different transmission cycles according geographical origin, biomes and/or urbanization degree. Financial support: FAPESP
- RESERVOIRS [299] P 686 - DETECTION OF LEISHMANIA INFANTUM CHAGASI IN WHOLE BLOOD OF SHEEP FROM AN ENDEMIC AREA IN SOUTHERN BRAZIL MARCONDES, M.1; LEUTENEGGER, C.2; ESTRADA, M.2; ROSA, F.A.1; LEITE, J.H.A.C.1; CAMARGO, D.G.1; BRAGA, E.T.1 1.SCHOOL OF VETERINARY MEDICINE, SÃO PAULO STATE UNIVERSITY, ARACATUBA, SP, BRAZIL; 2.IDEXX LABORATORIES, WEST SACRAMENTO, UNITED STATES.
Keyword:zoonotic visceral leishmaniasis; real-time pcr ; domestic reservoirs
Abstract: Domestic dog has been considered the main vertebrate reservoir for zoonotic visceral leishmaniasis (ZVL), a serious public health problem in Brazil. Although visceral leishmaniasis (VL) has been diagnosed in other domestic species, the occurrence of VL in cattle and ovines has been poorly investigated. The aim of the present study was to investigate the presence of Leishmania infantum chagasi in whole blood of cows and sheep living in the periurban area of Araçatuba, São Paulo State, Southeastern Brazil, an endemic area for VL. Blood samples were
collected from 101 sheep and 176 cows and submitted to real-time polymerase chain reaction (qPCR). DNA was extracted under standard protocols on a commercial platform. A housekeeping gene (18S rRNA) was used to determine DNA content and quality. The PCR test was based on IDEXX’s proprietary real-time PCR oligonucleotides (IDEXX Laboratories, USA). Highly conserved metalloproteinase gp63 gene sequences were aligned and a region selected for 2 primers and a hydrolysis probe designed using a commercial software. An additional 2 primers flanking the real-time PCR assay were designed for sequence verification purposes. Real-time PCR was run with standard primer and probe concentrations using a commercially available mastermix, and was performed using default cycling conditions on a commercial instrument. Average analytical sensitivity was ten molecules per reaction (reproducible in triplicate analysis). Analytical specificity was confirmed by sequencing a positive real-time PCR reaction. Analytical specificity was further confirmed by testing DNA extracted from 23 bacterial, protozoal and rickettsial isolates. Reproducibility (CV on CT values and on absolute quantified numbers), amplification efficiency, linearity, square values and signal to noise ratio parameters all passed the required criteria and thresholds. All cows samples were negative for the presence of Leishmania DNA, whereas the overall rate of Leishmania positivity in sheep was 96% (97/101). The implications of those results in the transmission of L. infantum chagasi in Brazil are not well understood. Nevertheless they emphasize a possible role of sheep in the maintenance of ZVL in endemic areas. Further studies are needed to elucidate the role of sheep in the Leishmania cycle, as this may be crucial for the design of control programmes for VL. - CLINICAL LEISHMANIASIS [301] P 687 - PRESENCE OF LEISHMANIA INFANTUM CHAGASI DNA IN THE BRAIN OF DOGS WITH VISCERAL LEISHMANIASIS CARDINOT, C.B.; MELO, G.D.; SILVA, J.E.S.; MACHADO, G.F.; NUNES, C.M.; MARCONDES, M. SCHOOL OF VETERINARY MEDICINE, SÃO PAULO STATE UNIVERSITY, ARACATUBA, SP, BRAZIL.
Keyword:visceral leishmaniasis; central neurvous system; qpcr
Abstract: There are few reports of neurological disorders in dogs with visceral leishmaniasis (VL), with or without the participation of other opportunistic agents. The aim fo the present study was to investigate, by Real Time PCR, the presence of Leishmania infantum chagasi DNA in the brain of dogs naturally affected by visceral leishmaniasis (VL). Twenty four naturally infected dogs, 20 symptomatic without neurological signs, 2 symptomatic with neurological disorders and 2 asymptomatic, were evaluated. All dogs were euthanized in compliance with a federal law. Samples from meninges, frontal cortex, thalamus, cerebellum, choroid plexus of lateral ventricles and fourth ventricle were collected and subjected to DNA extraction using a commercial kit. PCR forward (5'-CCTATTTTACACCAACCCCCAGT-3') and reverse (5'GGGTAGGGGCGTTCTGCGAAA-3') primers, that amplify a fragment of 116 bp of the kinetoplast minicircle DNA from Leishmania, were used. Real-Time PCR was performed using a commercial mastermix with SYBR-Green fluorophore. DNA was amplified in the brain of 23/24 (95.8%) dogs. The parasite load ranged from 2 to 16.964 amastigotes/mL with a median of 116 parasites/mL and mean and standard deviation of 1.166 ± 3.546 parasites/mL. The average number of parasites in dogs with VL with neurological disorders, in dogs without neurologic disorders and in asymptomatic dogs were, respectively, 2.010, 1.119 and 772 parasites/mL. These results demonstrate that the parasite has the ability to penetrate into the nervous system and should contribute to the development of neurological lesions.
- EPIDEMIOLOGY [304] P 688 - CHARACTERIZATION OF LEISHMANIA SPP SPECIES IN URBAN RESERVOIRS IN THE ENDEMIC AREA OF BAURU-S.P, BRAZIL DE LIMA, V.M.F.1; SANCHES, L.C.1; PEROSSO, J.1; MELO, L.M.1; DA SILVA, K.L.O.1; DE LIMA, B.D.2 1.UNIVERSIDADE ESTADUAL PAULISTA, ARAÇATUBA, SP, BRAZIL; 2.UNIVERSIDADE DE BRASÍLIA,, BRASÍLIA, DF, BRAZIL.
Keyword:dogs; pcr-rflp; leishmaniasis
Abstract: Leishmaniasis are diseases that are characterized by compromising the skin, mucous membranes and internal organs, according to the species of Leishmania and the host immune response. In the city of Bauru - SP, Brazil there is record of the occurrence of cutaneous and visceral leishmaniasis. The geographical distribution of leishmaniasis is limited to distribution of the vector, and the number of cases has been increasing in recent years, mainly due to environmental changes caused by humans, which increases human exposure to vector. Dogs are important in the epidemiological cycle of the disease due to large number of parasites in the skin, so it is important to study the species that infected dog. Ninety seven dogs with clinical symptoms compatible with leishmaniasis and diagnosis confirmed by the detection of antiLeishmania antibodies were studied. Leishmania spp DNA was detected in peripheral blood samples by PCR. To identify the species, parasite DNA from the blood was used to amplify the intergenic region ITS1 of the rRNA gene (5’ AGCTGGATCATTTTCCGATG 3’, 5’ TATGTGAGCCGTTATCCACGC 3’). A sample was considered positive for Leishmania DNA when a PCR-amplified fragment of 227-268 bp was detected. Shift PCR with an alternative oligo 3’ (5’ TGATACCACTTATCGCACTT 3’) was performed to confirm the Leishmania DNA amplification (295-338 bp). Restriction pattern analysis was applied to the amplified fragments with HaeIII enzyme digestion from PCR and shift PCR to identify the Leishmania species. Total DNA from Leishmania species (L. chagasi, L. braziliensis, L. amazonensis, L. major) and HeLa cells were used as positive and negative controls, respectively. Most dogs (70%) showed a pattern consistent with L. amanzonensis, L. chagasi was detected in 10% and unusually 8 % of the samples showed L. major. The predominant species observed in dogs in urban area in Bauru,S.P were L. amazonensis. Financial Support: FAPESP and FAP-DF
- EPIDEMIOLOGY [307] P 689 - RISK FACTORS FOR THE INCIDENCE OF INFECTION BY LEISHMANIA INFANTUM IN THE CITY OF TERESINA, PIAUÍ. ALVES, E.B.1; CARVALHO, F.A.A.2; CRUZ, M.D.S.P.2; COSTA, C.H.N.2; WERNECK, G.L.1
1.UNIVERSIDADE FEDERAL DO RIO DE JANEIRO, RIO DE JANEIRO, RJ, BRAZIL; 2.UNIVERSIDADE FEDERAL DO PIAUÍ, TERESINA, PI, BRAZIL.
Keyword:leishmaniasis visceral; risk factors, infections; cohort studies, epidemiology
Abstract: The geographic spread of visceral leishmaniasis (VL) is considered one of the most important public health concerns in Brazil. The paradigm of a typical rural disease has been overcome, its transmission cycle is clearly established in many urban and peri-urban areas. As the process of urbanization is relatively recent, there is little knowledge about the epidemiology of VL in this environment, particularly about risk factors for asymptomatic infection. This study evaluated
demographic, socioeconomic and environmental risk factors for the incidence of infection by Leishmania infantum in the urban area of the city of Teresina, PiauĂ, Brazil. This is a cohort study in which the subjects were selected from two settings: areas where there was some kind of intentional action with regard to the control of LV and areas without any type of intervention. At the baseline, participants answered a questionnaire about risk factors and infection with L. infantum was evaluated using the Montenegro skin test (MST). Those with a negative result were tested again 18 months later. Associations between variables and the incidence of infection were expressed by risk ratios (RR) and their respective 95% confidence intervals (CI), estimated by using Poisson regression with robust variance. We conducted a multivariate analysis stratified by type of setting (areas with and without intervention) using stepwise procedures for variable selection. The cumulative incidence of infection by L. infantum was 35% in 18 months, higher than those found in other similar studies. In areas with no intervention the presence of tile oven or deposit in the backyard, the lower number of people in household and a shorter time living in this house were associated with higher incidence of infection. In areas with intervention, a shorter time of dog ownership and lower level of literacy of household head were also associated with higher incidence. The results indicate the need for more longitudinal studies exploring the features of the peridomestic environment and household structure as potential factor modulating the risk of infection. This would contribute to a better understanding of the epidemiological dynamics and inform public health surveillance systems in order to establish appropriate control measures. - CONTROL PROGRAMS [315] P 690 - IMPROVE THE PROGRAM COVERAGE AND SURVEILLANCE SYSTEM TO ASSESS BURDEN OF LEISHMANIASIS IN KABUL, AFGHANISTAN BUTT, W.A.; SAFI, N.U. WORLD HEALTH ORGANIZATION, KABUL, AFGHANISTAN.
Keyword:improve program coverage; strengthening of surveillance ; increase case detection
Abstract: Abstract Background: Leishmaniasis is endemic In AFG. Most of cases are cutaneous caused by Leishmania tropica, transmitted human to human by sand fly Phlebotomus sergenti, and Leishmania Major, transmitted from animals to human by sand fly phlebotomus papatasi. Kabul, reporting more than 30% of annual incidence, is the world capital of CL ACL is more common in AFG which is generally characterized by a large and/or multiple cutaneous lesions with a variable tendency to self-cure, due to exposure to sand fly, most lesions occur on the face, which often cause social stigmatization in affected persons due to its disfiguring scar, especially on the face of young girls and women that will be culturally unaccepted for marriage The impact of CL is not only limited to the period of the active infection, but can also have a lifelong effect as for stigma and marginalization due to disfiguration and permanent disabilities. Injections of antimonite (SSG) is the first line drug available in AFG for Leishmaniasis treatment which should be available free of charge in primary health care centers NMLCP is the technical department of Ministry of Public Health which has the responsibility of Leishmaniasis control in AFG and WHO is the only partner supporting the strategic and operational needs of NMLCP The presentation illustrates the impact due to strengthening the surveillance system for Leishmaniasis through improvement of the coverage for service delivery and introduction of reporting forms indicating the treatment outcome of CL patients in AFG
Methods Intervention to expand the program coverage for service delivery, diagnosis and case management, and strengthen the Leishmaniasis surveillance system, based on the past epidemiological trends and endemicity levels These interventions were expansion of the program coverage by introduction and initiation of CL services, diagnosis and case management, through BPHS and application, for the first time ever in AFG, of data reporting forms illustrating treatment outcomes. These forms have been distributed through the public health facilities operating at the primary health level in Kabul AFG over two years, for pilot and first assessment Conclusion The introduction of the enhanced reporting forms, combined with the expansion of Leishmaniasis (CL and VL) program coverage, overall, resulted into: i) increasing the accessibility to effective diagnosis and treatment and increasing the number of reported cases that were remaining under reported in previous years; ii) reducing the number of cases misdiagnosed; iii) illustrating the treatment outcome of Leishmaniasis patients who receive the treatment at public health centers and rationalizing the use of leishmaniasis treatment, drugs AFG Afghanistan ACL Anthroponotic cutaneous leishmaniasis BPHS Basic Package of Health Services CL Cutaneous Leishmaniasis NMLCP National Malaria and Leishmania Control Program SSG Sodium Stibogluconate VL Visceral Leishmaniasis
- CONTROL PROGRAMS [322] P 691 - COST-EFFECTIVENESS OF TREATMENT STRATEGIES FOR VISCERAL LEISHMANIASIS IN SUDAN MEHEUS, F.1; ABDALLAH, A.2; BALASEGARAM, M.3; BALTUSSEN, R.4; MUSA, A.2; BOELAERT, M.5 1.UNIVERSITY OF CAPE TOWN, CAPE TOWN, SOUTH AFRICA; 2.INSTITUTE OF ENDEMIC DISEASES, KHARTOUM, SUDAN; 3.DRUGS FOR NEGLECTED DISEASES INITIATIVE, GENEVA, SWITZERLAND; 4.RADBOUD UNIVERSITY NIJMEGEN MEDICAL CENTRE, NIJMEGEN, NETHERLANDS; 5.INSTITUTE OF TROPICAL MEDICINE, ANTWERP, BELGIUM.
Keyword:cost-effectiveness; treatment; sudan
Abstract: Background: Despite their toxicity and long hospitalization (30 days), pentavalent antimonials, mainly sodium stibogluconate (SSG), are still the most frequently used first-line treatment for visceral leishmaniasis in Sudan, and East Africa in general. Over the past decade there have been a number of important drug developments including liposomal Amphotericine B (L-AmB), oral miltefosine (MF) and paromomycin (PM). Each of these compounds has its advantages and disadvantages in terms of their efficacy and cost; MF is teratogenic and has a long half-life, PM although low cost requires intramuscular injections by health workers while L-AmB remains
very expensive despite recent price decreases for the public sector. In this study we examine the cost-effectiveness of various treatment options for visceral leishmaniasis in Sudan. Methods: We assess from the societal perspective 7 different treatment alternatives, including 3 combination therapies: L-AmB with MF, L-AmB with SSG and SSG with PM, and 4 monotherapies: MF, L-AmB (10days), SSG and meglumine antimoniate. Facility and household costing surveys are carried out and combined with data from the literature, clinical trial data and a Delphi survey among VL clinical experts. Sensitivity analysis is done on variables with considerable uncertainty. Results/conclusions: Pending the results from a clinical trial, we will present average and incremental costeffectiveness ratios expressed in terms of cost per death averted. The results of the study will inform national and international policy makers about the most appropriate treatment strategies for VL from an economic perspective.
- CONTROL PROGRAMS [323] P 692 - THE ECONOMIC BURDEN OF VISCERAL LEISHMANIASIS IN SUDAN: AN ASSESSMENT OF HOSPITAL AND HOUSEHOLD COSTS MEHEUS, F.1; ABDALLAH, A.A.2; BALTUSSEN, R.3; YOUNIS, B.M.2; BALASEGARAM, M.4; KHALIL, E.A.2; BOELAERT, M.5; MUSA, A.M.2 1.UNIVERSITY OF CAPE TOWN, CAPE TOWN, SOUTH AFRICA; 2.INSTITUTE OF ENDEMIC DISEASES, KHARTOUM, SUDAN; 3.RADBOUD UNIVERSITY NIJMEGEN MEDICAL CENTRE, NIJMEGEN, NETHERLANDS; 4.DRUGS FOR NEGLECTED DISEASES INITIATIVE, GENEVA, SWITZERLAND; 5.INSTITUTE OF TROPICAL MEDICINE, ANTWERP, BELGIUM.
Keyword:household cost; hospital cost; sudan
Abstract: Introduction: Visceral leishmaniasis (VL) is a neglected parasitic disease that is fatal if left untreated and is endemic in eastern Sudan. In this study we estimated the direct and indirect costs of treatment for VL from the perspective of the provider and the household. Methods: We collected data in three public hospitals in Gedaref State, Eastern Sudan. Provider costs of VL diagnosis and treatment was estimated through a combination of step down and microcosting. Household direct and indirect costs were collected with a structured patient exit survey (n=75) at these facilities. Results: The median total cost for one VL episode was estimated to be US$ 450. Despite the free provision of VL drugs at public hospitals, households bore 53% of the total cost of VL with one episode of VL representing 40% of the annual household income. More than 75% of households incurred catastrophic out-of-pocket expenditures.
Conclusions: The length of treatment of 30 days led to important costs for both health providers and households. Alternative treatment regimens that reduce the duration of treatment are urgently needed.
- EPIDEMIOLOGY [342] P 693 - SEROSURVEILLANCE (IFAT) OF LEISHMANIA INFANTUM IN ITALY - 20052011 REPORT MIGLIAZZO, A.1; PIAZZA, M.1; LUPO, T.1; NUCATOLO, G.1; GALANTE, A.2; LO VERDE, V.2; VITALE, F.1 1.CENTRO REFERENZA NAZIONALE LEISHMANIOSI - ISTITUTO ZOOPROFILATTICO SPERIMENTALE DELLA SICILIA, PALERMO, ITALY; 2.AREA BIOLOGIA MOLECOLARE - ISTITUTO ZOOPROFILATTICO DELLA SICILIA, PALERMO, ITALY.
Keyword:seroprevalence; ifat; leishmania
Abstract: Leishmaniasis is a typical example of zoonosis world-wide diffused. The geographic distribution depends on the presence of sandfly vectors, and animal reservoirs. In Southern Europe canines are considered the main reservoir of infection and phlebotomies are the vectors. Different tests are routinely used for the diagnosis such as: IFAT, ELISA, microscopic examination on smears, or cultural isolation. In Italy Istituti Zooprofilattici Sperimentali (IZS) set up a network of collaborating laboratory that perform serological diagnosis and collect epidemiological data in each regions improving the official epidemiological report on “Leishmaniasis state of art” in Italy. According the policy of service quality and applying EEC Directives on inspection and evaluation of Good Laboratory Practices (OIE) the indirect immunofluorescent-antibody test (IFAT), is performed in our network since 1987 and thousands of animal sera are monitored every year. Serological data collected in all Italian regions by the IIZZSS network through 2005-2011 are showed. We used the serological test to construct a database useful to the epidemiological approach and to study the prevalence of the infection. Multispot microscope slides test made by Italian National Leishmaniasis Referecence Centre are employed and distributed to various laboratories. The IPT1 MON1 strain is cultivated and employed as antigen fixed on the microscope slides, to test the presence of antibody in the examined serum samples. The authors report the analysis of 498.589 samples and calculated the seroprevalence on the basis of the collected data (odds ratio 95%) for each region. Data, although is not defining a complete epidemiological map, represents the first collection of national data about the tests conduct in the IIZZSS laboratories network in the years 2005-2011. It reveal the evidence of the seroprevalence trend in different Italian regions. The cut-off value used for sera positivity was established in every Institute related to the prevalence regional status. Samples were filtered as much as possible to avoid the collection of data obtained on the same animals in different times. This work demonstrated the increase of the endemic infection status in Italian areas where only sporadic cases were previously reported. Moreover the growing number of examinations in all the laboratories, demonstrate the high attention level of the operators towards leishmaniasis. - EPIDEMIOLOGY [350] P 694 - LEISHMANIA SIAMENSIS AS THE CAUSE OF AUTOCHTHONOUS CUTANEOUS LEISHMANIASIS OF HORSES IN GERMANY - A NEW EMERGING DISEASE? KUHLS, K.1; RAUHUT, F.2; KÖHLER, K.3; LITZKE, L.4; SCHÖNIAN, G.2
1.MOLECULAR BIOTECHNOLOGY AND FUNCTIONAL GENOMICS, TECHNICAL UNIVERSITY OF APPLIED SCIENCES, WILDAU, GERMANY; 2.INSTITUTE OF MICROBIOLOGY AND HYGIENE, CHARITÉ UNIVERSITY MEDICINE BERLIN, BERLIN, GERMANY; 3.INSTITUTE OF VETERINARY PATHOLOGY, JUSTUS LIEBIG UNIVERSITY GIESSEN, GIESSEN, GERMANY; 4.CLINIC FOR HORSES (SURGERY), JUSTUS LIEBIG UNIVERSITY GIESSEN, GIESSEN, GERMANY.
Keyword:diagnostics; emerging infectious disease; leishmania siamensis
Abstract: In Germany, leishmaniasis is usually considered as being imported through travelling to or from endemic regions but rare putative autochtonous human cases and cases in different animal species are also described. In 2004, a granulomateous inflammation was found at the eye lid of a horse. The animal was born in Germany and kept in Bavaria and Hesse. After surgery resected tissue was examined histopathologically and a cutaneous leishmaniasis was diagnosed. The parasites have been identified as belonging to the genus Leishmania, but no homology was found to any of the known species at that time. Analysis of the DNA polymerase revealed an identical sequence to those of two strains from patients with skin lesions from Martinique. All three strains were found to be only distantly related to all other human-pathogenic Leishmania species. In 2008, a new Leishmania species referred to as L. siamensis has been described as the causative agent of visceral leishmaniasis in Thailand and more sporadic autochthonous human infections were reported from there since then. Cutaneous leishmaniasis was reported in horses and a cow from Switzerland and Germany in 2009, and sequencing of the ITS-1 rDNA revealed identity to L. siamensis. Another case of such infection of a horse was found 2011 in Florida, USA. By comparing the ITS-1 sequence of our equine sample isolated in 2004 with these new data we could identify it as L. siamensis. Moreover, our data support the close phylogenetic relationship of L. siamensis to the zoonotic species L. enrietti. The occurrence of L. siamensis in Central Europe raises many questions, e.g. whether this is a new emerging zoonotic disease, about the origin of this parasite, the mode of transmission, and the involved vector. Further investigations are necessary to establish whether these cases found on different continents point to a new global emerging zoonotic disease or whether it has been overlooked so far because of the sporadic occurrence of such cases. This includes a detailed study of possible cases, the putative transmission cycles and the involved reservoirs and vectors in all effected countries. This knowledge will be essential for respective surveillance and control measures. - VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [356] P 695 - ACTIVE SEARCH OF BREEDING SITES OF SAND FLIES (DIPTERA: PSYCHODIDAE) IN MARAMBAIA ISLAND, STATE OF RIO DE JANEIRO, BRAZIL. DE SOUZA, T.L.; DE LIMA, D.A.; DE ALMEIDA, A.B.; BENIGNO, C.V.; PONTES, C.S.; FIGUEIREDO, F.B.; DE SOUZA, M.B. FUNDAÇÃO OSWALDO CRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:sand flies; flotation technique; emergency trap
Abstract: Introduction: Sand flies are important insects to public health, as they are responsible for the transmission of Leishmaniasis. The control of parasitic diseases is difficult to implement, because they have numerous epidemiological and environmental factors that influence them. The immature stages of sand flies develop in soils with essential features and ideal for their life cycles, such as organic matter, humidity, shade and temperature. Information concerning potential breeding sites are fundamental to understanding the Leishmaniasis epidemiology and ecology. There are few studies with satisfactory results, but the search for immature, mostly, is accomplished by emergency traps, seated on soil. A flotation technique with sugar solution is
also used to get immature forms from the substrate. This study analyzes the microenvironments in the Marambaia Island, city of Mangaratiba, Rio de Janeiro, which provide favorable conditions for the development and success of immature stages of sand flies. This region receives people from various endemic areas of Leishmaniasis throughout Brazil and still has historic autochthonous case of the disease and positive serology in dogs. Materials and methods: It’s about a sectional study, which is conducted entomological research for breeding sites through traps emergency and direct search for immature forms with the flotation technique. According to density of adult insects and food source for females, was elected a region analysis that have features important for the development of immature stages, as a humidity, shade and organic matter. At this location, 10 emergency traps were placed, adapted from the trap used by Casanova (2001), monitored from 15 to 15 days. These undergo a rotation every 2 months, a period in which the soil is also collected and processed in saturated sugar. Results: Until now, accounting for 6 months of work, we found 13 specimens of adult sand flies caught in the emergency traps, including the species: L. intermedia, L. migonei, L. whitmani, L. schreiberi and L. capixaba. Two larvae were also found in soil. Conclusion: From the results, we conclude that the proposed methodology is effective and provides satisfactory results. It is worth emphasizing the importance of such studies for understanding the population dynamics of adult insects in certain regions. - EPIDEMIOLOGY [359] P 696 - AMERICAN CUTANEOUS LEISHMANIASIS ASSOCIATED WITH THE DISTRIBUTION OF THE VECTOR LUTZOMYIA (NYSSOMYIA) WHITMANI (DIPTERA: PSYCHODIDAE: PHLEBOTOMINAE), IN BRAZIL COSTA, S.M.1; CORDEIRO, J.L.P.2; RANGEL, E.F.3 1.INSTITUTO OSWALDO CRUZ/FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 2.CFMA/PRESIDÊNCIA/FIOCURZ, RIO DE JANEIRO, RJ, BRAZIL; 3.INSTITUTO OSWALDO CRUZ/ FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:lutzomyia (n.) whitmani; ecological niche modeling; distribution of the vector
Abstract: The Leishmaniases represent important public health problems in Brazil. The continuing process of urbanization and expansion of human activities on forested areas disrupt natural habitats, modifying the ecology of some species of Leishmania, sand fly vectors and reservoirs, and consequently changing the epidemiological pattern of American Cutaneous Leishmaniasis (ACL). The characterization of factors that influence the spatial distribution of sand fly species is a first-step for understanding of the ecology of ACL. Through the technique of ecological niche modeling (Ecological Niche Factor Analysis ENFA) was evaluated the environmental suitability for LTA and Lutzomyia (N.) whitmani, based on the classic concept of ecological niche of Hutchinson (1957). For data related to the occurrence of the disease, were used the municipal regions with records of endemic areas for ACL (N = 1882). For information of phlebotomines, were identified municipalities with occurrence of vector (N = 633). In this analysis, 11 Environmental Variables (VA) (09 bioclimatic, altitude and slope) were considered. Lutzomyia (N.) whitmani was identified by the model as a species that occurs in a particular condition in the study area, in the context of the environmental variables , and data suggest that the species is tolerant, with a range of niche not so close (M = 0.77, T = 0.71). Many evidences have indicating that this sand fly has capacity to survive environmental changes and to adapt to new ecological niches, tolerating effects of climatic alterations Analysis by VA showed that Lutzomyia (N.) whitmani "prefers" (more suitable habitat) and is more frequent in regions with less rainfall and warm temperatures. These characteristics are in accordance with previous analysis discussing the distribution of this sand fly vector in the Brazilian biomes, occurring in high frequency in Cerrado and Atlantic Rainforest, with greater amplitude (M = 0.39, T = 0.89). Support Financial: LTL/IOC; FIOCRUZ/FAPERJ
INDEX AUTHOR AARA, N. ABANADES, D.R. ABASSI, I. ABATE, H.L. ABBEHUSEN, M.M. ABBEHUSEN, M.M.C. ABBOUD, L.C.S. ABDALLAH, A. ABDALLAH, A.A. ABDEEN, Z. ABDEL HALEEM, A.A. ABDELADHIM, M. ABDESSALEM, C.B. ABDOLI, H. ABDOUN, A.O. ABEBE, T. ABENGOZAR, M.A. ABI ABDALLAH, D.S. ABID, H. ABRAHAM, T. ABRANTES, C.F. ABREU SILVA, A.L. ABREU, B.L. ABREU, E. ABREU, H.C.N. ABRIL, M. ACARDI, S.A. ACOSTA, L.A. ACUÑA, V.L. ADAMS, E. ADAMS, E.R. ADAUI, V. ADDYA, S. AEBISCHER, T. AFONSO, L.C.C. AFONSO, M.M.S. AFONSO, M.O. AFRIN, F. AGRA, M.C.R. AGRA, M.F. AGUADO, J.M. AGUDELO, D.
ABSTRACT O 171 P 013 P 093 O 216 P 713, P 715 P 223 P 038 P 392 P 691 P 692 O 243, O 290, P 455, P 684 P 288 O 160, P 039, P 958 P 958 O 318 P 610 P 410 O 133 P 461, P 464 P 475 O 159 P 517, P 580 O 003 P 145 P 507 P 812 O 151 P 233, P 554 P 825 O 045, O 097 O 011, O 380, P 904 P 704 O 060, O 258, P 332, P 361, P 458, P 794 P 151 P 586 O 139, P 144, P 382, P 397, P 438 P 333 O 162 P 138 P 725 O 219 P 466 P 782 P 440 O 016 O 380, P 904
AGUIAR SOARES, R.D.O. AGUIAR SOARES, R.D. AGUIAR SOARES, R.D.O. AGUIAR, B.G. AGUIAR, C.O.M. AGUIAR, M.G. AGUIAR, S.B. AGUIAR, V.B. AGUILAR, E. AGUILLERA, A.H. AHMADI, F. AHMED, B. AHMED, E.M. AINSLIE, K.M. AISSI, W. AJOUAD, M. AKHAVAN, A.A. AKOPAYANTS, N. AKOPYANTS, N.S. AL RIMAWI, F. AL DWIBE, H. AL HAZZA, I.M. AL JAWABREH, A. AL JAWABREH, H. AL KHALIFA, M.S. ALAGESAN, K. ALAKURTTI, S. ALAM, M.Z. ALAM, S. ALAM, S.M. ALARCON, G. ALBA, M. ALBAN, S. ALBANEZ, M. ALBANO AMORA, S.S. ALBERGANTE, L. ALBERICIO, F. ALBERIO, S.H.V.O. ALBEROLA, J. ALBERTINI, A. ALBUQUERQUE, A. ALBUQUERQUE, E.R. ALCAZAR, W. ALCOLEA ALCOLEA, P.J. ALCOLEA, P.J. ALCOVER, M.M. ALENCAR, A.A.
P 072, P 143 P 071, P 533 P 129 P 197, P 199 P 276 O 233, O 234 P 587, P 588 P 076 P 860 P 046, P 342 O 305 O 064 O 084, O 222, O 376 O 023 O 308 P 355 O 253 O 195, O 127, O 318 O 348 O 074, O 142, O 351 P 455 P 724 P 014 O 243, P 684 O 243, P 684 P 014 P 019 P 338 P 026, P 724 O 055 O 222 P 704 P 397, P 438 P 826 O 311 P 865, P 868, P 872, P 875 P 874, P 878, P 879 O 340 P 467 P 935, P 943 P 075, P 646 P 366 P 463, P 503 P 939 P 338 P 157 O 348 P 148, P 152 P 944, P 947 O 170, P 583
ALENCAR, C.H.M. ALEXANDER, N. ALEXANDRA, W.A. ALEXANDRE MOREIRA, M.S. ALEXANDRE MOREIRA, M.S. ALEXANDRE PIRES, G. ALEXANDRE, E.C. ALEXANDRE, J.L.A. ALEXANDRE, T.R. ALFANI, A.O.S. ALFONSO, E. ALFONZO, E. ALI, N. ALI, V. ALIANCA, A.S.S. ALIGIANNIS, N. ALIMOHAMMADIAN, M.H. ALIROL, E. ALKAN, M.Z. ALLA, L. ALLEGRETTI, S.M. ALLEN, R. ALLENSPACH, K. ALMEIDA ALVES, T.M. ALMEIDA NETO, W.S. ALMEIDA AMARAL, E.E. ALMEIDA SOUZA, F. ALMEIDA, A.F. ALMEIDA, A.J. ALMEIDA, A.P. ALMEIDA, A.S. ALMEIDA, B.F. ALMEIDA, C. ALMEIDA, E.L. ALMEIDA, G.G. ALMEIDA, J.A. ALMEIDA, J.F. ALMEIDA, L. ALMEIDA, L.F. ALMEIDA, M.A.C. ALMEIDA, M.C.M. ALMEIDA, M.S. ALMEIDA, N.A. ALMEIDA, N.J. ALMEIDA, R. ALMEIDA, R.A.
O 043, P 739, P 853 O 008 P 848 P 440, P 594 P 593 P 488, P 528, P 571 O 182, O 341 P 398 P 293, P 296, P 608, P 609, P 614, P 617 P 351, P 389 P 095 P 257 O 375 O 154 P 599, P 603 P 638, P 644 P 487 O 318 O 356 O 295 P 843 P 911, P 912 O 192 P 646 P 632 O 322 P 346, P 408 P 507 P 411 O 312 P 296, P 608, P 614 P 682 P 720 P 936, P 948, P 954 P 771 P 054 P 786 P 219, P 293, P 296, P 608, P 609, P 614, P 617 O 294, P 393 P 740 O 157, P 123 P 445 P 133 P 076 P 821 P 192 P 550 P 570 O 036, P 074, P 326 O 209, O 210, O 328 P 207
ALMEIDA, R.P. ALMEIDA, T.C. ALMEIDA, T.M. ALMEIDA, V.A. ALONSO, A. ALONSO, C. ALONSO, D.P. ALSAADI, M. ALVAR, J. ALVAREZ, E. ALVES FERREIRA, E.V.C. ALVES, A. ALVES, B.G.V. ALVES, C. ALVES, C.R. ALVES, E.B. ALVES, F. ALVES, F.P. ALVES, K.F. ALVES, M.A. ALVES, M.L. ALVES, M.L.R. ALVES, N.D. ALVES, N.M.E. ALVES, P.B. ALVES, P.H. ALVES, P.M. ALVES, R.J. ALVES, S.S. ALVES, T.M.A.
ALVES, W.A. ALZATE, J.F. AMARAL, A.C.F. AMARAL, D.N.D. AMARAL, E.E.A. AMAROUCH, H. AMATO, V.S. AMIRPOUR, S. AMIT, A. AMOAH, S. AMOR HAOUAS, N. AMORA, S.S.A. AMORIM, A.A.S.
P 323 P 250, P 456, P 481, P 740 P 073 P 421 P 265, P 411 P 639, P 719 P 148, P 152, P 157 P 113 O 354, P 057, P 173 P 450 O 106, O 202, O 016, O 230, O 380 O 267 P 198, P 312 P 476 P 516 P 629 P 629 P 065, P 248, P 550 P 689 O 365 P 588 P 538, P 553 P 488 P 915 P 292 P 917 P 194 P 069 P 865, P 868, P 872, P 874, P 875, P 878, P 879 P 609 P 481 P 504 P 747 P 297, P 623 P 643 P 698 P 516 P 885, P 889, P 891, P 894 P 871, P 886, P 888, P 890, P 897 O 283, O 307 P 507 P 445 P 571 P 545 P 016 P 269 O 168, P 435 P 701 P 063 P 487 P 908 P 678 P 439
AMORIM, C.F. AMORIM, I. AMORIM, I.F. AMPUERO, J.S. AMRI, F. AMRO, A. ANDERSON, B. ANDRADE FILHO, J.D. ANDRADE FILHO, J.D. ANDRADE NARVAEZ, F.J. ANDRADE NETO, V.V. ANDRADE, A.S.R. ANDRADE, B.B. ANDRADE, D. ANDRADE, D.R. ANDRADE, H.F.
ANDRADE, H.M. ANDRADE, J.D. ANDRADE, J.M. Andrade, J.M. ANDRADE, L.C. ANDRADE, L.D. ANDRADE, M.B. ANDRADE, M.C. ANDRADE, M.M.C. ANDRADE, M.S. ANDRADE, M.V. ANDRADE, P.H. ANDRADE, P.H.R. ANDRADE, P.P. ANDRADE, P.S. ANDRE, M.R. ANDRES, C. ANDREU, D. ANDREWS, N. ANDREWS, P. ANDRIC, B. ANDRIGUETTI, M.T.M. ANEES, M. AÑEZ, N. ANG, K.H. ANGEL, S.O. ANGULO, C. ANIBA, M.R. ANNAJAR, B.
P 865, P 868, P 872, P 875 P 678, P 874, P 878, P 879 P 309, P 310, P 317 P 606, P 839 P 134 P 355 P 092, P 455, P 684, P 724 O 348 P 798, P 812, P 846, P 893 P 818 P 647 P 120 P 552 O 294, P 416 P 446 O 051, P 136, P 187 P 022 P 622 P 206, P 213, P 537, P 562 P 066, P 200, P 214, P 349, P 795 P 057 P 442 P 339 P 099 P 149, P 498 P 118 P 496 P 398 P 293, P 614, P 789 P 824 P 520 P 362, P 376 P 842 P 176 P 190, P 249 P 628 P 877 P 475 O 348 O 340 O 013, P 426 P 748 O 124 P 836 O 285 P 341 P 438 O 345 P 724
ANNELIES, M. ANNUNZIATELLA, M. ANTONELLI, L.R. ANTONELLI, L.R.D.V. ANTONIALLI, S.A.C. ANTONIOU, M. ANVERSA, L. AOKI, J.I. AOUN, K. AQUINO, M.C.C. AQUINO, T.A. ARAGAO, M. ARAGAO, M.T. ARAMIN, S. ARANA, B.A. ARANA, F.E. ARANDIAN, M.H. ARANGO DUQUE, G. ARAUJO NETO, A.R. ARAUJO SANTOS, T. ARAUJO, A. ARAUJO, A.I.F. ARAUJO, A.M. ARAUJO, C.F. ARAUJO, F.A.P. ARAUJO, F.M. ARAUJO, I.B. ARAUJO, J.M. ARAUJO, J.M.P. ARAUJO, K.C. ARAUJO, L.H. ARAUJO, M.F.L. ARAUJO, M.I.A. ARAUJO, M.S.S. ARAUJO, M.V. ARAUJO, N.N.S.L. ARAUJO, P.S.R. ARAUJO, R.N. ARAUJO, R.S. ARAUJO, V.E.M. ARAUJO, V.P. ARCE, A. ARCE, L. AREVALO, J. ARGAW, D. ARGYROPOULOU, K. ARIELLA DERENGE, C.
O 146, P 451 P 048 P 165 P 253 P 808 O 135 P 462 P 637 P 355, P 484, P 656, P 790 O 119 P 402 P 428 P 546 O 151 P 740 O 216 P 605 P 605 O 195, O 127, O 318 O 034 P 572, P 619 P 322 O 051, P 013 P 840 P 293, P 296, P 608, P 614, P 617 P 054, P 056 P 282 P 854 P 935 P 939 P 719 P 880 P 815 P 176 P 211 P 395 P 631 P 601 P 141 P 516 P 194 P 292 P 488, P 528, P 571, P 593, P 594 P 921 P 149 P 239 O 317 P 935 P 816, P 945 P 900 P 521 P 769 P 008 O 139, P 144, P 397, P 438 O 351, O 254, O 267 O 202 P 487 O 320
ARISTIDES, S.M.A. ARJMAND, R. ARKIN, M. ARKIN, M.R. ARMAND, A. ARMELIM, I.M. ARONSON, N. ARORA, S.K. ARROYO, F.A. ARRUDA, C.C. ARRUDA, C.P. ARRUDA, E.S. ARRUDA, L.V. ARRUDA, M.M. ARRUDA, P. ARRUDA, S. ARSERIM, S.K. ASEFFA, A. ASELA, C. ASELA, C.X. ASHOURI, H. ASLAN, H. ASRAT, H. ASSIS LIMA COSTA, F. ASSIS, A.P. ASSIS, E.W.S.D.V. ASSIS, M.D.G.G. ASSIS, T.S.M. ASSUNCAO, R.S. ATA, A. ATASOY, A. ATELLA, G.C. ATHAUDA, I. ATTIA, H. ATTIPOE, I. AUGUSTO, J. AULNER, N. AURELIANO, D.P. AVELAR, D. AVELAR, D.M. AVERSI FERREIRA, T.A. AVILA, G. AYALA URDAPILLETA, A.A. AYALA, M. AYALA, M.S. AYAZ, S. AYEH KUMI, P. AZADMANESH, K.
P 554 P 301, P 913 O 306 O 285 P 624 P 955 O 160 P 114 P 918 P 202 P 521 P 093 P 093 O 296, P 929 P 884 P 480, P 527 O 295 P 934 O 364 O 365 P 621 O 160, O 161, O 320, O 026 P 627 P 091, P 341 P 759 P 803, P 807 P 760, P 762 P 358 P 664 P 272 O 321 P 166 P 670 P 058, P 059, P 922 P 946 O 372 O 284 P 431 P 145 P 700, P 703, P 764 P 828 O 380 P 105 P 709 P 536 O 124 P 946 O 239
AZEREDO COUTINHO, R.B.G. AZEVEDO JUNIOR, J.W.C. AZEVEDO, C.G. AZEVEDO, E.G. AZEVEDO, F.S. AZEVEDO, L. AZMI, K. B, V.M. B. DE JESUS, J. B. LUCHEIS, S. BABBA, H. BABIKER, D.E. BACCI, L. BACELLAR, M.O. BACELLAR, O. BACELLAR, O.A. BACHELDAR, E.M. BACHMAIER, S. BAEZ, P. BAEZA RAMIREZ, I. BAFICA, A. BAFICA, A.M. BAGAR, S.R.A. BAHARIA, R. BAHARIA, R.K. BAHIA, A.P.C.O. BAHIA, D. BAHIA, E.M. BAHIA, M. BAHIA, M.T. BAHRIANI, M. BAIAO, A. BAILEY, M. BAILONA, G.H.C. BAILOR, H.R. BAKHIET, S.M. BALASEGARAM, M. BALASEGRAM, M. BALDEVIANO, G.C. BALEELA, R. BALLART, C. BALNO, C. BALTAZAR, F.H. BALTUSSEN, R. BANDARA, S. BANDARRA, M.B. BANDEIRA, C. BANDEIRA, D.S.
O 274, O 165 P 468 P 725 P 606, P 839 O 082 P 845, P 847 P 179, P 804, P 811 P 392 O 290, P 455, P 671, P 684 O 238 P 243 P 863 P 140, P 905, P 908 P 288 P 481 P 021 O 272 O 278, P 023 P 294 O 308 P 101 P 825 P 595 O 291 P 141 P 353 P 043 O 089, P 041 P 381 P 393 O 030 P 630, P 931, P 932 O 180 P 795 P 591 P 179, P 804, P 811, P 847 P 061 P 856 O 092, O 027 P 288 P 627, P 691, P 692 O 356 O 351 P 835 O 102 P 944, P 947 O 131 P 054 P 691, P 692 P 670 P 177, P 190 P 643 P 681
BANDEIRA, E.L. BANDEIRA, M.C.A. BANDINI, G. BANDOPADHYAY, S.K. BANIC, D.M. BANIHASHEMI, M. BANKOTI, R. BANSAL, M. BANULS, A.L. BAÑULS, A.L. BAQUERO, O.S. BAQUI, M.M.A. BARALDI, S.R. BARATA, I.R. BARBAS, C. BARBIAN, K.D. BARBIERI, C.L. BARBO, M.L.P. BARBOSA DOS, M.L. BARBOSA JR, W.L. BARBOSA, A.D. BARBOSA, D.S. BARBOSA, F.M.C. BARBOSA, G.L. BARBOSA, I.R. BARBOSA, J.A.R. BARBOSA, J.C. BARBOSA, J.C.O. BARBOSA, J.E.R. BARBOSA, J.R. BARBOSA, M.G.V. BARBOSA, P.R.A. BARBOSA, S.R. BARBOSA, V. BARBOSA, W.O. BARCELLOS, N.M.S. BARCINSKI, M.A. BARHOUMI, M. BARHOUMI, W. BARKER, J.H. BARON, S. BARRA, A.
BARRAL NETTO, M. BARRAL, A.
P 006 P 673, P 734 O 075 P 802 P 210 P 340 O 371 P 449 O 366 P 140 P 305, P 306 P 937, P 938 P 216 P 489 P 864 P 373, P 461 P 029 P 175, P 350 O 030 P 955 P 261 P 498 P 552 P 708, P 723 P 156 O 377 P 830 P 767 P 924 P 925 P 926, P 927 P 767 P 489 P 929 P 583 P 956 P 865, P 868, P 872, P 874, P 875, P 878, P 879 P 309 P 842 P 509 P 033 P 044 P 403, P 517 P 654 O 302 P 944 P 024 O 238, O 291, P 038, P 093, P 118, P 132, P 187, P 721, P 844 O 238, O 326, O 250, O 291, P 038, P 118, P 132, P 166, P 187, P 198, P 223, P 283, P 343, P 618, P 844
BARRAL, M.A.D.P. BARREIRO, E.J. BARREIRO, E.J.L. BARREIROS, D.C. BARRETO, A.S. BARRETO, T. BARRETO, T.V. BARRIO, A.B. BARRIOS, M.A. BARROS, A.S. BARROS, J.A.C. BARROS, N.B. BARROS, T.L. BARROSO, P.A. BART, A. BART, O. BARTHOLOMEU, D.C. BARUD, H.S. BASAGLIA, F.C. BASOMBRIO, M.A. BASSO, M.A. BASTIEN, P. BASTOS, C.E. BASTOS, L.A. BASTOS, V.J.C. BATE, W. BATES, M.D. BATES, P. BATES, P.A. BATHIA, A. BATISTA, A.J.S. BATISTA, A.P. BATISTA, J.A.S. BATISTA, J.F. BATISTA, L.R. BATISTA, M.A. BAUGH, L. BAVIA, M.E. BAYIH, A.G. BEATTIE, L. BECKER, K.M. BECKMANN, A.M. BEGO, A. BEHAR, P.R.P. BEIJNEN, J.H. BEITING, D.P. BEJARANO, E.E. BELAUNZARAN, M.L.
P 721 P 571 P 488 P 833 P 576 P 310 P 245 O 038 P 914 P 892 P 760 P 108 P 780 O 038 P 485 O 255, P 061 O 163 O 257, P 035, P 181, P 244, P 486, P 491 P 433 P 231 P 485 O 038 P 772 O 014, O 221, O 351 P 883 P 391 P 675 P 055, P 697 P 946 O 332, O 042, O 318 O 158, O 194 P 946 P 576 P 555 P 795 P 489 O 198 P 462, P 781, P 803, P 807 P 509 P 138 O 107, O 348 O 039 O 100, P 833 O 117 O 340 P 523 O 093 P 295 P 870 O 078, O 111 O 241, O 087 P 951 P 334
BELAY, S. BELHAJ HMIDA, N. BELIZARIO, D. BELL, A.S. BELLO, A. BELLO, A.R. BELLO, F.J.G. BELO, V.S. BELOTI, C.C. BEN ABDA, I. BEN ABDERRAZAK, S.B. BEN AHMED, M. BEN KHALAF, N. BEN MESSAOUD, N. BEN SGHAIER, I. BEN ABID, M. BEN OTHMAN, R. BEN SGHAIER, I. BENASSI, J.C. BENCHIMOL, M. BENETI, S.A.A. BENHELEL, K. BENIE, G. BENIGNO, C.V. BENITEZ, A.D.N. BENKAHLA, A. BENSAID, M. BENTES, P. BENTO, E.C. BERCINI, M.A. BERG, M. BERGMANN, B.R. BERL, F.P. BERMUDEZ, H. BERNARD, H.I. BERNARDINETTI, U. BERNARDINO, A.M.R. BERRICH, M. BERRIMAN, M. Berriman, M. BERROSPE, P.E. BERROZPE, P. BERROZPE, P.E. BERTHO, A.L. BERTOLLO, C.M. BERTOLLO, D.M.B. BESCHIN, A. BETTAIEB, J.
O 216 P 942 P 153 P 909, P 910 O 152 O 342 P 217 P 065 P 723 P 683, P 708 P 540 P 355 P 586 P 153, P 958 O 306 O 363 P 355 P 484 O 345 P 484 P 539 P 103 P 631 P 355 O 125 P 695 P 713, P 715 P 059 P 501 P 189 P 781 P 870 O 143, P 382, P 384, P 385, P 386 P 162, P 304, P 407, P 545, P 547, P 559, P 574, P 575 P 371 P 380 P 008 P 957 P 427, P 523 O 124 O 002, O 143, P 672 O 298 O 344 P 778 P 814 P 806 P 228 P 238 P 150, P 237 P 632 P 857 P 101 P 942 P 916 P 305, P 306, P 922
BETTAÏEB, J. BETTS, M.R. BEVERLEY, S. BEVERLEY, S.M. BEZERRA DE MENEZES, J.G.P. BEZERRA, A.C.D.S. BEZERRA, C.B. BEZERRA, F.M.B.V. BEZERRA, G.F.B. BEZERRA, I.O. BEZERRA, I.P.S. BEZERRA, M.E. BEZUNEH, A. BHANDARI, V. BHANDERI, P.K. BHASKAR, B. BHASKAR, K.R.H. BHATIA, A. BHATTACHARJEE, H. BHATTACHARYA, P. BHATTACHARYA, S.M. BHATTACHARYYA, T. BHATTARAI, N. BHATTARAI, N.R. BHOWMICK, S. BHUIYAN, A.T. BIANCO, E.M. BICHAUD, L. BIFELD, E. BIGELI, J.G. BILCH, K. BIMAL, S. BINO, S. BIONDO, A.W. BISETTO JR., A.B. BISETTO JUNIOR, A. BISOL, J. BISWARO, L. BITTAR, R.C. BITTENCOURT, C.R. BIVONA, M. BLACKBURN, G.J. BLACKWELL, J. BLACKWELL, J.M. BLAKE, D. BLANCO, V.M. BLANDON, G.A.
O 125 O 087 O 297, O 348 O 074, O 075, O 142, O 351, P 256, P 277 O 129 O 052, P 112, P 115, P 116 P 212 P 678 P 303 P 565 O 112 P 436, P 729, P 730, P 732, P 733 P 956 O 116, P 208 P 897 O 216, P 610 P 363, P 364 P 753 P 003 O 266 O 093, P 610 O 004 O 026 P 388 O 265 O 143, O 078, P 053, P 672 O 111, O 067, P 383, P 386, P 679 O 154 O 154 O 109 P 638, P 644 O 194 O 105 P 676 P 410 P 063 P 599 P 295, P 611, P 843 P 395, P 852 P 776 O 096 P 826 P 854 P 500 P 269 P 561 O 187 P 053 P 133, P 264 O 343 O 352 O 268, P 011 P 646 O 147 O 099, P 737 P 589
BLANK, A.F. BLAYNEY, B. BLONDEL, A. BLOOM, J. BLUM, J. BOAKYE, D. BOAMPONG, J. BOAVENTURA, V. BOCCHI, M.R.
BOELAERT, M. BOELART, M. BOGAART, E.V.D. BOGDAN, C. BOGDAN, C.M. BOGGILD, A. BOGGILD, A.K. BOIREAU, P. BOITE, M.C. BOIX, E. BOLAS, A.S. BOLETI, H. BOLHASSANI, A. BONANO, V. BONFIETTI, L.X. BONFIM, T.D. BONGIORNO, G. BONGRAND, P. BORCHANI, L. BORCHICHI, S.R. BORDA, C.E. BORDIGNON, M.O. BORDONI, M. BORGES, A. BORGES, A.C.J. BORGES, D.T. BORGES, R. BORGES, V. BORGES, V.M. BORJA CABRERA, G.P. BORJA, L.S. BORST, P. BOSHART, M. BOSQUIROLI, L.S. BOSQUIROLI, L.S.S.
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BOSSCHAERTS, T. BOTELHO, H.A. BOTT, E. BOU HAYA, J.V. BOU HABIB, D.C. BOUDABOUS, R. BOUILLET, L.E.M. BOUILLET, L.M. BOUJAAMA, A. BOULET, G. BOULORIZAEDH, M. BOURATBINE, A. BOURDEAU, P. BOURDOISEAU, G. BOURREAU, E. BOUSSLIMI, N. BOUSSOFFARA, T. BOUTY, L.F. BOUTY, L.F.M. BOUVIER, G. BOWES, D.E. BOZZA, M. BOZZA, M.T. BOZZA, P.T. BRACHER, F. BRAGA, D.S.F.S. BRAGA, E.T. BRAGA, M.S. BRAGA, S.L. BRAILLARD, S. BRANCAL, H. BRANDAO FILHO, S.P. BRANDAO FILHO, S.P. BRANDAO, N.A.A. BRANDAO, R.C.F. BRANDAO, S.T. BRANNIGAN, J.A. BRAS GONCALVES, R. BRASIL, H.H. BRASIL, R. BRASOV, I.E. BRAZ, J.M. BRAZIL, R.P. BRAZUNA, J.C.M. BREITLING, R. BRENNAND, A. BRESCIANI, K.D.S.
O 342 P 798 P 334 P 725 P 183 P 908 P 434 P 135, P 242 P 067 P 654 P 452 P 591 P 355, P 656 P 484, P 790 O 221 O 221 O 273 P 790 P 153 P 499 P 502 P 403 O 265 P 118, P 136 P 446 O 051, P 013 O 275 P 399, P 414 P 162 P 054, P 056, P 686 P 143 P 143 P 533, P 816 P 636 P 596, P 642 O 293 P 659 P 850 P 906 P 789 O 009, O 128 P 609 P 219, P 293, P 296, P 330, P 335, P 585, P 608, P 614, P 617, P 650, P 940, P 941 P 613 P 902 P 821 O 152 P 159 P 209, P 211 P 160, P 955 P 518, P 607 P 261 P 057, P 065, P 746, P 818 O 296 P 384 P 101 P 402
BRETTMAN, E. BRILHANTE, A.F. BRILHANTE, M.A. BRIONES, M.R. BRITO, A.M.G. BRITO, J.F. BRITO, L.P.S. BRITO, M.E.F. BRITO, R.C.F. BRITO, R.S. BRODSKYN, C. BRODSKYN, C.I. BRUCE, K.K.M. BRUNK, B. BRUNO, F. BRUSTOLONI, Y.M. BRYCESON, A. BUENO, L.L. BUEREN, J.A. BUFFET, P. BUFFET, P.A. BULA, I. BUMB, R.A. BURCHMORE, R. BURCHMORE, R.J. BUREL, A. BURRUS, R.G. BURZA, S. BUSSOLOTTI, A.S. BUTAUD, T. BUTLER, C.D. BUTT, W.A. BYRD, J. C, J. C. MOREIRA, P.A. C. RODAS, L.A. CABEZAS, Y. CABRAL DE FRANCA, T.M. CABRAL, G. CABRAL, L. CABRERA, L. CABRERA, M. CABRERA, R. CACERE, C.R. CACERES, O.C. CADENA, H. CAETANO, R.C.S. CAFFARENA, E.R.
O 074, P 277 P 838 P 171 O 030 P 612 P 765 P 322 P 906 P 585 P 010, P 293, P 296, P 411, P 608, P 609, P 614, P 617, P 850 P 135 P 170 P 038, P 136 P 187 O 275, P 036 O 238, P 223 P 923 O 142 P 064, P 400 P 569, P 579 O 201 O 257, P 486, P 491 P 525 O 085, O 362, O 363, P 061 P 457 P 722 O 171 P 356 P 100 O 033 O 190 P 680 O 108, O 356, O 057, O 261 P 759 P 068 O 062 P 690 O 369 O 238 P 876 P 863 O 190 P 860 O 341 P 463 O 267 P 127, P 203, P 298, P 302 P 835 P 231 P 345 O 011, O 196, O 380, P 904, P 910 P 233 P 550
CAGUANO CAZA, W.A. CALABRESE, K.S. CALDART, E.T. CALDAS, I. CALDEIRA, R.D. CALDERON, W. CALEARE, A.O. CALEGARI SILVA, T.C. CALHEIROS, M.B. CALIARI, M.V. CALOGEROPOULOU, T. CALVO BADO, L. CALVO BADO, L.A. CALZADA, J. CALZADA, J.E. CAMARA, A. CAMARA, C.A. CAMARA, F.V. CAMARASA, M. CAMARASA, M.J. CAMARGO NEVES, V. CAMARGO NEVES, V.L. CAMARGO, D.G. CAMARGO, L.M.A. CAMELO, N.L. CAMERA, C.A. CAMPAGNARO, G. CAMPI AZEVEDO, A.C. CAMPILLO GIMENEZ, L. CAMPINO, L. CAMPOS SALINAS, J. CAMPOS SANTOS, E.C. CAMPOS, A.L.B. CAMPOS, D. CAMPOS, J.M. CAMPOS, M.B. CAMPOS, S.P. CAMPOS, T. CAÑADAS, K.E.B. CANAVACI, A.M.C. CAÑAVATE, C. CANAVEZ, F. CANCINO, B. CANDANEDO GUERRA, R.M.S.N. CANDLISH, A.A. CANESE, A. CANGUSSU, S.
P 918 P 139 P 192, P 507 P 713, P 715, P 716, P 787, P 826 O 180 P 864, P 867 P 535 P 625 O 037 P 885, P 891, P 894 P 886 P 888, P 889, P 890, P 897 P 316 O 310 O 197, P 540 P 544 P 146, P 895 P 009 O 180 P 593 P 640 P 865, P 872 O 307 P 529 O 377 P 869 P 686 P 345 P 907 P 492 P 357, P 666 O 276 O 330 O 219, P 163, P 453, P 463, P 503, P 805, P 887 P 417 P 563 P 935 P 343 P 143 P 167, P 169, P 193, P 212, P 424 P 568 O 240 P 918 P 165 P 018, P 253 O 215, P 934 P 082 P 722 P 685 P 450 P 919 P 310
CANNONE HERGAUX, F. CANTANHEDE, L.M. CANTILLO, O. CANTINHO JUNIOR, J.J. CANTO CAVALHEIRO, M.M. CANTO CAVALHEIRO, M.M.D. CANUTO, G.A.B. CAPITANI, C.O. CAPPARELLI, F.E. CAPPELLAZZO, A.C. CAPPIELLO, S. CARANHA, L. CARAZZOLLE, M.F. CARBAJO, D. CARCELEN, J. CARDIM, L.L. CARDINOT, C.B. CARDOSO, C.R. CARDOSO, F.A. CARDOSO, F.O. CARDOSO, J.M.O. CARDOSO, L. CARDOSO, L.S. CARDOSO, M. CARDOSO, R.M. CARDOSO, T.M. CARDOZO, R.M. CARGNELUTTI, D.E. CARIDHA, D.P. CARILLO, E. CARIOCA, S.M. CARLOS, S.M. CARLSEN, E.D. CARMO, R.F. CARN, G. CARNEIRO FEIJO, F.M. CARNEIRO, A.L. CARNEIRO, C.M. CARNEIRO, D.D.M.T. CARNEIRO, G. CARNEIRO, J.C. CARNEIRO, L.A. CARNEIRO, M. CARNEIRO, M.P.D. CARNEIRO, M.W. CARNES, J. CARNIELLI, J.B.T. CAROLLO, C.A.
P 262 P 747 P 179, P 804 P 709 P 319 P 120, P 346, P 408, P 523, P 563 P 421 P 373 P 499 O 157 P 404 O 292 P 876 O 030 P 467 P 113 O 039, O 100 P 771 P 687 O 238 P 764 O 296, P 929 P 139 O 091, P 067, P 072, P 714 O 293 P 476, P 503, P 726, P 777 P 141 O 166 P 428, P 546 P 021 O 038 P 104 O 114 O 293 P 813, P 892 P 297 O 053 O 374 O 365 P 865, P 868, P 872, P 874, P 875, P 878, P 879 P 570 O 257 O 091, P 067, P 071, P 072, P 129, P 434, P 714 O 039, O 100 O 116 P 677 P 422, P 855 P 900 P 071, P 434, P 533, P 816, P 903, P 954 O 156 P 022 P 113 P 349 P 521, P 543
CARPINTERO, J. CARRASCO, N. CARREGAL, V.M. CARREGARO, V. CARREIRA, L.M. CARREIRA, V.S. CARREﾃ前, H. CARRIJO, R.S.G.G. CARRILLO, E. CARRILLO, L.M. CARRILO, E. CARSON, C. CARTER, K. CARTER, K.C. CARTER, N. CARVALHAL, D.G.F. CARVALHEIRO, M. CARVALHO ANDRADE, A. CARVALHO DANTAS, I.M. CARVALHO, A.K. CARVALHO, A.M. CARVALHO, A.S. CARVALHO, A.W.S. CARVALHO, B.M. CARVALHO, C. CARVALHO, D.M. CARVALHO, E.
P 769 O 016 P 416 O 210, O 328 P 772 P 634 P 448 P 556 P 725 O 215, O 016, O 046, P 938 O 011, O 280, P 904 P 937 O 197, P 513 P 356 O 115 P 053 P 450 O 348 P 207 O 312, O 336, P 508 P 105 P 266 P 267 P 142, P 793 O 250 P 023, P 024 P 163 P 906 P 705 P 780 O 170, P 583 O 240, O 351, P 264
CARVALHO, E.M. CARVALHO, F.A.A. CARVALHO, F.G. CARVALHO, F.S. CARVALHO, G.M.L. CARVALHO, K.I. CARVALHO, K.I.L. CARVALHO, L. CARVALHO, L.O.P. CARVALHO, L.P. CARVALHO, P. CARVALHO, P.S.L. CARVALHO, R.M.A. CARVALHO, R.S. CARVALHO, S. CARVALHO, S.F.G. CARVALHO, S.M.S. CARVALHO, S.S.
O 233, O 277, O 050, P 128, P 204 O 241, O 272, P 294 P 480 O 278, O 166, O 169, P 021, P 023, P 024, P 087, P 134, P 141, P 337, P 423, P 527, P 590, P 785 P 133 P 689 P 906 P 928 P 655 P 800 P 193 P 142 O 240, O 278 P 192 P 204 P 294 O 241, O 272, O 087 P 023 P 243 P 362 P 721 O 170, P 583 O 182, P 185 O 070 P 496 P 953 P 261, P 456
CARVALHO, T.B.F. CARVALHO, Y. CASANOVA, C. CASARIL, A.E. CASTAGNINO, M. CASTANYS, S. CASTELAO, K.G. CASTELLI, G. CASTELLS, X. CASTELLUCCI, L. CASTELO BRANCO, R.C. CASTIGLIONI, P. CASTILHO MARTINS, E.A. CASTILHO, R.O. CASTILHO, T.M. CASTILHO, W.S. CASTILLEJO, S. CASTRO BORGES, W. CASTRO OLIVEIRA, R. CASTRO, A.A.P. CASTRO, E.A. CASTRO, E.V. CASTRO, H. CASTRO, J.M. CASTRO, J.S.M. CASTRO, L. CASTRO, L.G.M. CASTRO, M.C.A.B. CASTRO, N. CASTRO, R. CASTRO, R.A.O. CASTRO, R.A.O.E. CASTRO, R.O. CASTROVINCI, P. CASTROVINCI, P.A. CATARINO, C.M. CATCHPOLE, B. CATHERINE, R. CAUAS, E.M.C. CAUMES, E. CAVALCANTE DE OLIVEIRA, A. CAVALCANTE SILVA, L.H.A. CAVALCANTE, G.G. CAVALCANTE, T.V. CAVALCANTI, A.S. CAVALCANTI, M.P. CAVALCANTI, S.M.M. CAYLA, M.
P 209, P 211 P 098 P 746, P 748, P 841 P 057, P 823, P 832, P 846, P 893 P 707 P 413, P 417 P 601 P 064, P 400 P 944 P 133, P 264 P 436, P 729, P 730, P 732, P 733 O 035, O 052, P 112 P 373 O 129 P 376 P 520 P 180 P 080, P 718 P 944 P 126, P 143 P 197 P 856 O 096 P 236 P 098, P 441, P 444, P 742 P 761 O 335 O 070, O 188, O 059, P 262, P 471 P 728, P 834 P 817 P 283 P 341 P 411, P 566 P 429 P 311, P 317 P 509 P 126 O 082 P 807 O 320 O 161 P 738 O 213 O 052 P 616 P 457 P 198 P 571 P 864, P 867 P 856 O 184 P 335, P 906 P 293 O 205
CEBALLOS, A.L. CEDEÑO, D.L. CELES, F. CELES, F.S. CELESTE, B.J. CELESTE, J.L. CELESTE, J.L.L. CEN, S. CERDA, R.M.G. CERIZE, N.P. CESETTI, M.V. CEZAR, T.L. CHAARA, D. CHAGAS, A.P. CHAGAS, B.D. CHAKRABARTI, S. CHAKRAVARATY, J. CHAKRAVARTY, J. CHAKROUN, A. CHAKROUN, A.S. CHALGHAF, B. CHAMAKH AYARI, R.C. CHANCE, M.L. CHANDRAWANSA, P.H. CHANG, K.P. CHAOUCH, M. CHAPPUIS, F. CHARREL, R. CHATELAIN, E. CHAUDHARY, K. CHAUDHARY, R. CHAVES, C.S. CHAVES, L.F. CHAVES, S.A.M. CHAVES, S.P. CHAVEZ FUMAGALLI, M.A. CHAVEZ FUMAGALLIA, M.A. CHECA, R. CHELBI, I. CHEMKHI, J. CHEN, H. CHEN, S. CHENIK, M. CHERNI, S. CHICHARRO, C. CHILDERSTONE, M. CHIÑAZ PEREZ, A. CHIOCCOLA, V.L.P.
O 365 O 283, P 327, P 328 O 326 P 433 P 446 P 341 P 795 P 200 P 377 P 918 P 407 P 648 P 787 P 140 P 949, P 952 P 818 P 030 P 186 O 315, O 163, P 478, P 582 O 138 P 096 O 125 O 119 O 158 O 217 O 329, O 288, O 028, P 026 P 449 P 586 O 356 O 269 O 194 P 636, P 642 O 104 P 063 P 171, P 174 P 895 P 415, P 725, P 756 P 574 P 362 P 018, P 123, P 520 P 226 O 342 P 654 O 125 O 194, O 289 O 285 O 208, O 306, O 119 P 654 O 106, O 215, O 068, P 188, P 934 P 366 P 882 P 160
CHIRINOS, Y. CHIYO, L. CHLIF, S. CHOO, A.Y.L. CHOO, Y.L. CHOUDHURY, T.A. CHOUHAN, G. CHOURASIA, A. CHOWDHURY, R. CHOWDHURY, S. CHOWDHURY, T.A. CHOWDHURY, V. CHRISTODOULOU, V. CHROBAK, M. CHUSRI, S. CIANCAGLINI, P. CIARLINI, P.C. CILLI, M.L.C. CLARK, S.C. CLOKE, T. CLOS, J. CLOSS, E. CLOUTIER, S. COELHO NETO, G.T. COELHO FINAMORE, J. COELHO, A.C. COELHO, C.H. COELHO, E.A. COELHO, E.A.F. COELHO, E.F. COELHO, G. COELHO, G.M. COELHO, Z.C.B. COHEN, S. COIMBRA, E.S. COIMBRA, M. COLER, R.N. COLHONE, M.C. COLLA JACQUES, F.E. COLLAVERI, J. COLLOVATI, M. COLOMBO, F.A. CONCECAO SILVA, F. CONCEICAO SILVA, F. CONDE, C.A. CONDELO, H.S. CONRADO DOS SANTOS, W.L. CONSORTIUM, S.
P 259 P 826 O 125, P 305, P 306, P 942 O 367 P 110 O 314, P 665 P 466 O 015, P 478, P 749 P 753 P 359, P 365, P 401 O 222 P 753 O 135 O 105 P 662 P 108 P 398 P 808 P 319 O 133, P 410 O 001, O 006, O 105, O 149 O 133 O 101 P 212 O 051 P 405 P 253 P 226 P 191 P 375 P 123, P 253, P 362, P 376 P 018, P 520 O 157 P 629 P 145 P 174 O 003 P 459, P 568 P 887 O 092, O 093, O 027 P 108 P 748, P 841 P 746 P 600 P 615 P 505 O 122 O 237, P 037, P 090, P 164 P 560, P 573 P 083 P 522, P 524, P 526 O 345, P 058, P 059
CONSTANTINO, C. CONTER, C.C. CONTRERAS, M.A. CONVIT, J. COOMBS, G.H. CORBETT, C.E.P. CORBETT, C.P. CORDEIRO DA SILVA, A. CORDEIRO, J.L.P. CORDEIRO, J.P. CORDOVA, G.R. CORREA JUNIOR, J.D. CORREA OLIVEIRA, R. CORREA, I.A.A.Q. CORREA, J.R. CORREA, M. CORREA, P.R. CORREA, Z.J. CORSET, C. CORSET, M. CORTES BOITE, M. CORTES, L.M.C. CORTES, S. CORTESE, L. CORTEZ MARCOLINO, L.M. CORTEZ, A.M.C. CORTEZ, D.A.G. CORTEZ, L.R.C. CORTEZ, S.V. CORVO, M.L. CORWARE, K. CORWARE, K.D. COS, P. COSSIO, A. COSTA E SILVA, M.F. COSTA FERREIRA, M.E. COSTA FERREIRA, V.S. COSTA RIBEIRO, M.C.V. COSTA SILVA, M.F. COSTA SOUZA, B.L.S. COSTA VAL, A.P. COSTA, A.A. COSTA, A.J. COSTA, A.P. COSTA, A.S.N. COSTA, B.H. COSTA, B.L. COSTA, C.
P 713, P 716 P 554 O 252, P 899 P 951 P 797, P 909 P 053, P 450 O 033 P 142, P 146, P 167, P 169, P 212, P 291, P 793 P 193, P 422, P 424 O 330, O 372, O 293, P 079, P 476, P 519, P 726 P 696 P 699 P 918 O 081 P 242, P 533 P 588 P 556 O 361 P 065 P 424 O 133 O 133 P 711 P 065 O 219, P 163, P 463, P 503, P 805, P 887 P 048 P 354 P 897 P 889 P 848, P 885, P 886, P 890, P 894 P 871 P 421 P 848 P 871 P 955 O 312 O 133, P 410 P 437 P 452, P 460 O 099, O 258, P 737, P 859 P 069 P 738 P 166 P 236 P 738 O 276, P 643 P 208 O 116 O 186 P 765 P 788 P 759 P 764 P 685 P 758, P 764 P 191 P 304 O 349
COSTA, C.H.N. COSTA, D. COSTA, D.C. COSTA, D.L. COSTA, E.C. COSTA, E.M. COSTA, F.B. COSTA, G.F. COSTA, J. COSTA, J.F. COSTA, J.L.F. COSTA, J.M. COSTA, J.R.A. COSTA, K.F.L. COSTA, L. COSTA, L.D. COSTA, L.E. COSTA, L.H.C. COSTA, M.A. COSTA, M.D. COSTA, N.A. COSTA, P.L. COSTA, P.R.R. COSTA, R. COSTA, S.M. COSTA, T. COSTA, T.A. COSTA, T.F.R. COSTA, T.R. COSTA, V. COSTA, W.A. COTA, B.B. COTA, G.F. COTOVIO, M. COTRIM, P. COTRIM, P.C. COTTON, J. COTTON, J.A. COUBEU, S.B. COURA VITAL, W. COURTENAY, D.A.O. COURTENAY, O. COUTINHO COSTA VELOSO, O.M. COUTINHO SILVA, R.
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COUTINHO, O.M.V.C. COUTINHO, S.G. COUTO, L.S. COVAS, C.J.F. CRAVO, P. CREPALDI, P.H. CRISANTE, G. CRISTAL, J. CRISTAL, J.R. CRISTALDO, G. CRISTIANO, M.L. CRISTOVAO, E.C. CRISTOVAO, J.M. CROFT, S.L. CRUZ, A. CRUZ, A.K. CRUZ, A.S. CRUZ, E. CRUZ, I. CRUZ, J. CRUZ, K.P. CRUZ, M. CRUZ, M.D.S.P. CRUZ, M.D.S.P.E. CRUZ, M.E. CRUZ, M.E.M. CRUZ, M.S. CRUZ, M.T. CRUZ, T. CUENCA, M. CUERVO ESCOBAR, S. CUERVO, P. CUIN, A. CUISINIER, A. CULL, B. CUMAN, R.K.N. CUNHA DO NASCIMENTO, M.T. CUNHA JUNIOR, E.F. CUNHA NETO, E. CUNHA, C.F. CUNHA, G.D. CUNHA, G.M.R. CUNHA, G.V.D. CUNHA, J. CUNHA, J.C. CUNHA, J.C.L. CUNHA, J.L.
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CUNHA, J.L.R. CUNHA, J.M. CUNHA, P.N.A. CUNNINGHAM, J. CUPOLILLO, E. CUSTODIO, E. CUSTODIO, M.G.F. CUTOLO, A.A. CUYPERS, B. D, A.K. DA CONCEICAO, W.M. DA COSTA LIMA, T.D. DA COSTA SILVA, T.A. DA COSTA, A.G. DA COSTA, A.P. DA COSTA, C.M. DA COSTA, E.C. DA COSTA, J.B.N. DA COSTA, L. DA COSTA, L.B. DA COSTA, L.D. DA COSTA, R.V. DA COSTA, S.C.G. DA CRUZ, A.M. DA CRUZ, N.C.A. DA CRUZ, P.F. DA CUNHA, J.P. DA FONSECA, A.C. DA GRACA, G.C. DA MATTA, C.B.B. DA MOTTA, J.C. DA MOTTA, J.O.C. DA ROCHA, D.R. DA ROCHA, W.D. DA SILVA JUNIOR, V.A. DA SILVA, A. DA SILVA, A.D. DA SILVA, A.E. DA SILVA, A.F. DA SILVA, A.J.M. DA SILVA, C.V. DA SILVA, D.T. DA SILVA, E.D. DA SILVA, E.O. DA SILVA, E.S. DA SILVA, F.J. DA SILVA, F.M.M.
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DA SILVA, G.O. DA SILVA, G.P. DA SILVA, I.S. DA SILVA, J.C. DA SILVA, J.R. DA SILVA, J.S. DA SILVA, K.L.O. DA SILVA, L.C. DA SILVA, L.C.O. DA SILVA, L.H.P. DA SILVA, L.V. DA SILVA, M.F.L. DA SILVA, M.R. DA SILVA, S.M. DA SILVA, T.A.M. DA SILVA, V.M.G. DA SILVEIRA NETO, M.M. DA SILVEIRA PERES, L.L. DA SILVEIRA, J.F. DA CRUZ, A.M. DA SILVA, S.A. DABIRI, S. DABIRIAN, S. DADO, D. DAGGER, F. DAGNE, D.A. DAIFALLA, N.S. DALAGRANA, L. DALIGAULT, F. DAMAJ, S. DAMASCENA, N.P. DAMASCENO, A.R. DAMASCENO, L. DAMASCENO, L.M. DAN GOOR, M.E. DANCKAERT, A. DANDREA, A. DANTAS PEREIRA, P.D. DANTAS TORRES, F. DARABI, M.H. DARDONVILLE, C. DARGHOUTH, M. DARVISHI, M. DAS, A. DAS, M.L.
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DAS, P. DAS, P.K. DAS, S. DAS, V. DAS, V.N. DAS,, S. DASH, A.P. DASKALAKI, A. DAVID, C.C. DAVID, J.R. DAVIES, M. DAZA, J.A. DE ALMEIDA LEITE, J.R.S. DE ALMEIDA, A.B. DE ALMEIDA, A.C.O. DE ALMEIDA, A.F. DE ALMEIDA, A.P. DE ALMEIDA, A.P.F. DE ALMEIDA, E.L. DE ALMEIDA, L. DE ALMEIDA, M.C.C. DE ALMEIDA, R.P. DE ALMEIDA, T.M. DE AMORIM, I.F.G. DE ANDRADE PINTO, P.C.E. DE ANDRADE, A.J. DE ARAUJO, C.N. DE ARAUJO, E.A.C. DE ARAUJO, J.M.P. DE ARAUJO, K.C.L. DE ARAUJO, V.C. DE ARAUJO, V.C.P. DE ARRUDA, C.C.P. DE AZEVEDO, A.C.R. DE AZEVEDO, A.F. DE AZEVEDO, R.B. DE BARROS, N.B. DE BRITO, C.F.A. DE BRITO, M.E.F. DE BRITO, R.F. DE CAMARGO, R.A. DE CARVALHO, A.T. DE CARVALHO, G.Q. DE CARVALHO, G.S. DE CARVALHO, L. DE CARVALHO, L.C.P. DE CARVALHO, L.M.
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DE CARVALHO, M.R. DE CARVALHO, T.G. DE CASTRO FREITAS, T.M. DE CASTRO NETO, A.L. DE CASTRO, E.M. DE CASTRO, M.C.A.B. DE CELIS, H.A. DE CROP, M. DE DONCKER, S. DE FARIAS, D.M. DE FIGUEIREDO, H.R. DE FIGUEIREDO, L.C. DE FRANCA MENDES, F.G. DE FRANCA, S.H.F. DE FREITAS, E.O. DE FREITAS, J.C. DE FREITAS, L.A. DE JESUS, A.C. DE JESUS, A.R. DE JESUS, E.A. DE JESUS, J.B. DE JESUS, R.C.S. DE LA FUENTE, S. DE LA GUARDIA, C.I. DE LIMA COSTA, K.F. DE LIMA, B.D. DE LIMA, D.A. DE LIMA, H. DE LIMA, I.D. DE LIMA, V.M.F. DE LOS SANTOS, M.B. DE LUCA, P.M. DE LUCCA, H.R.L.R. DE LUCENA, N.C. DE MACEDO, L.B. DE MACEDO, M.S.F. DE MEDEIROS, J.F. DE MELLO, M.F. DE MELO NETO, O.P. DE MELO, E.V. DE MELO, G.D. DE MELO, I.M. DE MELO, M.F. DE MELO, S.D. DE MENDONCA, I.L. DE MENEZES, J.P.B. DE MIRANDA, M.B. DE MORAIS, I.B.
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DE MORAIS, O.O. DE MORAIS, R. DE MORAIS, R.C.S. DE MOURA, F.K. DE MOURA, S.A.L. DE MOURA, T.C.M. DE MOURA, T.R. DE MUYLDER, G. DE OLIVEIRA E CRUZ, D.M. DE OLIVEIRA NETO, M.P. DE OLIVEIRA SILVA, E.P. DE OLIVEIRA, A.F.J. DE OLIVEIRA, A.H.C. DE OLIVEIRA, A.J.F. DE OLIVEIRA, A.L.L. DE OLIVEIRA, A.N. DE OLIVEIRA, A.P. DE OLIVEIRA, B.C. DE OLIVEIRA, B.M.N. DE OLIVEIRA, C.I. DE OLIVEIRA, D.E. DE OLIVEIRA, E.F. DE OLIVEIRA, F.A. DE OLIVEIRA, G.M. DE OLIVEIRA, J.H. DE OLIVEIRA, L.B. DE OLIVEIRA, M.A. DE OLIVEIRA, M.F. DE OLIVEIRA, M.Y.K. DE OLIVEIRA, R.N. DE OLIVEIRA, T.F. DE OLIVEIRA, V.C. DE OLIVEIRA, W.A. DE PADUA MILAGRES, F.A. DE PAIVA CAVALCANTI, M. DE PAIVA, R.M. DE PAULA, C.D.R. DE PAULA, N.A. DE PAULA, R.C. DE RESENDE, D.M. DE SA, J.C. DE SA, W.T. DE SIMONE, S.G. DE SOUSA, A.C. DE SOUSA, C.B. DE SOUSA, F.D. DE SOUSA, L.C.
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DE SOUSA, L.L.F. DE SOUSA, M.D.G. DE SOUSA, M.R. DE SOUZA, B.M. DE SOUZA, C.A.C. DE SOUZA, C.C. DE SOUZA, C.L. DE SOUZA, C.M. DE SOUZA, C.S.F. DE SOUZA, E.B. DE SOUZA, F.A. DE SOUZA, J.V. DE SOUZA, M.A. DE SOUZA, M.B. DE SOUZA, N.A. DE SOUZA, O.M.F. DE SOUZA, T.L. DE SOUZA, W. DE VRIES, H. DE VRIES, H.J. DE VRIES, P.J. DE LUCA, P.M. DEACON, A. DEACON, A.D. DEBNATH, S. DEBONI, S.C. DEBORAH, D. DEBRABANT, A. DEBUS, A. DECUYPERE, S. DEDET, J. DEDET, J.P. DEEP, D.K. DEGROSSOLI, A. DEKONING, H. DEL VALLE, S.G. DELGADO, M. DELGADO, O. DELLAGI, K. DELPUTTE, P. DEMANT, P. DEMICHELI, C. DEMICHELI, C.P. DEMIR, M. DEMUYLDER, G. DEN BOER, M. DEN BOER, M.L. DEOTI, B.
P 892 P 436, P 732, P 733 O 224, P 358 P 108 P 782 P 066 P 782 P 735 P 139, P 192, P 415, P 865, P 868, P 872, P 875 P 231 P 139 P 240 P 511 P 695, P 698, P 712, P 824 P 097, P 761 O 044 P 695, P 712 O 310, P 103, P 352, P 399, P 414, P 432 O 098 P 151 O 078 O 274, P 005 O 279 P 111 P 333 P 870 O 297 O 297 O 132 O 111 O 135, O 014, O 221, P 140, P 905 P 098, P 908 P 364 P 168 P 101 P 367 P 859 P 417 O 259, P 542 P 058, P 059 P 452 O 049, O 086, O 260 O 311, P 393, P 606, P 839 O 081, O 082 P 272 O 306 O 203, O 023 O 202 P 309
DEPAQUIT, J. DERBALI, M. DESCHACHT, M. DESCHUTTER, J. DESCOTEAUX, A. DESEWU, K. DESJARDINS, M. DESJEUX, P. DESOTI, V.C. DESPONDS, C. DEY, R. DHAHRI, B. DHAKAL, S.S. DHALARIA, P. DHALIA, R. DHOM LEMOS, L. DI MARCO LO PRESTI, V. DI MUCCIO, T. DI BLASI, T. DIAO, L.Y. DIAS DE MELO, G.D. DIAS TEIXEIRA, K.L. DIAS, E.S. DIAS, F.C. DIAS, F.E.F. DIAS, G.V. DIAS, M.H.F. DIAS, R. DIAS, R.C.F. DIAS, S. DIAS, S.G. DIAS, T.L.M.F. DIAZ, B.L. DIAZ, E. DIAZ, N.L. DIAZ, R. DIETZE, R. DIKOLLI, E. DILERMANDO, J.A. DILGER, E. DILL, D.C.S. DILLON, R. DILLON, R.J. DINIS OLIVEIRA, R.J. DINIZ ATAYDE, V. DINIZ, A.G. DINIZ, M.M.C.S.L. DIOGO, E.B.
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P 369 P 163 P 013 P 427 O 291 P 860 P 271 P 255 P 139 O 180 O 142, O 351 O 251 O 221 P 243, P 474 P 040 O 302 P 094 P 210 O 078, O 111 O 115, P 356 P 165 P 613 P 404 P 131, P 251, P 283, P 479, P 569, P 579, P 801 P 838, P 851, P 893 P 638, P 644 P 020 P 375 P 907 P 263 P 344, P 345 O 136 P 427 P 883 O 037 P 893 P 108 P 949 O 129, P 081 P 538, P 553 P 523 P 427 P 657 P 511 O 151, P 250, P 261, P 576, P 740 P 097, P 649, P 902 P 223 P 069
DOS SANTOS, W.L. DOS SANTOS, W.L.C. DOSSET, M. DOURADO, F.V.G. DOUSTDARY, F. DOWNING, T. DOWSLEY FILHO, ,.M.A.S. DRAGAS, S. DRAHOTA, J. DRIEMEIER, M. DRINI, S. DRUMOND, K.O. DRUZIAN, A.F. DUAILIBE, R.F. DUARTE, A. DUARTE, M.C. DUARTE, M.I.S. DUBE, A. DUBOVSKY, J. Dujardin , J.
DUJARDIN, J. DUJARDIN, J.C. DUMAIDI, K. DUMETZ, F. DUNCAN, R. DUNN WALTERS, D. DUPANOVIC, B. DUPLAY, P. DUPLESSIS, C. DUQUE, M. DUQUE, S.M. DUREAU, R. DURVASULA, R. DUTHIE, M. DUTHIE, M.S. DUTRA, J. DUTRA, J.L.L. DUTRA, J.M. DUTRA, W.O. DUVAL, R. DUZ, A.L.C. DVORAK, V. E FERREIRA, R.C. E SILVA, A.A.S. E SILVA, J.S.F. E SILVA, R.P.
P 282 O 181, O 183, P 076, P 077, P 207, P 391, P 639, P 719 O 118 P 939 O 025 O 140, O 141, O 143, O 298, O 344, P 053, P 672 P 616 O 013 O 244 P 579 O 208 O 198 P 579, P 851 P 783, P 784, P 819 O 220, O 319, P 772, P 786 P 226 O 157, P 018, P 123, P 362, P 376, P 520 P 639 O 089, O 120, P 041, P 042, P 043, P 381, P 388, P 514, P 582 O 369 O 344 O 002, O 140, O 143, O 078, O 148, O 285, O 315, O 351, O 357, O 254, O 267, P 053, P 062, P 144, P 348, P 382, P 383, P 384, P 385, P 386, P 438, P 672 P 773 O 235, O 111, O 113, P 088, P 363, P 364, P 401, P 679 O 243 O 002 O 026 P 077 O 013, P 426 P 227 P 946 O 280 P 328 O 190 O 192, O 247 P 250, P 615 P 576 O 092, O 093, O 027 O 266 O 209 P 950 P 504 P 840 O 277, O 050 P 555 P 714 O 216, O 248, O 249 O 030 P 319 O 083 P 330, P 650
EATON, P. ECHEVERRI, L.F. EDGEL, K.A. EFSTATHIOU, A. EGEA, A.C. EGURROLA, J.A. EIDE, D. EJARA, E.D. EJGHAL, R. EJOV, M. EL AHMADI, Z. EL HASSAN, A.M. EL HESSNI, A. EL SAFI, S. EL SAFI, S. ELAMINE, W. ELEUTERIO, C. ELIAS, C.R. ELKHOURY, A.N. ELKHOURY, A.N.S.M. ELLIS, S. ELLIS, S.J. ELNAIEM, D. ELNAIEM, D.A. EMPKE, A. EMPKE, A.L.T. ENGEL, J.C. ENTRINGER, P.F. EQUBAL, A. ERDEM, H.A. EREN, R.O. EREQAT, S. ERMIS, Ö.V. EROL, N. ERTABAKLAR, H. ES SETTE, N. ESCACENA, C. ESCH, K. ESCOBAR, P. ESLAMI, G. ESPADA, C.R. ESPINOSA, D. ESPINOZA, J.R. ESPIR, T.T. ESSID, A. ESTAQUIER, J. ESTECHE, M.V. ESTEVAM, L.M.
P 561 P 327 O 351 P 680, P 835 P 372, P 406 P 787 O 164 O 070 O 262, O 263 P 015, P 016 O 094 O 363 O 343 P 015 O 254 O 265 O 138 O 312 P 658, P 767 P 489 O 380 O 040, O 223 O 262, P 366, P 627 O 356 O 075, O 297 O 218, O 020 P 873 P 745 O 285 P 166 P 599, P 603 P 287 O 052 O 035 O 290, P 684 O 321 O 321 O 295, O 321 O 253 P 769 P 884 P 497, P 500, P 510, P 556, P 560, P 573 P 001, P 621 P 404 O 139 P 245 P 258 P 916 O 330, O 372 P 628 P 703
ESTEVES, A. ESTIRADO, A. ESTRADA, M. ETHIER, C. EVANGELISTA, J.S.A.M. EVANGELISTA, M.G. EVERS, F. EWALD, M.P.C. EZZIKOURI, S. F. CHIERICI, N. FABER, D. FABIANO OLIVEIRA, L. FABIENNE, T. FABRA, M. FADUL, M.R. FAGUNDES SILVA, G.A. FAGUNDES, A. FAIOES, V.S. FAIRLAMB, A.H. FAIVRE CHARMOY, M. FAIZ, A. FAIZ, M.A. FAKIOLA, M. FALCAO, C.A.B. FALCAO, C.B. FALCAO, M.J.C. FALCAO, S.A.C. FALCHETTI, F. FARACO, A.A.G. FARAUT, F. FARIA, A. FARIA, A.C. FARIA, A.R. FARIA, D. FARIA, J.V. FARIA, K.F. FARIA, S. FARIAS ROTONDANO, T.E. FARIAS LUZ, N. FARIAS, J.C. FARINHA, A. FASAHAT, M. FASEL, N. FATHALLAH MILI, A. FATTORE, G. FAUCHER, B. FAUSTINO, S.K.M. FEIJENS, P.
O 336, P 508 P 769 P 686, P 852 P 336 P 502 P 472 P 713 P 829 P 016 P 863 P 332 O 159 O 326 P 113 P 952 P 234, P 238 O 122, P 824 P 408, P 427 O 153 O 297 O 109 P 366 O 343, O 352, P 133 P 547 P 407 P 622 O 326 P 845 P 520 O 014 P 309 P 069 P 537 O 277 P 523 P 807 P 753 P 842 P 118 O 198 P 163 P 621 O 035, O 074, O 052, O 351, P 112, P 115, P 116, P 218 P 501 P 825 O 014 O 322 P 460
FEIO, M.J.F. FELGER, I. FELICIANGELI, M.D. FENDRICH, R.C. FEREGUETTI, T.O. FERGUSON, M.A. FERJANI, B. FERMIN, Z. FERNANADEZ, M.S. FERNANDES, A.B. FERNANDES, A.C.S. FERNANDES, A.M.M.M. FERNANDES, A.P. FERNANDES, A.P.S. FERNANDES, A.P.S.M. FERNANDES, C.E.S. FERNANDES, C.J. FERNANDES, F.R. FERNANDES, J.R.M. FERNANDES, J.S. FERNANDES, L.S. FERNANDES, M.A. FERNANDES, M.F. FERNANDES, M.Z. FERNANDES, R.M. FERNANDES, R.P.M. FERNANDES, W.D. FERNANDEZ REYES, M. FERNANDEZ, A. FERNANDEZ, J. FERNANDEZ, J.L. FERNANDEZ, M.S. FERNANDEZ, R. FERNANDO, F.S. FERRARI, H. FERRARI, H.F. FERRARINI, S.R. FERRAZ, R. FERREIRA DA COSTA, E.S. FERREIRA SOARES, M.C. FERREIRA, A.L. FERREIRA, A.L.C. FERREIRA, A.M.T. FERREIRA, A.S. FERREIRA, C. FERREIRA, D.D. FERREIRA, E.C. FERREIRA, E.M.
P 561 O 255, P 061 O 259, P 910 P 826 O 224 O 075 P 958 P 040, P 379, P 380, P 443 P 806 P 720 P 246 P 678, P 865, P 868, P 872, P 874, P 875, P 878, P 879 P 018, P 165, P 226, P 253, P 375 P 145 P 191, P 214 P 057 P 754 P 393 O 311 P 545 P 141 P 179, P 804, P 811 P 847 O 170, P 583 P 504 P 283, P 893 P 278 P 481 P 504 P 893 P 475 P 914 P 835 P 008 O 045, O 095, O 097, P 778 P 680 P 190 P 056 P 634 P 448 P 559 P 237 O 096 P 158 P 818 P 416 P 552 P 057 P 325 P 378, P 420 P 370 P 788 P 731 P 795 P 703
FERREIRA, F. FERREIRA, F.C. FERREIRA, F.M. FERREIRA, G.E.M. FERREIRA, G.M. FERREIRA, I.G. FERREIRA, I.V.G. FERREIRA, J.B. FERREIRA, J.H.L. FERREIRA, J.L.P. FERREIRA, J.O. FERREIRA, L.A.M. FERREIRA, L.T. FERREIRA, P.A. FERREIRA, R.A. FERREIRA, R.G.M. FERREIRA, S.A. FERREIRA, T.R. FERREIRA, V.F. FERREIRA, W. FERREIRA, W.A. FERREIRO TERCEIRO, B.R. FERRER, J. FERRER, L. FERRERA, W. FERRIERES, V. FERRO, C. FERRO, M.C. FERRUA, B. FIALHO JUNIOR, L.C. FIECK, A. FIERRO, M.N.A. FIGUEIRA, C.P. FIGUEIRA, L.P. FIGUEIREDO, F.B. FIGUEIREDO, H.R. FIGUEIREDO, J.K. FIGUEIREDO, L.C. FIGUEIREDO, M. FIGUEIREDO, M.M. FIGUEIREDO, R.C.B.Q. FIGUEIREDO, S.G. FIGUEIREDO, W.M.E. FIGUEREDO, L.A. FIGUEREIDO, J.R. FILHO, A.A.P. FILHO, C.J.L.B. FILHO, D.M.
P 937, P 938 P 956 P 509 P 126 P 866 P 073, P 082, P 320 P 119 O 136 P 885 P 888, P 889, P 891, P 894 P 897 P 886, P 890 P 788 P 350 P 445 P 210 P 393 O 116 P 587 P 007 O 276 P 316 P 532 P 747 O 294, P 416 P 198, P 312 P 492 P 409 O 311 P 393 P 090 P 113 O 110 O 279 O 190 P 859 O 008 P 430 P 066 O 247 P 918 P 207 P 258 O 296, P 415, P 695, P 698, P 712, P 929 P 846 P 857 P 276 P 309, P 311, P 629 P 316 P 640 P 638, P 644 P 349 P 034 P 171, P 174 P 219 P 622 P 673, P 674, P 675, P 734, P 783, P 784, P 819 P 712 P 498
FILHO, E.P.S. FILHO, J.D.A. FILHO, J.D.S. FILHO, L.X. FILHO, O.A.M. FILHO, S.P. FILHO, V.P. FINKELSTEIN, L.C. FIORENTINO, E. FISA, R. FISCHER, L. FISCHER, M.R. FISHER, A. FITZPATRICK, S. FIUZA, J. FIUZA, V.O. FLOETER WINTER, L.M. FLOHE, L. FLORENTINO, P.V. FLORES LOPEZ, E.J. FLOREZ, A. FLOREZ, M.F. FOGLIA MANZILLO, V. FOLEY, J.E. FONSECA, A.C. FONSECA, A.F.Q. FONSECA, A.M. FONSECA, G.S. FONSECA, I.P. FONTELES, R.S. FORDELLONE, M.R.C. FORSHAW, A. FORTES DIAS, C.L. FORTIN, A. FOSTER, L.J. FOTOUHIARDEKANI, R. FRAGA, D.B.M. FRAGA, J. FRAGA, T.L. FRANCA COSTA, J. FRANCA DIAS, M.H. FRANCA SILVA, J.C. FRANCA, A.N. FRANCA, A.O. FRANCA, E.M.O. FRANCA, J.R. FRANCA, T.M.C. FRANCESQUINI, F.
P 516 P 800 P 362 P 612 P 292, P 496, P 516 P 633 P 731, P 735 P 210 O 123, O 292, P 604 O 366, P 084, P 722 O 320, P 038 O 333 P 543 O 102 O 026 P 759, P 821 P 788 O 129, P 081, P 465 O 188 O 305 P 461 O 286, P 531 O 347 O 283 O 212, O 292, P 048 P 852 P 731 P 532 P 795 P 496 O 220, O 319, P 490, P 772, P 786 P 673, P 674, P 675, P 734 P 444, P 742 O 192 P 759 O 338, O 342, P 460 O 114 P 180 P 701 O 044, P 391, P 522, P 526, P 833 O 254, P 062 P 283, P 569 P 343 P 194 P 795 P 214, P 759, P 765 P 567 P 851 P 479, P 801 P 869 P 520 P 581 P 291
FRANCISCATO, C. FRANCO, A. FRANCO, A.M.R. FRANCO, A.R. FRANCO, G.R. FREIRE, F.P. FREIRE, G.M. FREIRE, J.M. FREIRE, M.L. FREIRE, M.P. FREIRE, R.C.M. FREIRE, R.L. FREITAS, A.C.P. FREITAS, A.I. FREITAS, I.S. FREITAS, J.C. FREITAS, J.C.C. FREITAS, L.A.R. FREITAS, M.S. FREITAS, R.A. FREITAS, T.D. FREUDZON, M. FREZARD, F. FREZARD, F.J. FREZARD, F.J.G. Frezard, F.J.G. FRIESEN, J.A. FRY, L. FUCHS, J.R. FUENZALIDA, A.D. FUJIWARA, R. FUJIWARA, R.T. FUKUDA, M. FUMAGALLI, M. FUMAGALLI, M.A.C. FUMAGALLI, M.C. FUSTER, F. FUX, B. FUYA, P. FUZARI, A.A. FUZIBET, J.G. G. DE SOUSA, F.D. GABORIAUD KOLAR, N. GADELHA, S.M.S. GADISA, E. GAELLE, B. GAGO BADENAS, F. GAGO, F.
P 251 P 864 P 258, P 260 P 224, P 549 P 178 P 147 P 546 P 225 O 276 P 568 P 762 P 917 P 915 P 713, P 715, P 716, P 787, P 826, P 829 P 945 P 609 P 888, P 889, P 891, P 894 P 848, P 871, P 885, P 886, P 890, P 897 P 499 P 502, P 506 P 076 P 524 O 181 P 810 P 345 P 854 P 089 O 311, P 606, P 839 O 082, O 112, O 145, P 387, P 565 P 492 O 081 P 509 P 393 P 339 O 283 P 410 O 308 O 097 O 026 O 257, O 294, O 317, P 181, P 239, P 347, P 486, P 491 O 034 P 375 O 157 P 376 P 757, P 766, P 769 P 731 P 709 P 818 P 221 P 876 P 406 P 811 P 845, P 847 P 934 O 146, P 451 P 529 O 307, P 218, P 525
GAIDO, S.B. GALAÏ, Y. GALANTE, A. GALARDO, A.K.R. GALARZA, F. GALATI, E.A.B. GALATI, E.B. GALEANO, E. GALHARDO, J.A. GALINDO, W. GALISTEO, A.J. GALLEGO, M. GALLO, G. GALLO, L. GALUPPO, M.K. GALUTI, E. GALVAO, J.D. GALVAO, L. GALVAO, R.F.P. GALVAO, S.R. GALVEZ, R. GALVIS, F.O. GAMA, A. GAMA, M.E. GAMA, M.E.A. GAMARRO, F. GAMERO, M.D. GANGNEUX, J. GANGWAR, S. GANNAVARAM, S. GARATE, T. GARCEZ, L. GARCEZ, L.M. GARCIA BUSTOS, M.F. GARCIA HERNANDEZ, R. GARCIA PEREZ, A. GARCIA, A.L. GARCIA, B. GARCIA, E. GARCIA, F. GARCIA, G.R. GARCIA, L. GARCIA, M. GARCIA, M.H. GARCIA, M.H.O. GARCIA, R.D. GARDINASSI, L.G.A. GARG, M.
P 276 P 484 P 400, P 693 P 831 P 825 O 007, P 097, P 658, P 818, P 861, P 893, P 912, P 957 P 652, P 653, P 774, P 937, P 938 P 707 P 808 P 910 P 389 P 944, P 947 P 050 P 472 P 004, P 031 P 937, P 938 P 828 O 180 P 939 P 856 O 342 P 938 P 476 P 167 P 212 P 385, P 413, P 417 P 379, P 443 O 303, O 190, P 122 P 329 O 368, O 026 P 494 O 042, P 513 O 197 P 660, P 935, P 943, P 952 P 104 O 038 P 385 P 413 P 367 P 438 O 375 O 215 O 068 P 148, P 152 P 323 O 254 P 527 O 199 P 956 P 439 P 323 O 076
GARGANO, V. GARLAPATI, R.B. GARNAOUI, A. GARNAOUI, A.M. GAROFALO MINA, K.L. GARRIDO, L.M. GASHOUT, A. GASIMOV, E. GASPANI, S. GASPAR, M. GASPAR, M.M. GASPAROTTO, J.C. GAUDENCIO, K. GAUNA, A. GAUR, U. GAUTAM, S. GAUTUM, S. GAVA, R. GAWHARI, F. GAZZINELLI GUIMARAES, P. GAZZINELLI, R.T. GEA, S. GEBRE MICHAEL, T. GEDAMU, L. GEERTRUYDEN, J.V. GEHRKE, S.S. GELANEW, T. GEN, F. GENES, C. GENESCA, M.A.M. GENEST, P. GENNARI, S.M. GENOIS, M. GENTILE, A.G. GEORGETTI, E.D.S. GEORGIKOPOULOU, K. GERALDO, M.M. GETTI, G. GHAEDI, K. GHALIB, H. GHANEI, M. GHARBI, A. GHAWAR, W. GHEDIRA, K. GHERARDINI, P.F. GHERSI, B.M. GHIYA, B. GHODS, R.
O 187 P 651 O 119 O 208, O 236, P 039 P 918 P 747 P 724 O 094 O 203 O 182 O 312, O 336, P 471 P 236, P 849, P 858, P 862 P 659 P 380 O 302 O 206, O 207, P 086 O 299 P 160, P 601, P 631, P 911 P 450 P 486 P 165 P 035, P 145, P 191, P 213, P 537, P 562 P 104 O 248, P 029, P 750, P 751 O 117 O 012, O 316 P 482 P 308 P 313 P 218 P 392 O 107 P 685, P 809 P 336 O 038 P 856 O 310 P 156, P 172 O 279, P 111 P 299, P 300 P 610 O 318 O 125, O 289 O 125, P 922, P 942 P 059 O 346 P 680 O 171 O 318
GHOLAMI, E. GHOSH, J. GHOSH, P. GHOSH, R.S. GIACOIA GRIPP, C.B. GIAMMARRESI, M. GIDWANI, K. GIL SOARES, L.H. GIL ZAMBRANO, G.C. GILMORE, D. GIMBLET, C. GIMENEZ, A. GIMENEZ, D.S.B. GIMENEZ, G. GIOANNINI, T.L. GIRALDO, L.E.R. GIRAUD, E. GIRONA LLOBERA, E. GITHURE, G. GIUDICE, A. GIUNCHETTI, R. GIUNCHETTI, R.C. GIUSTA, C.J. GIZZARELLI, M. GODINHO, J.L.P. GODINHO, N.D.V. GODOY, E. GODOY, R.E. GOES, J.J. GOLDMAN PINKOVICH, A. GOLDSCHMIDT, M.H. GOLLOB, K.J. GOMES ALVES, A.G. GOMES SILVA, A. GOMES, A.A.D. GOMES, A.F.S. GOMES, A.H.S. GOMES, A.M.O. GOMES, A.R.F. GOMES, B. GOMES, C.C. GOMES, C.E.M. GOMES, C.M. GOMES, C.M.C. GOMES, D. GOMES, D.C. GOMES, E.V.R.
O 025 O 235, P 088 O 266 O 109, O 021 P 228 P 017 O 015, P 361, P 749 P 898 P 379, P 443 O 160, P 958 O 272 O 270 P 745 P 334 O 073 P 462 O 332, O 047, O 193, P 791 P 084, P 722 P 101 P 423, P 785 O 293 P 194 O 091, P 067, P 071, P 072, P 129, P 197, P 199, P 292, P 434, P 533, P 714 P 191 O 292 O 310, P 432 O 126, P 428 P 628 P 097 P 889, P 891 P 848, P 871, P 885, P 888, P 894, P 897 P 886, P 890 O 131 O 087 O 277, O 050 O 188 P 215, P 234, P 235, P 237, P 238, P 269, P 551 P 852 P 939 P 007 P 160, P 955 P 559 P 897 P 888, P 891 P 886, P 889 P 871, P 885, P 890 P 894 P 898 P 193, P 422, P 424 P 278 O 083, O 126, P 095, P 530, P 546, P 648 P 142, P 146, P 167, P 169, P 212, P 291 P 212 P 304 O 156 P 857
GOMES, F. GOMES, G.S. GOMES, K. GOMES, L. GOMES, L.H.M. GOMES, L.I. GOMES, M.S. GOMES, P.A.T. GOMES, R. GOMES, R.A. GOMES, S. GOMES, V.A. GOMEZ NIETO, L.C. GOMEZ OCHOA, P. GOMEZ, A. GOMEZ, C.P. GOMEZ, M. GOMEZ, M.A. GOMIDE, L.B. GONCALVES NETO, J.R. GONCALVES, A.L. GONCALVES, A.M. GONCALVES, D.D. GONCALVES, E.G.R. GONCALVES, E.L. GONCALVES, F.T. GONCALVES, G.M. GONCALVES, L.M. GONCALVES, R. GONCALVES, R.G. GONCALVES, S.A. GONCALVES, S.C. GONCALVES, V.T. GONDIM, F. GONDIM, F.C. GONGORA, R. GONTIJO, C.M.F. GONTIJO, N.F. GONTIJO, R.C. GONZAGA, W.F. GONZAGA, W.F.K.M. GONZALEZ LOMBANA, C. GONZALEZ REY, E. GONZALEZ, D. GONZALEZ, E. GONZALEZ, K. GONZALEZ, L. GONZALEZ, M.
P 850 O 220, O 319 P 950 P 759 O 264 O 319, P 490 P 260 P 358 O 276, P 903 O 257, P 491 P 541 O 160, O 161, O 320 P 440 P 563 P 703 P 113 O 110 O 365 P 806 O 270 P 825 P 227 P 157 O 258 O 147 O 029, O 099, P 089, P 100, P 737 P 731 P 767 P 761 P 158 P 713, P 715 P 212 P 755 P 867 P 108 P 471 O 312 P 660 P 921 P 945 P 335 P 330, P 585 P 650 P 528 P 600 P 407, P 575 P 100 P 812 O 276, P 225, P 762, P 798, P 800 P 239 P 606 O 317 O 276, P 225 P 156 P 172, P 175 O 272 P 417 P 766 P 757, P 766 P 146 P 248 O 108, O 261
GONZALEZ, N. GONZALEZ, Y.P. GORCII, M. GORDON, M. GORE SARAVIA, N. GOTO, H. GOTO, Y. GOTUZZO, E. GOULART, L.R. GOULD, I.T. GOULD, M. GOUNDRY, A. GOUVEIA, C. GOUZELOU, E. GOYAL, N. GOYAL, N.G. GRADONI, L. GRAFF, J.W. GRAFOV, A. GRAFOVA, I. GRAMICCIA, M. GRANO, F.G. GRAVINA, M.C.S. GRAZIANI, D. GRAZINOLLI, M. GREGGIANIN, G.F. GREKOV, I. GRIEWANK, K. GRIFFITHS, S. GRIMALDI, G. GRIMMER, G.H. GRISARD, E.C. GROGI, M. GROGL, M. GROPPI, S. GROSS, E. GRYMBERG, P. GUACHAMIN, P.E.Y. GUAN, R. GUARDO, M. GUCCIONE, J. GUDERIAN, J. GUEDES JUNIOR, J.C.O. GUEDES, C.E.S. GUEDES, H.L. GUEDES, H.L.M. GUEDES, P. GUEDES, R.P.O.
O 270 O 170, P 583 P 905, P 908 O 100 O 099, P 737, P 859 O 354, P 002, P 091, P 266, P 267, P 341, P 590 O 090, O 370, O 174, O 177, O 178 O 267 O 157, P 123 O 045, P 778, P 806 P 792 P 101 O 071 P 699, P 936 O 135 O 076, P 003, P 329 O 309 O 212, O 232, O 292, O 320 O 073, O 087 P 549 P 549 O 232, O 123, O 254, O 292, P 604, P 947 P 032, P 049, P 196 P 392 O 126, P 095, P 428 P 831 P 439 O 049 O 333 O 162 P 145 P 183 P 515 O 114 O 362, O 363, O 289, P 518, P 607 P 704 P 655 P 178 P 918 P 470 O 380 P 048 P 250, P 576 O 209, O 092, O 027, P 610 P 526 O 036, P 074, P 326, P 391 P 304 O 116, O 156, P 162, P 575 O 180 P 080, P 718
GUEDRI, E. GUEGUEN, S. GUERBOUJ, S. GUERFALI, F. GUERFALI, F.Z. GUERRA, B. GUERRA, J. GUERRA, J.A. GUERRA, J.A.O. GUERRA, J.M. GUERRA, P.V. GUERRA, R.N.M. GUERREO, R. GUERRERO, I. GUHA, R. GUIGUEN, C. GUILLEN, C. GUIMARAENS, L.H. GUIMARAES COSTA, A.B. GUIMARAES, A. GUIMARAES, E.L.A.M. GUIMARAES, L.H. GUIMARAES, L.N.A. GUIMARAES, P.N. GUIMARAES, S.S.V. GUIMARAES, T.T. GUIMARAES, V.F. GUIZANI TABBANE, L. GUIZANI, I. GUNDAMPATI, R.K.K. GUO, H. GUPTA, C.M. GUPTA, D. GUPTA, D.S. GUPTA, G. GUPTA, K. GUPTA, R. GUPTA, S. GUPTA,, R. GUSHI, L.T. GUSTIN, C. GUTERRES, S.S. GUTIEREZ, C.T. HABBOUL, Z. HAGIWARA, M.K. HAILU, A.
O 363 O 214, O 215, P 068 P 096 O 205 P 058, P 059, P 232 P 559 P 189 P 307 O 170, P 583 P 489 P 034, P 171, P 174 P 907 P 257 P 944 O 235, P 088 P 122 P 084, P 722 P 480 O 301, O 304 O 158 P 945 O 169, P 134 O 233, O 166, P 133, P 423, P 527, P 590, P 785 P 204 O 277, O 050 P 337 P 851 P 579 P 768 P 935 P 492 P 659 O 345 O 138, P 096, P 184, P 403, P 501, P 517, P 580, P 586 P 045 O 075 P 130 O 104, P 602 P 315 P 598 O 308, O 339 O 371 P 388 P 596, P 642 O 089 P 173 P 058 P 558 P 559 P 951 P 355, P 484 P 266 O 133, P 410 O 216, O 356, O 262, O 263, P 029, P 116, P 308 O 248
HAILU, W. HALABALAKI, M. HALLIDAY, A.J. HAMANO, S. HAMARSHEH, D.O.O. HAMARSHEH, O. HAMDI, S. HAMILTON, J.G.C. HANAFI BOJD, A.A. HANCOCK, R.E.W. HAND, T.W. HANNOLD, C. HAOUAS, N. HAQUE BHUIYAN, A.T.R. HAQUE, R. HARADA, S. HARALAMBOUS, C. HARBIGE, L. HARDOIM, D.J. HARIGUA, E.S. HARLEY, D. HARRABI, M. HARTKOPF, A.C.L. HARTL, D. HARTLEY, M. HARTMANN, S. HASHEMINIA, S.J. HASHIGUCHI, Y. HASKER, E. HASSAN, S.M. HASSANI, K. HASSEINE, L. HASSOUNA, N.B. HATEFI, Z. HAUYON LA TORRE, Y. HAVELKOVA, H. HAY, C.L. HAYDOCK, A. HEALY, A. HEINE, C. HEJAZI, H. HEJAZI, S.H. HEJAZI, S.H.H. HELMER CITTERICH, M. HELYNCK, O. HENARD, C.A. HENDRICKX, S.
O 133 O 216 P 487 O 158 O 084 P 455 P 092, P 684, P 724 O 253, P 015, P 016, P 092 P 746 O 127 P 377 O 121 P 518 P 140, P 905 P 366 O 084, O 109, O 021, O 176, P 366 O 179 O 135 P 111 P 192 P 403 O 018 P 305, P 306 P 131 P 019 O 035, O 052, P 112, P 115 P 320 P 299, P 300 O 038 O 012, O 015, O 316, O 357, O 055, O 163, O 067, P 749 O 109, O 021 O 029 P 221 O 208, O 236, O 306, P 039, P 153 P 621 O 250 O 086 O 049 O 053 O 348 P 461 O 166 P 301 P 620 P 300, P 913 P 621 P 299 O 346 O 284 O 053 P 452
HENRIQUES, S. HERAI, R.H. HERINGER, J.F. HERMIDA, M.D. HERMOSO, T. HERMOSO, T.J. HERNANDEZ MONTES, O. HERNANDEZ RIVERA, M.P. HERNANDEZ, C. HERNANDEZ, C.G. HERNANDEZ, L. HERNANDEZ, S. HERRERA VELIT, P. HERRNANDEZ MONTES, ,.O. HEUKELBACH, J. HIAMAMOTO, R.M. HICKMAN, M.R. HIDALGO, J.O.Q. HIDE, M. HIDER, R.C. HIGA JUNIOR, M.G. HIGINO, T.M.M. HIRAMOTO, R.I.M. HIRAMOTO, R.M. HOCHBERG, L. HOFMEYER, K. HOLDER, A.A. HOLETZ, F.B. HOLZMULLER, P. HOMBACH, A. HOMBACH, A.L. HOMEDES, J. HOMMEL, M. HORACIO, A.M.L. HOROWITZ, M.R. HORTA, M.A.P. HORTA, M.F. HORTA, M.F.M. HOSEIN, S. HOSEINI, S.G. HOSSAIN, M.S. HOSSEINI, M. HOU, Y. HOUGHTON, R. HOWARD, R.F. HOYOS, C. HOYOS, C.L. HROBARIKOVA, V.
P 453 O 030 P 551 O 183, P 207 P 380, P 443 P 474 P 040 P 595 P 595, P 882 P 600 P 781 P 462, P 803, P 807 O 342 P 757 P 245 P 882 O 043, P 739, P 853 O 296 O 114 P 918 O 366 P 482 P 851 P 644 P 631 P 395 P 489, P 540, P 601, P 767, P 857 O 289 O 092, O 027 O 152 P 020 P 159 O 006 O 001 O 110 P 366 P 567 P 293 P 708 P 102 P 178 P 646 P 620 O 084 O 318 P 055, P 697 O 194, O 289 O 093, P 610 O 266 P 485 O 038 O 249
HU, R. HU, R.V.V. HUBER, K. HUBERT, B. HUDA, M. HUEB, M. HUITEMA, A.D. HULANGAMUWA, S. HUMMER, K. HUPPERT, A. HURRELL, B.P. HURWITZ, I. HUSSAIN, M. IBRAHIM, I.C. IBRAHIM, M.E. ICEVIC, M. IGLEZIAS, S.D. IGLEZIAS, S.D.A. IGUCHI, G.U. IKEDA, F.A. IMAMURA, H. IMAMURA, R. INACIO FILHO, J.D. INACIO, R.R. INAOKA, D.K. INBAR, E. INFRAN, J.M. INFRAN, J.O.M. INIESTA, L. INIESTA, V. INSTITUT PASTEUR INTERNATIONAL NETWORK,L. IPE, A. IPE, I.M. IRETON, G. IRETON, G.C. IRISO, A. ISHIKAWA, E.A. ISHIKAWA, E.A.Y. ISHIMI, C.M. ISIKGOZ TASBAKAN, M. ISLAMUDDIN, M. ISOLA, E.L. ISOMURA, K. ITALIA, J. ITO, M.M. ITOH, M. IZIDORO, M.A.
O 098, P 458 P 151 P 399 O 221 P 796 P 158 O 078 P 670 O 160 O 216, O 065 O 326 O 192, O 247 O 124 O 051 O 343 O 013 P 160 P 955 P 823, P 832, P 846 P 395 O 002, O 143, O 298, O 344, P 672 O 168 P 346 P 090 P 164 O 179 O 072, O 297 P 823, P 846 P 832 P 084, P 722 P 113 P 663 P 667 P 667 P 610 O 266 P 757, P 766, P 769 P 422, P 424 P 060, P 275, P 810 P 893 P 117, P 287 P 466 P 334 P 313 P 450 P 747 O 173 P 538
IZQUIERDO, A.Y. JACOBUCCY, J.F. JAEGER, T. JAFARI, R. JAFFE, C. JAFFE, C.L. JAGANNADHAM, M.V. JAISANKAR, P. JAISWAL, A.K. JAISWAL,, A.K. JAKOB, U. JALALI ZAND, N. JAMIESON, S. JAMIL, K. JAMJOOM, M. JANINO, M.P.D. JANKEVICS, A. JANOTTI, A. JANSEN, H. JANUARIO, C.I. JAOUADI, K. JAQUET, D. JARA, M. JARDIM FILHO, W.R. JARDIM, A. JAROSIKOVA, T. JASKE, E.L. JASPER, L. JAVADI, A. JEAN CLAUDE, D. JEANDEL, P.Y. JEBRAN, F.A. JEDDI TEHRANI, M. JERONIMO, S. JERONIMO, S.B. JERONIMO, S.M. JERONIMO, S.M.B. JERONIMO., S.M.B. JESUS, A.M.R. JESUS, A.R. JESUS, R.S. JHA, A.N. JIA, P. JIANG, R. JIMENEZ MARCO, T. JIMENEZ RUIZ, A. JIMENEZ, A. JIMENEZ, M.
P 918 P 489 O 188 O 195, O 127, O 318, P 913 O 216 O 032, O 254 P 045 P 359 P 041, P 042, P 514 O 120, P 043 O 089 O 059 O 318 P 133, P 264 O 109 P 946 P 546 P 384 P 321, P 322, P 572 O 338, O 342, P 460 P 516 P 140, P 905, P 908 P 361 P 144, P 397, P 438 P 729, P 730 O 256, O 131 O 049 P 029 O 289 P 299, P 300, P 913 O 146, P 451 P 221 O 064 O 318 P 884 P 273, P 289 P 286 O 352 P 147 P 128, P 255, P 278 P 285, P 522 P 896 P 323 P 133, P 250, P 264, P 456 P 422, P 424 P 137 O 373 O 353 P 084, P 722 O 307, P 218 P 525, P 529 O 361, O 364, O 164 O 046 O 244, O 245 P 757, P 763, P 766
JIMENEZ, M.I. JIN HONG, W. JOCHIM, R.C. JOHNSON, A. JOJOA, J. JONES, M.A. JONES, S.L. JORGE, C.L. JORGE, L. JORGE, T.A. JOSHI, S. JOSHI,, S. JUCA, A.A. JULIAO, G.R. JUNG, J.Y. JUNIO, A. JUNIOR, C.B. JUNIOR, C.F.S. JUNIOR, E.R. JUNIOR, E.S. JUNIOR, E.T.A.M. JUNIOR, F.D. JUNIOR, J.G.C. JUNIOR, M.B. JUNIOR, M.G.H. JUNIOR, M.S.C.L. JUNIOR, S.F. JUNIOR, V.R. JURADO, D.S. JUSI, M.M.G. K. KAAK, O. KAFFELA, M. KAJILURU, L.P. KALABRESE, K.S. KALANTARI, M. KALIL, S.P. KALLAS, E.G. KAMHAWI, S. KAMMOUN REBAI, W. KAMMOUN, W.R. KANAMURA, C.T. KANASHIRO, E.H.Y. KANG, A. KANSAL, S. KAPLAN, M.H. KARAKUS, M. KARAPETYAN, M.
P 157 O 324 P 029 O 369 P 737 O 283, P 328 P 749 P 081, P 789 O 110 P 699 O 120, P 043 O 089 P 729, P 730 P 920 O 329 P 309, P 310 P 195 P 179, P 804, P 811 P 510, P 556 P 728 P 492 P 020 P 864, P 867 P 568 P 354 P 579 P 131, P 479, P 579 P 234 P 623 P 803, P 807 P 589 P 249 P 130 P 184 P 580 P 602 P 626 P 301 P 419 P 142 O 029, O 159, O 160, O 161, O 318, O 320, O 025, O 026, P 248 P 039 O 236 P 955 P 160 P 577 O 247 O 015, O 163 O 339 O 295 O 100
KARKI, P. KAROUI, H. KARUNAWEERA, N. KASBARI, M. KASSAHUN, A. KASTELLAO, K.G. KATAKURA, K. KATO, H. KATO, K. KATO, K.C. KATZ, S. KAUFMANN, F. KAYE, P.M. KAZIMOTO, T.A. KEAN HOOI, K.A. KEDRA, D. KEESEN, T. KEESEN, T.L. KEESEN, T.S. KEESEN, T.S.L. KEL, A. KELLY, H. KENDJO, E. KENNEDY, L. KENT, A. KENT, A.D. KER, H.G. KESHAVARZ VALIAN, H. KESSLER, A. KETELUT, N. KHAJEIAN, M. KHALIL, E. KHALIL, E.A. KHAMESIPOUR, A. KHANAL, B. KHANDELWAL, K. KHANRA, S. KHARE, P. KHARE,, P. KHATAMI, S. KHATRI, M.L. KHOURI, R. KIAFAR, 5. KIDAR, A. KIKA, B. KIM, O.M. KIMA, P.E. KINGHORN, A.D.
O 111 P 517 O 217, P 027, P 028, P 668, P 669 O 221, O 124 O 216, O 248 P 631 P 026 P 026 O 311 O 081, O 082 P 350 O 297 O 162 O 340 P 865, P 868, P 872, P 874, P 875, P 878, P 879 O 306 O 205, P 232 P 255 P 286 P 128 O 050, P 273 P 059 O 109 P 457 O 213 O 098 P 151 P 458, P 794 P 129, P 434, P 903 O 211 P 367 P 253 O 195, O 127 P 627 O 356 P 692 O 211, O 080, O 318, P 085, P 299, P 300 O 002, O 015, P 384, P 672 O 171 P 802 O 120, P 043, P 388, P 514 O 089 O 239 O 123 O 291, P 618 P 340 P 905 P 295 P 534 O 005 O 308
KIRSZTEIN, O. KISSINGER, J.C. KITA, K. KITAHARA OLIVEIRA, M.Y. KJAER, V.K. KLATT, S. KOBETS, T. KOBOR, M.S. KOBS, T.L. KOCHHAR, S. KOHIDAI, L. KOHL, K. KÖHLER, K. KOKICI, M. KOLARICH, D. KOLES, N. KOLLI, B.K. KOLOURA, I. KONG, P. KONTHUR, Z. KOPYDLOWSKI, K.M. KOREN, R. KORENAGA, M. KORLACH, J. KOSTALOVA, T. KOTHA, S. KOUROU, K. KOWALEWSKI, M.M. KRAIEM, M. KRAJA, D. KRATOCHVILOVA, T. KRAVAR GARDE, L. KRAWCZAK, F.S. KRAYTER, L. KREISHMAN DEITRICK, M. KREISHMAN DETRICK, M. KRIEGER, H. KRISHNAPPA, H. KROPF, P. KRSTICEVIC, F.J. KRUMBIEGEL, G.B. KUAMAR, D. KUHLMANN, F.M. KUHLS, K. KULSHRESTHA, A. KUMAR, A. KUMAR, D. KUMAR, G.
O 216 O 200 O 179 O 234 P 080 P 019 O 049, O 086, O 260 O 353 P 898 O 109, O 021 P 017 O 035 P 694 P 295, P 611 P 019 O 160 P 449 P 487 P 519 P 019 O 363 O 147 O 038 O 107 P 029 O 308 P 318 P 814 P 501 P 295, P 611 P 717 P 544 P 700 P 671 O 362, O 363 O 289 P 747 P 596 O 237, O 133, P 410, P 437 P 121 P 164 P 582, P 597 O 074 O 102, O 137, O 139, P 694, P 711, P 877 P 364 O 109, O 021 O 026 P 154, P 495 P 284
KUMAR, M.R. KUMAR, N. KUMAR, R. KUMAR, V. KUMAR, V.N. KUREY, I. KUSHAWAHA, P.K. KUSHAWAHA,, P.K. KUTLUBAY, Z. KUYPERS, K. KWAKYE NUAKO, G. LA GIGLIA, M.A. LAAMRANI, A. LABRUNA, M.B. LACERDA, H.G. LACEY, C.N.J. LACHAUD, L. LADEIRA, L.O. LAFORGE, M. LAGE, D. LAGE, D.P. LAGE, P. LAGE, P.S. LAGO, E. LAGO, E.L. LAGO, J.H.G. LAHAV, T. LAHMEDI, R. LAI A FAT, R. LAI A FAT, R.F. LAKHAL NAOUAR, I. LAKSHMI, B.S. LAL, C. LAL, D. LAL, S. LAM, L.L. LAMBERT, M. LAMBERTZ, U. LAMI, P. LAMMEL, E.M. LAMOTHE, J. LANA, M. LANA, R.S. LANDGRAF, V.O. LANG, T. LANGHOR, I. LANGUE, R. LANNA, M.F.
P 450 O 057 O 206, O 207, O 299, O 265, P 086 P 063 P 130 O 049, O 260 P 388 O 089 P 360 P 460 P 946 P 050 O 253 P 700 P 128 P 285 O 162 P 457 P 562 O 330 P 375 P 226, P 520 O 157, P 018, P 123, P 362, P 376 P 375 P 226, P 520 P 018, P 376 O 278, P 024, P 423, P 785 P 134 P 641 O 348 O 138 O 098 P 151 O 026 O 088 O 057 P 667 P 154 O 353 O 303 O 054, P 180 O 221, P 140 P 334 P 221 P 067 P 758, P 764 P 479 O 047, P 430 P 448 P 098 P 197, P 199
LANZA, J.S. LAOUINI, D. LARA SILVA, F.O. LARANJEIRA, D.F. LARANJEIRA, E.T.S. LAROSA, R. LARRAGA, V. LAUKENS, K. LAULETTA LINDOSO, J.A. LAUNAY, D. LAUNOIS, P. LAURANCE, J. LAURENT, M.D. LAURENTI, M.D. LAUTELA, J.A. LAUTENSCHLA, H.R. LAVITSCHKA, C.O. LAWYER, P. LAWYER, P.G. LAZO ESCALANTE, M. LAZO, M. LE CACHEUX, C. LEAL JUNIOR, F.G. LEAL NETO, O.B. LEAL, G.G.A. LEAL, S.M. LEAO, E.F. LEATHERBARROW, R.J. LECOEUR, H. LEE, G.I. LEEDS, S. LEEFLANG, M. LEGENTIL, L. LEITE, A.B. LEITE, A.C.L. LEITE, C.G. LEITE, H.S. LEITE, J.C.S. LEITE, J.H.A.C. LEITE, J.P.V. LEITE, M.D.X. LEITE, M.O. LEITE, R.S. LEITE, S.T.V.C. LELES, D. LEMAIRE, J. LEMESRE, J.
P 492 P 058, P 059, P 922 P 759, P 765 O 181, P 524 P 209, P 211 P 658 O 348, P 148, P 152, P 157 P 385 P 231 P 596, P 636, P 642 O 273 O 092, O 027 P 193, P 422 P 424 P 142, P 146, P 167, P 169, P 291, P 394, P 793, P 855 P 212, P 736, P 771, P 852 P 431 P 793 P 861 O 297 O 161 O 121 P 245 P 535 O 164 O 181 P 664 P 071 P 497, P 510, P 556 P 902 O 152 O 047 P 148 O 114 O 060 O 190 P 528, P 594 P 541, P 564 P 812 P 755 O 183 P 054, P 056, P 686 P 362 P 833 P 740 P 416, P 552 P 810 P 840 P 058 P 159
LEMICHEZ, E. LEMOS, E.M. LEMRANI, M. LEON GUERRERO, D. LEON, L. LEON, L.L. LEOPOLDO, P.T.G. LESCANO, A.G. LESSA, M. LESSA, M.M. LEUNG, D.T. LEUTENEGGER, C. LEVCHENKO, A. LEZAMA DAVILA, C. LIA, R.P. LIAO, L. LIARTE, D.B. LIBURKIN, T. LICIO, C.S.A. LIEBEL, S.M.R.S. LIGEIRO, D. LIMA JUNIOR, M.S.C. LIMA JUNIOR, D. LIMA, A.C. LIMA, A.L. LIMA, A.M. LIMA, A.P.C.A. LIMA, B.D. LIMA, B.S. LIMA, C. LIMA, C.A.A. LIMA, E.F. LIMA, F.P. LIMA, I.S. LIMA, J.G.B. LIMA, J.P.M.S. LIMA, J.R. LIMA, J.T.R. LIMA, J.V.F. LIMA, J.W.O. LIMA, L.D.R. LIMA, L.M. LIMA, L.R. LIMA, L.V. LIMA, M.A. LIMA, M.C. LIMA, M.S. LIMA, M.S.C.S.
P 430 P 349, P 496 O 253, P 015, P 016, P 092 P 417 P 427 P 523 P 250, P 261, P 456 O 351 P 680, P 835 P 133 P 871, P 889, P 891 P 886, P 888, P 890, P 897 P 885, P 894 P 278 P 686, P 852 O 371 O 308 O 010 P 055, P 697 P 099, P 256 O 072 P 509 P 236 O 341 P 801 O 328 P 759, P 765 P 499, P 502, P 506, P 677 P 147 P 263 O 071 P 263 P 222 O 126, P 095 P 850 O 293 P 209 P 842 P 731 O 181, P 524 O 036 P 285 P 278 P 809 P 422, P 424 P 813, P 815, P 892 P 876, P 880 P 169, P 855 P 065 P 571 P 488 P 193, P 422 P 424 O 108, O 057, O 261 P 933 O 043, P 739, P 853 P 775
LIMA, N. LIMA, P.M. LIMA, R.B. LIMA, S.C. LIMA, S.S. LIMA, V.M.F. LIMA, W.P. LIMAM, S. LINDOSO, J.A. LINDOSO, J.A.L. LINO, M.T.B. LIOTTA, D.J. LIPIK, V. LIPOLDOVA, M. LITZKE, L. LIU, D. LLANES ACEVEDO, I.P. LLANES, P. LLANOS CUENTAS, A. LLANOS, A. LLEONART, R. LLULL, J. LO VERDE, V. LOBO, C.H. LOCATELLI, F.M. LOCKWOOD, D.N. LONARDONI, M.V.C. LONGCHAMP, J. LOPES DO CARMO, L.A. LOPES MORI, F.M.R. LOPES, A.P. LOPES, D.M. LOPES, I.D. LOPES, J.D. LOPES, L. LOPES, M.V. LOPES, R. LOPES, R.M. LOPES, U.G. LOPES, X.G.C. LOPEZ GONZALVEZ, A. LOPEZ SANCHEZ, A. LOPEZ, A. LOPEZ, L. LOPEZ, Y. LORDELO, M. LORENZ, B. LORIA CERVERA, E.N.
O 356 P 262 P 664 O 167, P 479 P 519 P 740 P 645 P 394, P 402 P 395 P 736 P 407 P 586 O 227, P 472 P 371 P 241 P 577 P 504 O 045, O 097 P 549 O 049, O 086 O 260 P 694 O 373 P 188 P 009 O 267, P 144, P 245, P 397, P 438, P 535 O 254 P 009 P 075 P 400, P 693 P 622 O 038 O 085 P 233 P 053 P 698, P 712 P 787 P 726 P 141 P 896 P 172, P 175 P 156 P 805 P 559, P 625 O 182, O 336, P 508 O 312 O 037, O 275, P 155, P 183, P 222, P 304 P 896 P 373 P 461 P 464, P 467 O 280 O 361, O 380 O 364 P 025, P 046, P 047 O 238 O 333 P 647
LOSADA BARRAGAN, M. LOURDES, R.A.E. LOUTET, M. LOUZIR, H. LOVE HOMAN, L. LOVELL, A.M. LU, W. LUBBAD, M.Y. LUBY, S.P. LUCAS, C.M. LUCHEIS, S.B. LUGO, D. LUGO, D.A. LUISI JUNIOR, C.M. LUN, Z. LUPO, T. LURA, T. LUTGEN, P. LUVIZOTTO, M.C.R. LUZ, E. LUZ, N. LUZ, N.F. LUZ, S.L.B. LYE, L. LYNEN, L. LYONS, P. LYRA, M.R. M, S.L. M, S.L.L. M. PEREIRA, D.C. MACEDO SILVA, S.T. MACEDO, B.M. MACEDO, D.S. MACEDO, E.C. MACEDO, L.A. MACEDO, S.R.A. MACHADO DE ASSIS, T. MACHADO DE ASSIS, T.S. MACHADO COELHO, G.L. MACHADO COELHO, G.L.L. MACHADO, A.L. MACHADO, A.R.S.R. MACHADO, C.M.T. MACHADO, E.M.M. MACHADO, F.C. MACHADO, G.F. MACHADO, L.F.M. MACHADO, L.M.
O 325 P 035 O 003, P 377 O 236, O 363, P 039, P 958 O 302 P 289 P 110 P 014 O 018, O 062, O 172, P 741, P 796 O 351 P 745 P 298, P 302 P 203 P 247 P 055, P 697 O 187, P 064, P 400, P 693, P 837 O 194 P 469 P 634 P 635 O 096, P 098, P 776 P 136, P 343 O 051 P 920 O 074, O 142, O 351, P 116, P 277 O 262, O 263 O 343 P 237 P 597 P 483 P 863 P 352 P 526 P 888, P 891 P 897 P 885, P 886, P 889, P 890, P 894 P 468, P 770 O 275 P 325 O 264 P 768 P 567 P 214, P 795 P 021 P 201, P 534 P 347 P 533 P 512 P 176, P 229, P 247, P 252 P 032, P 049, P 056, P 196, P 687 P 165 P 191, P 253 P 145
MACHADO, N. MACHADO, P. MACHADO, P.A. MACHADO, P.R. MACHADO, P.R.L. MACHADO, R.Z. MACHELARD, A. MACHINI, F.K. MACIEL REZENDE, C.M. MACIEL, B.L. MACIEL, J. MACKENZIE, B. MACKENZIE, C.D. MACORIS, M.L.G. MACRÌ, D. MADDUGODA, M.P. MADEIRA, I. MADEIRA, M.F. MADHUBALA, R. MAES, I. MAES, L. MAGALHAES, A. MAGALHAES, A.C.S. MAGALHAES, A.S. MAGALHAES, F.B. MAGALHAES, L.K. MAGALHAES, M.A.F. MAGALHAES, R.D.M. MAGALHAES, V. MAGALHAES, V.P. MAGILL, A. MAHAJAN, R. MAHFUZ, F. MAHMOUDI, A.R. MAIA ELKHOURY, A.N. MAIA, C. MAIA, F.C. MAIA, J.M. MAIA, R. MAIA, R.G. MAJI, M. MAJUMDAR, S. MAJUMDER, H.K. MAJUMDER, S. MAKOWIEKI, M.E. MAKURATH, M.A. MALAVIYA, P. MALEKI, 3.
P 476 P 204, P 480, P 527 P 459 O 277, O 050 O 169, P 134 O 166, P 337, P 423, P 785 P 249 O 273 P 282 P 538, P 553 P 255 P 919 O 070 P 448 P 748 O 187 P 430 P 310 O 122, P 824 P 090, P 415 O 088 P 062 O 146, P 451, P 452, P 460, P 636, P 642 P 023 P 939 P 264 P 592 P 271 P 583 P 725 P 018 P 087 P 516 O 289 O 108, O 057, O 261 O 064 O 318 O 229 O 219, P 453, P 887 P 640 P 726 P 577 P 492 O 154 P 584 P 359, P 365 P 584 P 869 O 283 O 012, O 015, O 316, O 357 P 340
MALHEIRO, A. MALHEIROS, A.F. MALIK, M.D. MALONE, J.B. MALTA, H. MALTA, M. MALVIYA, P. MANAFI, H. MANAGIDO, M.B. MANCERO, T. MANDAL, G. MANDAL, S. MANNA, L.L.M. MANNA, M. MANNAER, A. MANNAERT, A. MANS, D. MANS, D.R. MANSINHO, K. MANTECA ACOSTA, M. MANTECA ACOSTA, M. MANTILLA, J.C. MANUEL, C. MANZANO, J.I. MARCELINO, A.P. MARCET, P. MARCILI, A. MARCO, J.D. MARCONDES, C.B. MARCONDES, M. MARCOS, S.F. MARCUS, J. MARGIOTTA, A. MARIA, A.L. MARIALVA, E.F. MARIANTE, R.M. MARIN, D. MARINHO JUNIOR, J.F. MARINHO JUNIOR, J.F. MARINHO, C.C. MARINHO, P. MARINO, E. MARION, E. MARKIKOU OUNI, W. MARKIKOU, W. MARKING, U. MARKUS, R.P. MARLOW, M.A.
P 258 P 809 P 671 P 901 P 343 P 786 O 163 P 701 P 750, P 751 O 380 O 004, P 070, P 107, P 248 P 070, P 107 P 425 P 802 O 344 O 002, O 143, P 672 O 098, P 458, P 794 P 151 P 805 P 806, P 822 O 097, P 778 P 560, P 573 P 122 P 413 P 764 O 252 P 685, P 809 O 038, P 485 O 358 P 054, P 056, P 394, P 395, P 686, P 687, P 736, P 771, P 793, P 852 P 209, P 211 P 277 P 064 P 754, P 755 P 924 P 923, P 925, P 926, P 927 O 301 P 859 P 850 P 906 P 585 P 795 P 278 P 769 O 366 O 208 P 039 O 023 O 048 P 866
MARQUES PAULO, E. MARQUES, C. MARQUES, C.S. MARQUES, L.H.S. MARQUES, M. MARQUES, M.C.S. MARQUES, M.J. MARQUES, P.R. MARQUEZ, E.S. MARQUEZ, R. MARQUIS, H. MARR, A.K. MARRA, A.K. MARTEL, C. MARTELETO, B. MARTELETO, B.H. MARTIN MARTIN, I. MARTIN, D. MARTIN, O. MARTIN, R. MARTIN, V. MARTINEZ FLOREZ, A. MARTINEZ, A. MARTINEZ, A.M.B. MARTINEZ, D. MARTINEZ, D.Y. MARTINEZ, E. MARTINEZ, M.A. MARTINEZ, M.F. MARTINEZ, R. MARTINKOVIC, F. MARTINS FILHO, O. MARTINS FILHO, O.A. MARTINS MELO, F.R. MARTINS, A. MARTINS, A.C. MARTINS, A.C.C. MARTINS, C. MARTINS, D.A. MARTINS, D.R. MARTINS, D.R.A. MARTINS, E.P. MARTINS, J.C.D. MARTINS, L.E. MARTINS, L.F. MARTINS, M.S. MARTINS, N.V.D.N. MARTINS, T.F.C.
P 228, P 241 O 331 O 220, O 319, P 490, P 772, P 786 P 816 P 900 P 453 P 543 P 434, P 533, P 538, P 553, P 816 P 408 P 441, P 444, P 742 P 652, P 653, P 774 O 147, O 258 O 159 O 003, P 377 O 353 O 338 P 292 P 194 O 244, O 245, P 763 P 596 O 193, P 791 O 035 P 494 O 214, P 068 P 075 P 769 P 208 P 144 O 267 P 535 P 578 P 882 O 097, P 778, P 814 P 707 O 135 O 293 O 276, P 069, P 194, P 197, P 199, P 643 O 043, P 739, P 853 P 887 P 037 P 090 P 164 O 182 P 147 P 278 P 285 P 955 P 764 P 520 P 351 O 039, O 100 P 949 P 472
MARTINS, T.M. MARTINS, T.Y. MARTINS, V. MARTINS, V.P. MARTINS, V.T. MARTIS, N. MARTY, P. MARUYAMA, S.R. MARZOCHI, K.B.F. MARZOCHI, M.C.A. MARZOUKI, S. MASATAKA, K. MASCARENHAS, N.M.F. MASHAYEKHI, 4. MASPERO, R.C. MASSAFERA, R. MASSON, J. MASTRANGELO, A.V. MASUCCI, M. MATHEOUD, D. MATHIAS, F.A. MATHIAS, F.A.S. MATLASHEWSKI, G. MATOS GUEDES, H.L. MATOS, D.C.S. MATOS, M.F.C. MATOS, P.C.M. MATOS, V.T.G. MATRANGOLO, F.S.V. MATSUMOTO, Y. MATTA, V.L. MATTOS, M.S. MAURICIO, I. MAURICIO, I.L. MAZUMDER, S. MAZZONI, C. MC.MASTER, R.W. MCCAFFREY, A.P. MCCALL, L. MCCAROLL, J. MCGAHIE, D. MCKERROW, J. MCKERROW, J.H. MCLEAN, D. MCLIDEN, J.H. MCMAHON PRATT, D. MCMASTER, R.W. MCMASTER, W.R.
P 408 P 462 P 375 P 165 P 226 P 018, P 362, P 376, P 520 P 221 O 014, O 366, P 221, P 430 P 323 O 122, P 824 P 824 O 122 P 958 P 485 P 716 P 340 P 956 P 738 P 336 O 095 P 837 O 324 P 072 O 091 P 067, P 129 O 155, O 348, O 349, P 670 P 208 O 274 P 131 P 251, P 479 P 864 P 579 P 099 P 313 O 090, O 370, O 174, O 176, O 177, O 178, P 313 P 793 P 269 O 106, P 711 O 102 O 154 P 320 P 377 O 073 O 155 P 901 O 214 O 306 O 285 O 268, P 749 P 278 P 180 O 003 O 353
MEDEI, E.H. MEDEIROS, A.C.R. MEDEIROS, J.F. MEDEIROS, M.A. MEDEIROS, Z. MEDEIROS, Z.M. MEDINA, V. MEDLEY, G. MEDRANO, F. MEHEUS, F. MEHTA, R.D. MEIRA, C.S. MEIRELES, J. MEJIA, E. MEJRI, T. MEKONEN, W. MEKURIA, A.H. MELBY, P.C. MELENDEZ, V. MELLO, M.N. MELO JUNIOR, O.A.O. MELO, A.L.A. MELO, F.L. MELO, G.D. MELO, G.M.A. MELO, J.A.T. MELO, L.M. MELO, M. MELO, M.A. MELO, M.B. MELO, M.M.
MELO, M.N. MELO, N. MENDES AGUIAR, C.O. MENDES SOUSA, A.F. MENDES, A.R. MENDES, B.P. MENDES, M.S.T. MENDES, T. MENDES, T.A.O. MENDES, V.G. MENDEZ, G.A. MENDEZ, J. MENDEZ, S. MENDONCA, A.L.P. MENDONCA, D.S.
P 559 P 293, P 296, P 608, P 609, P 614, P 617 P 345 P 005 P 149, P 368, P 369 P 493, P 498 P 836 O 041 O 016 O 067, P 691, P 692 O 171 P 082 P 601, P 631, P 911 O 220, O 319, P 490 P 259 P 517, P 580 P 410 P 610, P 751 P 447 P 518 O 139, P 711 O 311 O 276 P 761 P 493 P 369 P 368 P 687 P 440 P 195 O 185, P 396, P 688 P 629 P 842 P 532 P 516 P 606, P 839 O 276, O 112, O 091, O 294, P 051, P 066, P 072, P 200, P 213, P 225, P 244, P 393, P 416, P 492, P 565, P 673, P 674, P 675, P 734, P 783, P 784, P 819 O 350 P 238 O 317 P 846 P 102 P 095, P 648 P 486 P 181 P 139 P 008 P 518, P 607 O 159 P 358 P 269
MENDONCA, I.L. MENDONCA, L.Z. MENDONCA, M.S. MENDONCA, S.C. MENDONCA, S.C.F. MENDOZA LEON, A. MENESES FILHO, A.J.S. MENESES, C. MENEZES, J. MENEZES, J.A. MENEZES, J.P.B. MENEZES, T. MENNA BARRETO, R. MENTEN, J. MENZ, I. MERELLES, S.L. MESA, C.V. MESQUITA LIMA, R.T. MESQUITA RODRIGUES, C. MESQUITA, J.T. MESTRE, G.L.C. MHADHBI, M. MICHALICK, M. MICHALICK, M.S. MICHALSKY, E.M. MICHEL, G. MICHELS, P.A. MICHIELIN, O. MIECZKOWSKI, P. MIEREZ, M.L. MIGLIAZZO, A. MIGOTTO, A. MIGUEL, D.C. MIKROS, E. MILAGRES, F.A.P. MILANI, B. MILANI, N. MILES, M. MILES, M.A. MILLER, E. MILLER, E.N. MILLS, J.E. MILON, G. MINERVINO, A.H.H. MINHARRO, S. MIRA, M. MIRANDA VERASTEGUI, C. MIRANDA, D.E.O.
O 198 P 197, P 199 P 883 P 150 O 274, O 165, P 005 P 474 O 322 O 159, O 160, O 161, O 320 P 146, P 169 P 731, P 735 P 167 P 204 P 124 O 262, O 269 P 601, P 631, P 928, P 957 O 044, P 526 O 280, P 589 P 370 P 124 P 370 P 389, P 390 O 184 P 586 P 311 P 606, P 839 P 788 P 567, P 759, P 765 P 754 P 430 P 598 O 035 O 030 P 666, P 779 O 187, P 400, P 693, P 837 P 351 O 282, P 477 P 419 P 406 P 572 P 619 O 203 P 342 P 711 O 137, O 265, P 308 O 102 O 216, O 065 P 133 O 152 O 284 O 047 P 809 P 856 P 189 P 535 O 267, P 245 O 128, P 940, P 941
MIRANDA, H.C. MIRANDA, J.C. MIRANDA, N. MIRET, J. MIRHENDI, H. MIRO, G. MIRTHA, M. MIRZAEI, F. MISHRA, A. MISHRA, A.K. MISRA, P. MITRA, G. MITSUKA BREGANO, R. MITSUKA BREGANO, R. MITTMANN, J. MITTRA, B. MIYASHIRO, S.I. MIYAZAKI, R.D. MKADA, I. MKANNEZ, G. MOCHLY ROSEN, D. MODABBER, F. MODOLELL, M. MODOLO, J.R. MOFFI, P. MOHAMATH, R. MOHAMED, H.S. MOHAMMED, R. MOKNI, M. MOLINA, N. MOLINA, R. MOLINARO, C.M. MOLLA, L.M. MONACO, N.Z. MONDAL, D. MONDEGO, J. MONDRAGON, K. MONROY OSTRIA, A.M. MONTALVO, A.M. MONTAﾃ前, N.A. MONTASSIER, H.J. MONTE NETO, R.L. MONTE NETO, R.L.D. MONTE NETO, R.L.D. MONTEIRO, E.L.O. MONTEIRO, E.M. MONTEIRO, G. MONTEIRO, G.G.
P 943 O 250 P 024, P 844 P 178 O 270, P 704, P 707 O 318 O 342, P 769 P 628 P 621 P 137, P 661 P 449 P 381 O 108, O 261 P 826 P 787, P 829 P 012, P 354 O 348 P 091, P 266 P 754, P 755 O 138 P 058, P 059 O 282 O 365 O 133, P 410 P 863 O 259 O 092, O 027 O 343 O 262, O 263 P 916 O 363, P 125 P 822 O 244, O 245, P 157, P 757, P 763, P 766 P 415 P 631 P 823 O 209, O 084, O 018, O 021, O 062, O 172, O 266, P 366, P 741, P 802 O 030 O 011, O 196, O 380, P 904 P 595, P 882 O 254, P 062 O 254 P 190 P 339 O 112, P 565 O 145 P 729, P 730, P 733 P 214 P 758, P 764 P 522 P 147 P 286
MONTEIRO, G.R. MONTEIRO, G.R.G. MONTEIRO, J.C. MONTEIRO, M. MONTEIRO, M.B.A. MONTEZZO, L.C. MONTGOMERY, J.M. MONTI ROCHA, R. MONTILLA MATOS, M.L.A. MONTOYA, A. MONTOYA, E.A. MONTOYA, R. MOON, K. MORA, M.C. MORADIN, N. MORAES, J.L.P. MORAES, M.O. MORAES, P.N. MORAES, R.C.S. MORAES, S.C. MORAES, V.S.S. MORAIS, G.B. MORAIS, L. MORAIS, M.H.F. MORAIS, M.H.G. MORAIS, M.R.P.T. MORAIS, R.C. MORAIS, S.M. MORALES, J. MORANNE, O. MOREA, G. MOREIRA, B.P. MOREIRA, C.P.S. MOREIRA, D. MOREIRA, D.R.M. MOREIRA, D.S. MOREIRA, I.M. MOREIRA, J.S. MOREIRA, M.A. MOREIRA, M.A.B. MOREIRA, M.L. MOREIRA, M.R. MOREIRA, N.D. MOREIRA, O.C. MOREIRA, P.A. MOREIRA, P.A.C. MOREIRA, P.R.R. MOREIRA, R.M.
P 255 P 273 P 285 P 128, P 289 P 845 P 476 P 261 P 448 P 680 P 349 P 342 O 342 P 327 O 380 P 180 O 038 P 485 O 324 P 673, P 674, P 734 P 783, P 784, P 819 O 184 P 807 P 906 P 820 P 808 P 502 P 884 P 533, P 900, P 945, P 954 P 731 P 735 P 875 P 335 P 622 P 910 P 221 P 104 P 216 P 632 O 372 P 541 P 564 P 339 P 664 P 037 P 090, P 164 P 634 P 928 P 194 P 292 P 209 P 067 O 091, P 072, P 129, P 240 P 083 P 880 P 815 P 177, P 190 P 939
MORELLI, K.A. MORENO MATEOS, D. MORENO, D. MORENO, J. MORENO, M. MORENO, M.A. MORGADO, F.N. MORGADO, M.G. MORIZOT, G. MORKOWSKI, S. MORTARA, R.A. MORTAZAVI, H. MOSER, B. MOSORE, M. MOSQUEDA, L.A. MOSSER, D. MOTA, M.D.S.S. MOTA, V.Z. MOTALAB, M.A. MOTAMEDIRAD, M. MOTOIE, G. MOTTRAM, J. MOTTRAM, J.C. MOU, Z. MOURA JUNIOR, I.L. MOURA NETO, J.P. MOURA, A.A.A. MOURA, A.C.A. MOURA, A.C.J. MOURA, A.L. MOURA, A.P.V. MOURA, F.B.P. MOURA, F.K. MOURA, V.A. MOURI, O. MOUTA CONFORT, E. MOUTA CONFORT, E. MOWBRAY, C.E. MOYO, D. MUKHERJEE, A. MUKHERJEE, B. MUKHERJEE, S. MUKHOPADHYAY, A. MUKHOPADHYAY, R. MUKHTAR, M. MUKHTAR, M.M. MULLEN, A. MULLEN, A.B.
O 136 P 529 O 307 O 215, O 016, O 046, O 293, O 068, P 934, P 937, P 938 P 859 P 342 O 375 P 148 P 037 O 237 P 228 O 363, P 457 O 194, O 289 O 030, O 305, O 335, P 192 P 085 O 140, O 141 P 946 P 779 O 240, P 204 P 949 P 459 P 796 O 239 P 082, P 911 P 222 O 033, O 071 O 373 P 851 P 719 P 622 O 128, P 940, P 941 O 186 P 181 P 200 P 813, P 815 P 892 P 880 P 572, P 619 P 321, P 322 P 457 O 122, P 090, P 415 P 824 P 636 O 287 O 340 P 336 P 401 P 401 P 314, P 315 O 004, P 070, P 107, P 248 P 401 O 349, P 610 O 138 P 288 P 356 O 115 P 450
MULLER, I. MÜLLER, K.E. MUNARI, D.P. MUNASINGHE, A. MUNDER, M. MUNIER LEHMANN, H. MUNIZ, A. MUNIZ, A.C. MUNIZ, C.R. MUÑOZ, D.L. MUNRO, P. MUNSTERMANN, L. MURILLO, J.D. MURRAY, P.J. MURTA, M.F. MURTA, S.M.F. MUS, A. MUSA, A. MUSA, A.M. MUSA, B. MUSKUS, C. MUSKUS, C.E. MUTHUSAMY, N. MUXEL, S.M. MYBURGH, E. MYLER, P. MYLER, P.J. MYNX, P. MYRIANTHOPOULOS, V. MYRSELI, T. NABI, S.G. NABIL, B.H.H. NABIPOUR, Y. NABUT, L.B. NADAL, J.M. NÆSS, A. NAGEM, R. NAGEM, R.A.P. NAIFF, M.F. NAKAGHI, A.C.H. NAKAMURA, C.V. NAKAO, R. NAKHASI, H. NAKHASI, H.L. NAMAN, C.B. NAOUAR, I. NARA, T. NARDY, V.
O 133, P 410 O 237, P 437 P 230 P 177 P 398 P 670 O 133, P 410 O 284 O 250 O 278 P 436, P 729, P 730, P 732, P 733 O 280, P 327, P 328, P 469, P 589 P 430 O 008 O 280 O 132 P 256 P 099, P 339, P 442 P 627 P 691 O 343, O 356, P 692 O 343 O 254 O 286, O 347, O 164, P 531, P 589, P 951 O 369 O 048, O 129, P 081, P 465 O 071 O 142, O 054, O 349 O 107, O 348 O 142 P 406 P 843 O 222 O 084, O 376 O 289 P 621 P 441, P 444, P 742 P 075 P 230 O 257 P 018 P 258 P 249 P 625 P 026 O 368 O 026 O 308 O 236, P 039, P 153 O 179 P 187
NARVAEZ, M.A. NARVAEZ, M.L.A. NASCIMENTO SILVA, M. NASCIMENTO SILVA, M.M. NASCIMENTO, A.A. NASCIMENTO, A.A.S. NASCIMENTO, B.W.L. NASCIMENTO, D.V. NASCIMENTO, E.G. NASCIMENTO, E.T. NASCIMENTO, F.C. NASCIMENTO, J.C. NASCIMENTO, L.M.D. NASCIMENTO, M.B. NASCIMENTO, M.D.D.S.B. NASCIMENTO, M.P. NASCIMENTO, M.S. NASCIMENTO, M.S.L. NASCIMENTO, M.T. NASCIMENTO, P.P. NASEREDDIN, A. NASKAR, K. NASSEREDIN, A. NATARAJAN, G. NATARAJAN, G.R. NATEGHI ROSTAMI, M. NAURATH, P.E. NAVARATNE, C. NAVARRO, A.L. NAVARRO, I.T. NAVAS, A. NDAO, M. NDUNGU, J. NEEDHAM, J. NEGRAO, F. NEGRAO, F.J. NEGREIROS FILHO, O. NEGREIROS, A.S. NEGRI, A. NEGROMONTE, G.R.P. NEIRA, L.F. NEITZKE ABREU, H.C. NERLAND, A.H. NETO DA SILVA, M.A.C. NETO, C.P. NETO, F.F. NETO, J.F.R. NETO, L.S.
P 040 P 443 P 380, P 474 O 039 O 100, P 833 O 317 P 239 P 708, P 798 P 226 P 391 P 896 P 289 P 165 P 253 P 788 P 270 P 592 P 436, P 729, P 730, P 732, P 733 P 201, P 534 O 180, O 210, O 328 P 253 O 304 P 147, P 289 O 243 P 684 O 032, O 290 P 088 P 455 O 369, O 026 O 368 O 211 P 930 P 670 P 489 O 096, P 441, P 444, P 713, P 715, P 716, P 742, P 787, P 826, P 829 O 147, P 089 O 256 P 366 O 289 P 283 P 893 P 856 P 468, P 618, P 770 O 307 P 883 P 497 P 236, P 849, P 858, P 862 P 230 P 436, P 729, P 730, P 732, P 733 P 176 P 255, P 286 P 128 P 398
NETO, M.B. NETO, O.P.M. NETO, R.B. NETO, R.D.M. NETTO, M.B. NEVES, B. NEVES, F.T.P. NEVES, L.X. NEVES, P.C.B.C. NEWTON, R. NGUNDAM, F. NICODEMO, A.C. NICOLETE, R. NIETO, J. NIETO, P.D. NIHASHI, N.R. NILGES, M. NIVEYRO, C. NOBUSUE, K. NOGUEDA TORRES, B. NOGUEIRA DE MELO, G.A. NOGUEIRA FREITAS, Y.B. NOGUEIRA, B.M.F. NOGUEIRA, F.C. NOGUEIRA, F.S. NOGUEIRA, P.M. NOGUEIRA, R.F. NOGUEIRA, R.S. NOIRI, E. NONATO, F.R. NORONHA, A. NORONHA, E.F. NORWOOD, J. NORWOOD, J.A. NOTREDAME, C. NOTTON, T. NOVAIS, F. NOVICOV, M. NOVO, S.P.C. NOVOZHILOVA, N.M. NOZAWA, S. NUCATOLO, G. NÜHS, A. NUNES PINHEIRO, D.C. NUNES PINHEIRO, D.C.S. NUNES, C.M. NUNES, D.E.C. NUNES, F.F.C.
P 036, P 198 P 020 O 169 P 492 P 022, P 223 O 372 P 565 P 143 P 512 O 360 O 114 O 168 P 435 P 108, P 325, P 747 O 215, O 068, P 934 P 901 O 179 P 403 P 008 O 084 P 595 P 233 P 865, P 868, P 872, P 874, P 875, P 878, P 879 P 358 P 040 P 928 O 275 P 150 P 551 P 269 O 173, O 174, O 176, O 177, O 178 P 487 O 238 O 296, P 929 O 289 O 362 O 205, P 232 O 075, P 277 O 241, O 087 P 485 P 840 O 075 P 189 P 693 O 149 P 499 P 677 P 502, P 506 P 394 P 540, P 687, P 780 P 918 P 496
NUNES, J. NUNES, J.O. NUNES, J.V.S. NUNES, M. NUNES, T. NUNES, V.L.B. NYLEN, S. NYUGEN, B. OCAMPO, C. OCAMPO, C.B. OCAMPO, R. OCAMPOS, H. OCHOA, R. OCHOA, R.A. OCONNOR, A. ODDONE, R. OGAWA, G.M. OGHUMU, S. OJEDA, J. OKWOR, I. OLIVA CORREIA, C. OLIVA, G. OLIVEIRA FILHO, R.D. OLIVEIRA NASCIMENTO, J.M. OLIVEIRA SILVA, C.V. OLIVEIRA NETO, M.P. OLIVEIRA SILVA, A. OLIVEIRA, A. OLIVEIRA, A.F.J. OLIVEIRA, A.G. OLIVEIRA, A.P. OLIVEIRA, A.P.M. OLIVEIRA, B.B. OLIVEIRA, B.C. OLIVEIRA, B.C.M. OLIVEIRA, C.I. OLIVEIRA, C.V.S. OLIVEIRA, D.F. OLIVEIRA, E. OLIVEIRA, E.B. OLIVEIRA, E.F. OLIVEIRA, E.J. OLIVEIRA, F. OLIVEIRA, F.A. OLIVEIRA, F.C.B. OLIVEIRA, F.M. OLIVEIRA, G.G.S. OLIVEIRA, H.C.
P 285 P 851 P 034 P 453 O 341 P 838 O 206, O 207, O 239, O 299 O 241 P 859 O 008 P 328 P 707 O 286, P 531 O 347, P 589 P 884 O 139, P 877, P 881 P 345 O 339 O 270, P 707 O 373 P 525 O 212, O 292, O 320, P 048 P 678 P 283, P 569 P 576 P 269 O 233, O 167, P 234, P 238, P 274, P 551 O 276 P 493 O 330 P 926, P 927 P 846 P 057, P 823, P 832, P 893 P 010, P 411 P 847 O 185 P 010, P 411 P 402 P 036 P 132, P 844 P 106 P 827 O 264, P 358, P 374 P 601 P 846, P 861 P 903 O 160, O 161, O 320, P 958 P 250 P 261, P 456 P 714 P 780 O 183, P 006, P 077, P 182, P 271, P 522 P 391 P 020 P 655
OLIVEIRA, I.Q. OLIVEIRA, J. OLIVEIRA, J.C. OLIVEIRA, J.S. OLIVEIRA, K.R. OLIVEIRA, L.A.M. OLIVEIRA, L.V. OLIVEIRA, M. OLIVEIRA, M.A. OLIVEIRA, M.A.P. OLIVEIRA, M.M. OLIVEIRA, M.P. OLIVEIRA, M.R.F. OLIVEIRA, N.J.D. OLIVEIRA, P.O. OLIVEIRA, P.S. Oliveira, R.B.d. OLIVEIRA, R.C. OLIVEIRA, R.M. OLIVEIRA, S.D. OLIVEIRA, S.M.D.V.L. OLIVEIRA, T.M.F.S. OLIVEIRA, T.S. OLIVEIRA, W.W. OLIVIER, M. OLLIER, B. OLOBO, J. OLORTEGUI, C.D.C. OLSEN, A. OMMEN, G. ONO, M.A. OOMMEN, J. OPPERDOES, F. ORDOBAS, M. ORE, M. ORIN, C. ORTA, J. ORTA, J.F. ORTEGA, M.E. ORTIGAO, M.R. ORTIZ, R. ORUE, A. OSADA, Y. OSHIRO, E.T. OSMAN, M. OSMAN, O. OSORIO, E.Y. OSORIO, W.G.
P 522 P 805 P 133, P 264 P 419 P 376 P 807 P 135, P 240, P 242 O 170, P 583 P 343 P 945 P 613 P 135, P 242 O 234, O 167, P 274, P 479 O 304 O 296 P 135 P 579 P 593 P 683 P 067 P 743, P 744 P 507, P 626 P 555 P 579, P 851 P 539 P 249 P 073 P 119 O 272 O 029, O 327, O 329, O 338 O 213 O 356, P 627 P 347 O 172 O 006 P 873 O 352 P 384 P 769 P 835 P 544 P 070 P 248 P 203 P 224 P 578 P 474 O 370 P 202, P 569, P 823, P 832, P 846, P 893 O 162 O 265 P 447 P 698
OSPINA, V.E. OSTOLIN, T.L.V. OSTRITZ, A. OSTYN, B. OSTYN, B.J. OTOMURA, F. OTRANTO, D. OUAISSI, A. OUALHA, R. OUELLETTE, M. OVALLOS, F.G. OVANDO, M.O. OWEN, N. OWENS, K. OYAFUSO, L.K. OZAKI, C.Y. OZBEL, Y. ÖZBEL, Y. OZENSOY TOZ, S. PAAPE, D. PACHECO, A.D. PACHECO, L.V. PACHECO, S.G. PACIENZA LIMA, W. PADMANABHAN, P.K. PADRON NIEVES, M. PADRON, M.D.C. PADUA, R.M. PAES LEME, F.O. PAES, R.A. PAGAN, A.J. PAGLIARI, C. PAGLIONI, D.N. PAGNIEZ, J. PAIN, A.M. PAIXAO, F.R. PAIXAO, L.S. PAL, T.K. PALATINK DE SOUSA, C.B. PALATUCCI, A.T. PALMA, M.S. PALMEIRO, M.R. PAN, L. PANDEY, K. PANIAGO, A.M.M. PANSINI, L.N.N. PANTOJA, K.M.B. PAPADAKI, A.
P 560 P 126 P 699 O 078, O 111, O 113, O 015, O 315, O 055 O 357 P 738 O 010, P 330 O 330, P 519 P 184 O 145, O 150, P 079, P 336 P 861, P 912, P 937, P 957 O 054 O 100 O 075, O 351, P 277 P 577 P 637 O 295, O 321, O 174, P 117 O 135 O 295, O 321, P 117, P 287 O 077, O 152 P 056, P 736 P 522 P 344 P 600 O 101 P 017, P 331, P 338 O 144 P 362 O 186 P 090 O 121 P 639 P 759 P 700 P 159 P 700 P 788 P 020, P 182 P 030 P 470 P 048 P 108 P 037, P 090, P 164 O 308 O 056 O 057, O 061, P 599 P 251, P 579, P 851 P 349 P 810 P 318
PAPADOPOULOU, B. PAPARCONE, R. PAPIEROK, G. PAPIEROK, G.M. PAPPENFUSS, T.L. PAQUET, M. PARANHOS FILHO, A.C. PAREDES, A.O. PARENTE, D.M. PARINANDI, N. PARKINSON, T. PARRADO, R. PARRAS, M.A. PARREIRA, B.L. PARREIRAS, P.M. PASA, S. PASCHOAL, M. PASCOAL XAVIER, M.A. PASCUAL, Y. PASQUALI, A.K.S. PASSERO, L.F.D. PASSOS, J. PASSOS, R.O. PASSOS, S. PASSOS, S.T. PATEL, B.N. PATHAK, N. PATI, H. PATRICK, R. PAUL CHOWDHURY, B. PAUL, C. PAULA, C.D.R. PAULINO, T.P. PAVELY, R. PAVINSKI BITAR, A. PAYS, E. PAZ, G.F. PEACOCK, C.S. PEARSON, R.D. PEDRO, N.F. PEDRO, W.A. PEDROSA, C.M.S. PEDROSA, M.O. PEDROSA, R.B. PEDROSO, R.B. PEINE, K. PEIXOTO, A.A. PEIXOTO, S.A.T.
O 101, O 103, O 025, P 070, P 312 O 212, O 292 P 159 P 068 O 339 O 338 P 808 P 907 O 322 O 308 O 152 P 438 O 097 P 322, P 572, P 619 P 516 O 295, O 321 P 378 P 069 O 276 P 542 P 716, P 826 P 142, P 291 P 641 O 188, P 262 P 576 O 272, O 278, P 204 O 240 P 728 P 602 P 596 O 297 P 584 O 146, P 451 P 095 P 297, P 623 O 193 O 159 P 101 P 700, P 703 O 367 P 110 P 278 P 289, P 896 P 857 P 780 P 848, P 871 P 006 P 892 P 554 O 308 P 818 O 170, P 583
PEIXOTO, V.S. PEÑA, M.S. PENAFORTE, K.M. PENHA, L.L. PENNISI, M.G. PERALES, J. PEREIRA DA FONSECA, I. PEREIRA CARVALHO, R. PEREIRA CHIOCCOLA, V.L. PEREIRA, A. PEREIRA, A.A.S. PEREIRA, B.A.S. PEREIRA, B.S. PEREIRA, C. PEREIRA, C.A. PEREIRA, C.C. PEREIRA, C.W. PEREIRA, D.H.D. PEREIRA, G.C. PEREIRA, G.G. PEREIRA, H.F. PEREIRA, J. PEREIRA, J.C. PEREIRA, L. PEREIRA, L.I.A. PEREIRA, L.O.R. PEREIRA, L.R. PEREIRA, L.S. PEREIRA, M.A. PEREIRA, M.H. PEREIRA, M.M.C. PEREIRA, R.M.S. PEREIRA, T.J.C.F. PEREIRA, V.F. PEREIRA, V.R.A. PERES, A.C. PEREZ VALERO, R. PEREZ VICTORIA, J.M. PEREZ, A.A. PEREZ, J.E. PEREZ, J.G. PEREZ, J.L. PEREZ, M. PERIM, M.C. PERKINS, M.D. PEROSSO, J. PEROTE, M. PERRI, S.H.V.
P 889, P 891 P 848, P 885, P 886, P 888, P 890, P 894, P 897 P 044 P 683 P 263 P 837 P 474 O 331, O 341 P 238 P 601 P 007, P 082, P 505, P 631, P 955 P 658, P 911 O 262 P 812 P 065 P 550 P 786 O 170, P 583 P 194, P 292 P 891 P 888 P 191 P 526 O 030 O 322 O 336, P 471 P 160 O 156 P 038 O 083 P 404 O 167, P 479 O 184 P 223 O 331 P 239 O 317 P 270 P 183 O 198, O 322, P 645 P 539 P 564 P 010, P 541 P 205, P 220, P 265, P 411, P 566 P 480, P 527 P 047 P 025, P 046 P 417 P 778 P 904 P 680 P 379 O 008 P 828 P 366 O 185, P 396, P 688 P 769 P 402
PERSICHETTI, M.F. PERTEGUER, M.J. PERUHYPE MAGALHAES, V. PERVITSKY, D. PESCARINI, J.M. PESCHER, P. PESSANHA, J.E.M. PESSOA, F.A.C. PETERKA, C.R.L. PETERS, N.C. PETERS, S. PETERSEN, A.L.O.A. PETERSEN, C. PETERSON, A. PETITDIDIER, E. PETRI, S.C.S. PETRONI, T.F. PFEILSTICKER, T.R. PIANTEDOSI, D. PIAZZA, F.M. PIAZZA, M. PICADO, A. PICONE, A. PIETRA, R.S. PIMENTA, P.F. PIMENTEL, J.D. PIMENTEL, J.P.D. PIMENTEL, M.I. PIMENTEL, M.I.F. PINHEIRO, C.G.M. PINHEIRO, F.G. PINHEIRO, L. PINHEIRO, L.S. PINHEIRO, M.P.G. PINHEIRO, M.S.B. PINHEIRO, P.G.M. PINHEIRO, S.B. PINHO, F.A. PINHO, M.C.S.O. PINNA, R.A. PINTO, A.J.W. PINTO, B.F. PINTO, E.F. PINTO, E.G. PINTO, I.S. PINTO, J. PINTO, M.C. PINTO, M.D.C.
P 837 P 494 O 276, P 069, P 643 P 518 P 747 O 205, P 232 P 821 P 345, P 920, P 923, P 924, P 925, P 926, P 927 P 681 O 121 O 171 P 106, P 326 P 884 P 094 P 159 P 371 P 745, P 873 P 900 P 048 O 093 P 064, P 400, P 693 O 055 O 092, O 027 P 643 P 103 P 956 P 258 P 150 O 237, P 237 O 165 P 006, P 020, P 182 P 224, P 258, P 260 P 309 P 120 P 915, P 917 P 649 P 006 P 828 P 091 P 266, P 267 O 129 P 210 P 316 P 358 O 156, P 215, P 235 P 409, P 482, P 612 P 818 P 262 P 700, P 703 P 492
PINTO, M.R. PINTO, N.F.S. PINZAN, C. PIPERO, P. PIQUEMAL, D. PIQUET PELLORCE, C. PIRAJA, G.V. PIRANGY, F.R.F. PIRES, F.R. PIRES, M.A. PIRES, S.F. PIRMEZ, C. PIROVANI, C.P. PISHGOOEI, 2. PISSARRA, H. PIVETTA, O.S. PLEBAN, K. POCHE, D. POCHE, R. POHLMANN, A.R. POIRIER, G. POLAT, E. POLATOGLOU, E. POLETTO, F. POLITOU, A. POLMAN, K. POMARES, C. POMBA, C. POMPEU, M.M.L. PONTE SUCRE, A. PONTES DE CARVALHO, L.C. PONTES, C.S. PONTES, E.R.J.C. PONTES, N. PONTES, N.N. PONTES, S.M. POPOV, V. PORROZZI, R. PORTER, C.H. PORTES, M.G.T. PORTO, A.L.F. PORTUS, M. POSTIGO, M. POTET, J. POZZOLO, E.M. PRADO, A.P. PRADO, A.P.D. PRADO, W.S.
P 557 P 842 P 198 P 611 O 345, P 059 P 122 P 863 P 583 P 538, P 553 P 476 P 066, P 349 P 206 O 234, O 167, P 274 P 099 P 340 P 772 P 957 P 482 P 651 P 651 P 654 P 558, P 559 P 336 P 360 P 101 P 558, P 559 P 318 P 144 O 014, O 366, P 221, P 430 O 319 P 034, P 174 P 331, P 338 O 183, P 006 P 695, P 698, P 712 P 251 P 884 P 255, P 273, P 278, P 286 O 051, P 013 O 053 O 184, O 325 O 252, P 899 P 869 P 640 P 944, P 947 P 157 O 203 P 826 P 748 P 841 P 864, P 867
PRAJAPATI, V.K. PRASASD, N. PRATA FONSECA, A.M.D.R.B. PRATAP, J.V. PRATES, D.B. PRATLONG, F. PRATTI, J.E.S. PRESBER, W. PRESTES, S.R. PREVEL, F. PRICE, H.P. PRINA, E. PROFETA DA LUZ, Z.M. PROTEASA, G. PRUZINOVA, K. PUIG, M. PULIDO, S.A. PULIDO, X. PULLUKCU, H. PUPLAMPU, N. PURCELL, N. PURI, S. QUARESMA, J. QUARESMA, P.F. QUEIROZ, A. QUEIROZ, A.C. QUEIROZ, D.C. QUEIROZ, D.P.S. QUEIROZ, D.S. QUEIROZ, I.T. QUEIROZ, J.W. QUEIROZ, L.H. QUEIROZ, M.F.M. QUEIROZ, P.V. QUEIROZ, P.V.S. QUIJADA, J.D. QUINNELL, R. QUINTAL, A.P. QUINTAL, A.P.N. QUINTANA, M.G. QUINTAO, L.C. QUINTELA, G. QUINTELA, L.P. QUINTELLA, L.P. QUIROGA, M.M. QVIT, N. R SOBRINHO, A.C. RABAN, R.
O 315 O 113, P 364 O 171 P 616 P 449 O 326, P 118, P 844 O 135, O 014, O 221, O 351, P 140, P 272, P 905, P 908 P 162 O 135 P 583 O 052, P 112 O 077 O 284 O 374 O 035 O 248 P 450 O 283, P 328 P 467 P 117, P 287 P 946 P 366 P 596, P 642 P 493 P 225 P 846 O 276 O 240, O 166, P 423, P 785 P 593 P 440, P 488, P 528, P 594 O 317 P 543 P 202 P 521 O 227 P 278 P 147, P 289 P 780 P 820 P 147 P 285 P 257 O 008, O 042, O 197, P 513 P 176 P 161, P 229, P 247, P 512 O 045, O 095, O 097 P 950 P 036 P 090 P 164 P 949 O 282 P 897 P 654
RABELLO, A. RABELLO, A.L.T. RABHI, I. RABHI, S. RABIDAS, V.N. RACHID, M.A. RACINE, G. RAFATI, S. RAHMAN, K.M. RAHMAN, M. RAHMAN, M.M. RAHMAN, M.W. RAHMAN, R. RAI, A. RAI, K. RAI, M. RAI, R. RAIZER, J. RAMALHO ORTIGAO, J.M. RAMALHO ORTIGAO, M. RAMANATHAN, V. RAMASAMY, G. RAMASAWMY, R. RAMDAS, S. RAMESH, V. RAMILO, D. RAMIREZ, L.E. RAMOS JUNIOR, A.N. RAMOS SANCHES, E.M. RAMOS SANCHEZ, E.M. RAMOS, A.P. RAMOS, A.S. RAMOS, C. RAMOS, F. RAMOS, G.S. RAMOS, M. RAMOS, P.K. RAMOS, P.K.S. RAMOS, R. RAMOS, S.R. RAMOS, W.R. RANASINGHE, P.S. RANASINGHE, S. RANGEL, E.F. RANGEL, M. RANGEL, M.U. RANGEL, O. RANSOM, J.
O 264, P 069, P 228, P 358, P 374, P 768, P 903 O 224 P 587, P 588 O 345, P 059 O 345 O 056 P 051 P 079 O 239, O 118, O 025, P 109 O 018, O 062, O 172, P 741, P 796 P 796 O 222, O 376 P 796 O 109, P 366 P 514 O 111, P 386 O 206, O 207, O 113, O 315, O 063, P 154, P 478, P 483, P 495 O 279 P 893 O 246 O 192 P 596 O 107, O 142, O 054, O 348, O 349 P 189, P 264, P 307 O 098 O 024 P 364 O 220 P 781 P 803, P 807 O 043, P 739, P 853 P 637 P 002, P 091 P 535 P 445 P 887 O 038, P 485 O 311 P 825 P 422 P 193 P 424 P 169 P 854 P 755 P 920 P 670 P 668 O 349 O 155 O 378, P 097, P 696, P 699, P 705, P 725 P 250 P 740 P 489 O 363
RANUCCI, T.R. RAO, M. RAOUANE, M. RASO, P. RASSI, Y. RASTOGI, R. RATNAM, J. RAUHUT, F. RAVEL, C. RAVINDRAN, R. RAVINETTO, R. RAYCHAUDHURI, S. RAZURI, H.R. RE, M.I. REA, M.J. REA, S. READY, P.D. REAL, F. REALE, S. REBBESTAD, K. REBELO, J.M.M. REED, S. REED, S.G. REED, S.J. REGINA SILVA, S. REGINALDO DE SOUSA, M.L. REGINALDO SOUSA, M.L. REGINALDO, M.L. REGIS, W.B. REGIS, W.C. REGIS, W.C.B. REGNAULT, B. REGO, F.D. REIFUR, L. REIMAO, J.Q. REINER, N. REINER, N.E. REINHOLD CASTRO, K.R. REIS CUNHA, J. REIS, A. REIS, A.B. REIS, C.R. REIS, I.A. REIS, L.C. REIS, L.E.S. REIS, P.G.
P 858, P 862 P 596 P 790 P 629 O 318 P 314 O 306 P 694 O 221, O 124, O 255, P 140, P 272, P 457 O 154 O 262 O 194, O 289 P 680 P 208, P 407 P 357, P 666, P 779 P 050 O 191 O 030, O 305, P 155 O 187, P 400 P 230 P 783, P 784, P 819 P 673, P 674, P 675, P 734 O 161 P 576, P 615 O 209, O 090, O 092, O 093, O 293, O 027, O 177, P 313, P 610 O 266, P 250 O 178 O 092, O 027 P 214, P 567 P 878, P 879 P 874 P 865, P 868, P 872, P 875 P 362 P 226 P 376 P 123 P 018 P 520 O 345, P 059 P 225, P 800 P 098 P 505 P 370 P 390, P 418, P 419, P 477 O 054 P 180 P 233 P 236, P 849, P 858, P 862 P 486 O 293 O 091, P 067, P 071, P 072, P 129, P 135, P 143, P 197, P 199, P 240, P 242, P 434, P 496, P 522, P 533, P 714, P 816, P 903 O 103 P 700, P 703, P 900 P 002 P 129, P 434, P 533, P 816 O 081 O 112, O 145, P 565
REIS, V.W. REITHINGER, R. REMPEL, S.S. RENARD, P. RENTERIA, J.A.C. RENTSCH, D. REQUENA, J.M. RESENDE, D.M. RESENDE, L.A. RESENDE, M. RESENDE, M.O. RESSNER, R. RESTREPO, A.M. RESTREPO, C.M. REZENDE, A.M. REZENDE, M.T. REZENDE, S.A. RHAJAOUI, M. RIBEIRO SAMPAIO, R.N. RIBEIRO ALVES, M. RIBEIRO DE SENNA, M.C. RIBEIRO DIAS, F. RIBEIRO GUIMARAES, M.L. RIBEIRO ROMAO, R.P. RIBEIRO, A.J. RIBEIRO, J.B.P. RIBEIRO, J.G. RIBEIRO, J.M. RIBEIRO, K.A.N. RIBEIRO, L.C. RIBEIRO, L.M. RIBEIRO, M. RIBEIRO, M.L. RIBEIRO, M.S. RIBEIRO, P.F.M. Ribeiro, R.A.N. RIBEIRO, R.R. RIBEIRO, S.J.L. RIBEIRO, T.G. RIBEIRO, T.T. RIBEIRO, V.M. RIBEIRO, Y.J.S. RIBOLLA, P.E.M. RICE, R. RICO, C.I. RICO, E. RIDOFF, M. RIERA, C.
P 567, P 795 P 513 P 408, P 563 P 058 P 918 O 072, O 131 P 062 P 135, P 242 P 434 P 197, P 199 O 372 P 293 O 160 O 280, O 365, P 327, P 328, P 469, P 589 P 009 P 135 P 242 P 067, P 072 P 509 O 082, P 126, P 197 P 092 O 083, O 126, P 095, P 105, P 428, P 439, P 530, P 546, P 648 O 184 P 069 P 404 P 083 P 215, P 235 P 261 P 726 P 476 P 095, P 439 P 108 P 029 P 831 P 158 P 823, P 832, P 846 P 441, P 444, P 742 P 550 P 431 P 838 P 683 P 606, P 839 P 433 P 226, P 376 P 018, P 520 P 874, P 879 P 194 P 630, P 931, P 932 P 292 P 436, P 729, P 730, P 732, P 733 O 354, P 057, P 173 O 289 P 573 P 525 O 193 P 084, P 722, P 947
RIGG, C. RIJAL, S. RIJAL, S.R. RILEY, L. RINCON, M.G. RIOS VELASQUEZ, C.M. RIOS, L.A. RIOS, R. RITMEIJER, K. RIVAS, L. RIVAS, M. RIYAD, M. RIZK, Y.S. RIZZO, N.R. ROA, L. ROATT, B.M. ROBERT GANGNEUX, F. ROBERT, P. ROBERTS, S. ROBERTS, S.C. ROBLEDO, M.B. ROBLEDO, S.M. ROBLES, J.J.T. ROCHA DOS SANTOS, C.F. ROCHA NETO, G.B. ROCHA, A.A.A. ROCHA, A.G. ROCHA, A.M.S. ROCHA, F.A. ROCHA, I.C.M. ROCHA, K.F. ROCHA, L.C. ROCHA, M. ROCHA, M.A.W. ROCHA, M.C. ROCHA, M.N. ROCHA, M.O.C. ROCHA, O. ROCHA, R.L. ROCHA, R.R. ROCHA, V. ROCHAEL, N.C. RODRIGUES NETO, J.F. RODRIGUES SILVA, R. RODRIGUES, A. RODRIGUES, A.M. RODRIGUES, A.M.S.
P 895 O 002, O 140, O 143, O 078, O 285, O 015, O 344, O 357, O 067, O 269, P 053, P 382, P 383, P 384, P 386, P 672, P 679 O 111 O 166 P 443 P 923, P 924 P 328 O 258 O 023, O 226, O 262 O 281, P 373, P 461, P 464, P 467, P 475 P 542 O 253 P 202, P 521, P 543 P 605 P 919 P 067, P 071, P 072, P 129, P 143, P 197, P 199, P 239, P 434, P 533 O 303, O 190, P 122 P 207 O 130 O 132 O 365 O 280, O 283, O 286, O 361, O 365, P 327, P 328, P 469, P 589 O 364 P 918 P 933 P 933 P 493 P 854 P 567, P 731, P 795 P 256 P 900 P 787 P 260 P 472 P 296 P 577 P 637 P 632 P 209, P 211 P 310 O 017, P 279, P 280, P 281 P 496 P 372, P 487 O 304 P 255 O 276 O 331, O 341 P 209, P 211 P 428
RODRIGUES, A.P.R. RODRIGUES, B.S. RODRIGUES, C.M. RODRIGUES, D.B. RODRIGUES, E.H.G. RODRIGUES, F.M.D. RODRIGUES, G.M. RODRIGUES, I.A. RODRIGUES, J.C.F. RODRIGUES, J.S. RODRIGUES, K.M.P. RODRIGUES, L.M. RODRIGUES, L.O. RODRIGUES, M.A. RODRIGUES, M.M. RODRIGUES, M.S. RODRIGUES, O. RODRIGUES, P.O. RODRIGUES, S.A. RODRIGUES, V. RODRIGUES, V.T. RODRIGUES, W.B. RODRIGUEZ VEGA, C.A. RODRIGUEZ CORTES, A. RODRIGUEZ, C. RODRIGUEZ, J. RODRIGUEZ, N.E. RODRIGUEZ, N.M. RODRIGUEZ, O.L. RODRIQUEZ, O.L. ROGERS, M. ROGERS, M.B. ROGERS, M.E. ROHE, M. ROHOUŠOVA, I. ROHOUSOVA, I. ROHRLICH, P.S. ROJAS, A. ROJAS, E.M. ROJAS, L.J. ROLAO, N. ROMA, E.H. ROMAN ESCUTIA, M. ROMANHA, A.J. ROMANO, A. ROMAO, A.B.D. ROMAO, R.R.P.R. ROMERO, G.A.S.
P 622 P 206 O 157 P 243 P 623 P 297 P 293, P 296, P 608, P 609, P 614, P 617 P 212 P 729, P 730 P 445 O 310, P 103, P 352, P 399, P 414, P 432 P 755 O 167 P 141 P 876, P 880 O 182, P 185, P 508 P 633 P 898 O 331 P 202 P 612 P 297 O 330 P 715 P 243 P 646 P 075 P 578 P 573 P 013 P 624, P 914 P 127, P 203, P 254, P 257, P 578 P 302 P 298 O 332, O 042, P 791 O 298 O 193 P 019 O 245 O 244, P 717 P 029 O 118 P 836 P 727 O 147 P 453 O 121 P 113 P 515 O 297 P 263 O 156 O 359, O 296, P 921, P 929
ROMERO, I. RONET, C. ROOS, D. ROOS, D.S. ROSA CRUZ, C.F. ROSA CRUZ, M.F. ROSA, D.S. ROSA, F.A. ROSA, J.R. ROSA, M.D.S.S. ROSA, M.E. ROSALES, M. ROSANDISK, M.L. ROSAS, J. ROSELINO, A.M. ROSELINO, A.M.F. ROSENTHAL, E. ROSSI BERGMANN, B. ROSSI, C.N. ROSSI, M. ROSTAN, O. ROUAULT HARDOIN, E. ROUAULT, E. ROWTON, E. ROY, G. ROY, K. ROY, L. ROY, S. ROYO, M. ROZZA, D.B. RUBIANO PEREA, L.C. RUBIANO, L.C. RUBINO, V. RUFFOLO, B.B. RUGANI, J.M.N. RUGGIERO, G. RUI LUVIZOTTO, M.C. RUIZ SANTAQUITERIA, M. RUIZ, D. RUIZ, J.C. RUTHEL, G. SA, R.G. SA, V.M.H. SA, W.T. SAADI, Y. SABAJO, L. SABAJO, L.O. SABATE, D.
P 515 O 035, O 074, O 273, O 351, P 112, P 115 O 142 O 087 P 652, P 653, P 774 P 774 P 652, P 653, P 774 P 193 P 054, P 056, P 686 O 097 P 445 P 134, P 337 O 099, P 737, P 859 P 150 P 542 P 201, P 534 P 546, P 648 P 221 O 116, P 208, P 555, P 558, P 600 P 394 O 035, O 052, P 112 O 303, P 122 O 284 O 047 O 161 P 336 P 030 O 067 O 235, P 030, P 088, P 382, P 401, P 802 P 467 P 177, P 268 P 737 O 099, P 859 P 048 P 829 P 225 P 048 P 448, P 928 O 307 P 132 P 135 P 242, P 256 O 087 P 067 P 572, P 619 P 885, P 894 P 889, P 891 P 888 P 501 O 098 P 151 O 110
SABERI, S. SABINO, K.C. SABINO, K.C.C. SABIO, P.B. SABOIA VAHIA, L. SABROZA, P.C. SACKS, D. SACKS, D.L. SACRAMENTO, L. SADLOVA, J. SAENZ, E. SAFI, N. SAFI, N.U. SAGICA, F.D.E.S. SAHA, P. SAHAL, D. SAHASRABUDDHE, A.A. SAHEKI, M.N. SAID, R.F. SAID, R.F.D.C. SAKRU, N. SAKURAI, T. SALA, T.N. SALAH, A.B. SALAM, M.A. SALAUN, P. SALAZAR, A.M. SALAZAR, L.M. SALAZAR, V.A. SALCEDO, J.M.V. SALDAÑA, A. SALDANHA, A.C. SALDARRIAGA, O.A. SALES, A.D. SALES, K.G.S. SALES, R.H.F. SALGADO, A. SALGADO, K. SALGADO, V. SALIBA, J.W. SALIBA, S.W. SALIH, M.A. SALJOUGHIAN, N. SALMON, D. SALOMON, M.C. SALOMON, O. SALOMON, O.D.
P 301, P 913 P 307 P 408 O 007 O 325, P 243 P 124 P 743, P 744, P 756, P 954 O 206, O 207, O 299, O 297 O 075, O 121 O 210 O 249 O 251 P 835 O 379 P 690 P 949 P 802 P 466 P 042 O 104, P 130, P 284, P 602 P 090 P 164 P 788 P 821 P 272 P 026 O 129, P 465 O 236, O 236, O 362, O 363, O 125, O 289, P 039, P 125, P 153, P 305, P 306, P 916, P 922, P 942 O 266 P 387 P 446 O 164 P 475 P 747 P 146, P 895 P 132 P 447 P 622 O 128 P 633 O 375, P 797 P 087, P 423, P 785 P 187 P 358, P 374 P 374 O 343 O 025 P 101 P 104 P 825 O 045, O 095, O 097, P 121, P 778, P 792, P 806, P 814, P 822 P 773
SALOTRA, P. SALTO, E. SAMANT, M. SAMJHAWAN, A. SAMPAIO FILHO, A.P. SAMPAIO, E.P. SAMPAIO, R.N.R. SAMPAIO, S.M.P. SAMSON, M. SAN MARTIN, J.V. SANCHES, F. SANCHES, L.C. SANCHEZ TEJEDA, G. SANCHEZ GOMEZ, M. SANCHEZ MURCIA, P. SANCHEZ MURCIA, P.A. SANCHEZ, A.P.C. SANCHEZ, C. SANCHEZ, J.F. SANCHEZ, M.A. SANCHEZ, M.C.A. SANCHEZ, M.V. SANDERS, M. SANJOBA, C. SANTA BARBARA, M.C.A. SANTABARBARA, M.C.A. SANTAMARIA, C. SANTANA, A.L. SANTANA, A.M. SANTANA, A.P. SANTANA, G. SANTANNA, C.M.R. SANTAREM, A.A. SANTAREM, N. SANTIAGO, A.S. SANTIAGO, M.E.B. SANTIAGO, R.C. SANTINI, M.S. SANTINI, S. SANTORO, M.M. SANTOS GOMES, G. SANTOS GOMES, G.M. SANTOS MATEUS, D. SANTOS OLIVEIRA, J.R. SANTOS, A.M.A. SANTOS, B.A. SANTOS, B.N. SANTOS, C.
O 113, O 022, O 171, O 026, P 363, P 364, P 671 O 016 O 101 P 794 P 677 O 234 P 570 P 748 O 303, P 122 O 016 P 291 P 396, P 688 P 882 O 325 P 525, P 529 O 307 P 918 O 215, O 046 P 835 P 203 O 375, P 127, P 254, P 257, P 259, P 342, P 578 P 341 P 104 P 053 O 090, O 370, O 174, O 177, O 178 P 579 P 579 O 106 P 481 P 481 P 740 P 187 P 528 P 095 P 079 O 293 P 099 P 398 P 548 O 045, O 095, O 097, P 778, P 806 P 792, P 814 P 825 P 376 O 336, O 341, P 772 O 182, O 331, P 185, P 508 O 182, O 331, O 341, P 185, P 508 P 228 P 241 P 498 P 858, P 862 P 073, P 119 P 110
SANTOS, C.N. SANTOS, C.R.O. SANTOS, C.S. SANTOS, D.A. SANTOS, D.M. SANTOS, E.L. SANTOS, F.F.M. SANTOS, F.K.M. SANTOS, F.N. SANTOS, G.J. SANTOS, G.J.L. SANTOS, H.D. SANTOS, I.A. SANTOS, I.K.F.M. SANTOS, I.O. SANTOS, J.L.B. SANTOS, J.L.C. SANTOS, L.C.S. SANTOS, L.G. SANTOS, M.B.L. SANTOS, M.F. SANTOS, M.F.C. SANTOS, M.L.B. SANTOS, M.R.A. SANTOS, M.S. SANTOS, O.D. SANTOS, P.A. SANTOS, P.L. SANTOS, P.R.B. SANTOS, P.V.W.G. SANTOS, R.C. SANTOS, R.S. SANTOS, R.S.S.S. SANTOS, T.A. SANTOS, T.A.R. SANTOS, T.C. SANTOS, T.R. SANTOS, Y.C.V. SARAH, H. SARAIVA CORREA, D. SARAIVA, E.M. SARAIVA, L. SARAVIA, N. SARAVIA, N.G. SARDAR, A.H. SARDINHA, F. SARNOSKI JUNIOR, S. SASSI, A.
P 632 O 168, P 435 O 238 P 645 O 367 P 036 P 504 P 433, P 446 P 619 P 775 P 815 P 415, P 712 P 499, P 677 P 502, P 506 P 856 P 655 P 323 P 409 P 813, P 892 P 532 P 526 P 639 P 952 O 319, P 490 P 057, P 893 O 151, P 250, P 456, P 576 P 945 P 440, P 488, P 528, P 594 P 145 P 939 P 549 P 767 P 456 P 953 P 201, P 534 P 795 P 867 P 834 P 817 O 275 P 541 P 564 P 077, P 207 P 634 P 810 P 451 O 146 P 461, P 464 O 301, O 304, P 183, P 246, P 378, P 420 P 812 O 029, O 351, O 258 O 147, P 089 O 256 P 189 P 098 P 184
SATOSKAR, A. SATOSKAR, A.R. SATOW, M.M. SAVAGE, P. SAVAGE, P.B. SAWALHA, K. SAWALHA, S. SCALONE, A. SCHÄFER, C. SCHALLIG, H. SCHALLIG, H.D. SCHALLIG, H.D.F.H. SCHECHTMAN, D. SCHEIRER, J. SCHER, R. SCHESKE, L. Schettini, D. SCHLEICHER, U. SCHLISSELBERG, D. SCHNORR, D. SCHNUR, L. SCHNUR, L.F. SCHOENIAN, G. SCHONIAN, G. SCHÖNIAN, G. SCHOONE, G. SCHOONE, G.J. SCHOUNTZ, T. SCHRAMM, V.L. SCHRIEFER, A. SCHROOTEN, W. SCHUBACH, A. SCHUBACH, A.O. SCHWANKE, K. SCHWEDERSKY, R.P. SCHWEIGERT, A. SCHWEYNOCH, C. SCIO, E. SCODELLER, E.A. SCOFIELD, A. SCOFIELD, K.M. SCOPEL, K.K.G. SCOPELLITI, D. SCORZA, J.V. SCOTT ALGARA, D. SCOTT, P. SCOULICA, E. SEBLOVA, V.H.
O 026 O 308, O 339, O 368, O 369, O 171, P 094 P 577 P 578 P 259, P 342 P 455 P 684 O 232, O 292, P 604, P 947 O 149 O 098, O 258, O 293, P 332, P 458, P 726, P 794 P 151 P 777 O 282 O 289 P 481, P 504 O 258 P 683 O 105, O 064, O 132 O 072 O 278 O 290 P 671 O 137, O 139, O 140, O 141, O 290, P 711 P 364 O 102, O 105, O 135, O 255, P 026, P 092, P 671, P 694, P 724, P 877 O 258, P 332, P 361 P 586 P 647 P 470 O 351, O 166, O 169, P 087, P 337, P 423, P 785 O 267 P 237 O 237, P 037, P 824 O 274, O 122, P 090, P 150 P 422 P 208 P 032, P 049, P 196 O 139, P 711 P 643 P 104 P 864, P 867 P 788 P 568 P 064 P 727 P 298 O 240, O 241, O 272, O 087, P 023, P 204 O 312, O 336 O 248
SEBY, F. SEDEﾃ前, M. SEIFERT, K. SEKAR, A. SEKI, M. SELVAPANDIYAN, A. SEMIAO SANTOS, S.J. SENA FILHO, J.G. SENA, C.A.P. SENA, J. SENA, J.M. SENE, I.S. SENEVIRATNE, K. SERAFIN, L.C. SERAKIDES, R. SERAKIDES, R.S. SERGENT, O. SERRA E MEIRA, P.C.L. SERRA, Q.D. SERRA, T. SERRANO, M.L. SERUFO, A.V. SERVIS, C. SESANA, A.M. SEVA, A. SEVA, A.P. SEVERO, M.M.S. SEVILLA, S. SEYED, N. SGHAIER, R.M. SHAHBAZI, F. SHAIK, J. SHAMS, A. SHAMSIMEYMANDI, M. SHAMSIMEYMANDI, S. SHANKAR, R. SHAPIRO, J.T. SHAREGHI, N. SHARIFI, I. SHARMA, B. SHARMA, M. SHARMA, S. SHARP, A. SHARPE, A.H. SHAW, C. SHAW, C.D. SHAW, J.J. SHAW, M.M.L.
O 080 P 084, P 722 O 079 O 107, O 348 O 370 O 026 P 777 P 842 P 481 P 948 O 114 P 831, P 933 P 052 O 198, P 275, P 290 P 670 P 456 O 276 P 516 O 190 P 812 P 823 P 947 P 494 P 165 P 145, P 191, P 253 O 035 P 213, P 562 O 046 P 937, P 938 P 870 P 769 O 118 P 058, P 059 O 195, O 127 O 074 P 621 P 701 P 701 O 015 P 801 O 318 O 118, P 591 O 356 O 004, P 107, P 248 O 113 P 450 P 094 P 356 P 450 P 053 P 949 O 134, O 242, O 323, O 066, P 658, P 746, P 789 O 323
SHAWKI, A. SHCUBACH, A.O. SHELTON, A. SHIFERAW, W. SHIMABUKURO, P.H.F. SHIO, M.T. SHIPMAN, C.S. SHIRZADI, M.R. SHKURTI, K. SHORTE, S. SHUKLA, S. SILBER, A.M. SILPAPOJAKUL, K. SILVA BARCELLOS, N.M. SILVA JUNIOR, C.F. SILVA SANTOS, L.A. SILVA ARAUJO, M.S. SILVA CARDOSO, L. SILVA COSTA, D.C. SILVA LOPEZ, A. SILVA PEREIRA, R.A. SILVA SANTOS, G. SILVA, A.A. SILVA, A.C. SILVA, A.F. SILVA, A.G. SILVA, A.L.A. SILVA, A.M. SILVA, A.P. SILVA, A.Q. SILVA, A.R. SILVA, A.R.S. SILVA, C.I.M. SILVA, C.J. SILVA, C.L. SILVA, C.P.C. SILVA, C.V. SILVA, D. SILVA, D.A. SILVA, D.C.M. SILVA, D.N. SILVA, E.B. SILVA, E.D. SILVA, E.L. SILVA, E.S. SILVA, E.V.L.P. SILVA, F. SILVA, F.F.
O 070 P 164 O 308 O 216 P 760, P 762, P 800 O 329 O 100 O 195, O 127 P 295, P 611 O 284 O 063 P 473 P 662 O 081 P 080, P 718, P 747, P 847 P 902 O 276 P 166 P 090, P 164 P 338 P 099 O 291 P 898 P 411, P 541, P 564, P 566 P 090 P 569 P 139 P 192, P 626 P 155 P 664 O 169 P 212 P 852 P 547 P 296, P 609, P 614 P 898 P 761 P 176 P 786 P 440 P 626 P 846 P 564 P 149 P 498 P 781 P 683, P 798 P 209, P 211 P 176 P 346
SILVA, F.J. SILVA, F.L.D. SILVA, F.O. SILVA, F.O.L. SILVA, F.O.L.E. SILVA, F.S. SILVA, G. SILVA, G.A.F. SILVA, G.M.C. SILVA, H.C.A. SILVA, I.D.R. SILVA, I.S. SILVA, J. SILVA, J.A. SILVA, J.C. SILVA, J.C.F. SILVA, J.E.S. SILVA, J.F. SILVA, J.H.T. SILVA, J.M. SILVA, J.P. SILVA, J.R.A. SILVA, J.S. SILVA, J.S.F. SILVA, K.B. SILVA, K.G. SILVA, K.L.O. SILVA, K.M. SILVA, K.M.S. SILVA, K.R. SILVA, L. SILVA, L.A. SILVA, L.A.C. SILVA, L.C. SILVA, L.C.S. SILVA, L.F. SILVA, L.O.R. SILVA, L.S. SILVA, L.T. SILVA, M.A. SILVA, M.A.L. SILVA, M.B.A. SILVA, M.D.C.F. SILVA, M.E. SILVA, M.E.B. SILVA, M.G. SILVA, M.L.F. SILVA, M.L.R.
O 128, P 940, P 941 P 949 P 323 O 069, P 052, P 275 P 758 P 788 P 550 P 825 P 569 O 037 P 678 P 424 P 799 P 785 P 945 P 290 P 795 P 758 P 764 P 049 P 032, P 196 P 687 P 650 P 915 P 060 P 720 P 445 O 210, O 328 O 181, P 077, P 524 P 530 P 828 P 940 P 650, P 941 P 396 O 185 P 468, P 770 P 764 P 468, P 770 P 618, P 721 P 311 P 303 P 210 P 907 P 907 P 867 P 865, P 868, P 872, P 874, P 875, P 878, P 879 P 815 P 192 P 091 P 335 P 759 P 788 P 149, P 493 P 368, P 369 P 581 P 581, P 860 P 885 P 888, P 889, P 891 P 886 P 637 P 297, P 623 P 939
SILVA, M.M.M. SILVA, M.R.B. SILVA, M.S. SILVA, M.V. SILVA, O. SILVA, P.H.S.E. SILVA, R. SILVA, R.A. SILVA, R.M. SILVA, R.P. SILVA, R.P.E. SILVA, R.T. SILVA, S. SILVA, S.B.A. SILVA, S.O. SILVA, T.C. SILVA, T.L.V.E. SILVA, T.M.S. SILVA, T.O. SILVA, T.R.R. SILVA, V.C. SILVA, V.L. SILVA, V.M. SILVA, V.P.M. SILVEIRA LEMOS, D. SILVEIRA LEMOS, D. SILVEIRA, A.M. SILVEIRA, F.T. SILVEIRA, H.M.C. SILVEIRA, T.G.V. SILVEIRA, T.L. SILVEIRA, V.M. SILVESTRE, R. SIMA, M. SIMBAIA, M.G.S. SIMOES DIAS, B.R. SINGH, A. SINGH, A.K. SINGH, B. SINGH, D. SINGH, D.P. SINGH, K.P. SINGH, N. SINGH, N.S. SINGH, O.P. SINGH, R.
P 915 P 917 P 613 O 170, P 583 P 297 P 803, P 807 P 670 P 810 O 070 P 678 P 522 P 489, P 793 P 335 P 585 P 512 P 423 P 309, P 317 P 767 O 294, P 200, P 244, P 673, P 674, P 675, P 734, P 783, P 784, P 819 O 139, P 711 P 625 P 222 P 209 P 639 P 594, P 640 P 593 P 489 P 762 P 195, P 561 P 415, P 712 P 921 P 285 P 917 P 138 P 197 P 546 P 864, P 867 P 142, P 146, P 167, P 169, P 193, P 212, P 291, P 422, P 424, P 855, P 902 P 583 P 554 P 268 P 149 O 330, O 372, O 293, P 079, P 726 O 049 P 029 P 918 O 036, P 074, P 326 O 206 O 315, O 058, O 063 O 315 O 015, P 011, P 478, P 749 P 154 O 061 P 667 P 603 O 189 O 206, O 207, O 299, O 268, P 154, P 749 P 667 O 207, O 015, O 265, O 268, P 011, P 086 P 749 P 364 O 078, O 111
SINGH, R.P. SINGH, S. SINGH, S.P. SINGH, T. SINHA, P.K. SIPAHI, O.R. SIQUEIRA FORMIGA, C.C. SIQUEIRA, L.L.D.N. SIQUEIRA, L.L.N. SIREKBASAN, S. SIRIMANNA, G. SIRIWARDANA, Y. SIRIYASATIEN, P. SISSAY, Y. SIVINI, M.A. SJURSEN, H. SKALTSOUNIS, A. SLAPNICKOVA, M. SLEIGH, A.C. SLIM, E. SMIRLIS, D. SMITH, B.A. SMITH, D.F. SMITH, P.L. SNOUSSI, M.A. SOARES, B.R. SOARES, C.R. SOARES, D.C. SOARES, D.F.M. SOARES, F.V. SOARES, G. SOARES, H.S. SOARES, K.A. SOARES, M.B.P. SOARES, M.I.S. SOARES, M.M.C.N. SOARES, M.R.A. SOARES, R. SOARES, R.M. SOARES, R.O.A. SOARES, R.P. SOARES, R.P.P. SOARES, S.C. SOARES, S.R.C. SOBRAL, A.A. SOBRINHO, A.C.R. SOBRINHO, L.S.V. SOBRINO, A.C.R.
O 012, O 015, O 316, O 357, O 055, O 163, P 749 O 093 O 055 P 449 P 186 O 057 P 117, P 287 P 322 P 915 P 917 P 360 P 027, P 669 O 217, P 027, P 028, P 668, P 669 P 662 O 133 P 664 P 230 P 406, P 487 O 086 P 741 P 908 P 318, P 372, P 406, P 487 O 298 O 077, O 162 O 152, O 251, O 298 O 363 P 942 P 844 P 368, P 369, P 493 P 246 P 378, P 420 P 935, P 943, P 949, P 952 P 945 P 549 O 291 P 685, P 809 P 105 P 487 P 372 P 141 P 857 O 198, P 060 P 775 P 629 P 809, P 937, P 938 P 065 P 412 O 275 O 051 P 431 P 241, P 371 P 756 P 885, P 889, P 894 P 886 P 888 P 891 P 394 P 890
SOCCOL, V.T. SOHRABI, Y. SOLANO GALLEGO, L. SOLANO GALLEGO, L. SOLCA, M.S. SOLEIMANI, H. SOLER, R.C. SOMENZARI, M.A. SOONG, L. SOORI, T. SORDO, L. SOREMEKUN, S. SØRNES, S. SOSA BIBIANO, E.I. SOSA, L. SOSA, N. SOSSAU, D. SOTERIADOU, K. SOTERIADOU, K.S. SOTO, A.M. SOTO, M. SOTO, P.A. SOUB, A.C.S. SOUSA NASCIMENTO LEITE, K.S. SOUSA BATISTA, A.J. SOUSA, A. SOUSA, A.M. SOUSA, A.Q. SOUSA, B. SOUSA, F.D.G. SOUSA, G.R. SOUSA, L.C. SOUSA, L.O. SOUSA, M.C.S. SOUSA, O. SOUSA, P.B. SOUSA, R. SOUSA, S. SOUSA, S.A. SOUTHWORTH, D. SOUTO PADRON, T. SOUTTER, F. SOUZA NOGUEIRA, F. SOUZA FAGUNDES, E.M. SOUZA, A.C.N. SOUZA, A.P.A. SOUZA, B.L.S.C.
P 236 O 096, P 826 O 049, O 086, O 260 O 213 P 646 P 391 P 526 O 044 O 195, O 127 P 577 P 398 O 053, P 016 P 085 P 934 O 042 P 230 P 647 P 707 O 362 O 132 P 406, P 487 O 135 O 380 P 132, P 166, P 223 P 018, P 093, P 226, P 375 P 624 O 083 P 158 P 558 O 188 P 731, P 735 P 813, P 892 P 887 P 815 P 660 P 880 P 867 P 530 P 009 P 307 O 166, P 087 O 293, P 726 P 594 O 059 P 246 O 213 P 474 P 643 P 851 P 132, P 844 O 156
SOUZA, B.V.C. SOUZA, C. SOUZA, C.M. SOUZA, C.S.F. SOUZA, D.A. SOUZA, D.M. SOUZA, F.A. SOUZA, G.O. SOUZA, H. SOUZA, H.C. SOUZA, I.S. SOUZA, J.B. SOUZA, J.V. SOUZA, L.D. SOUZA, L.E. SOUZA, L.M. SOUZA, M.A. SOUZA, M.F.V. SOUZA, P.A.D.N. SOUZA, P.A.N. SOUZA, P.R. SOUZA, R.S. SOUZA, V.A. SOUZA, V.B. SOYZA, P. SPÄTH, G. SPÄTH, G.F. SPONTON, A. SRIVASTAVA, P. SRIVASTAVA, R. STABELI, R. STAGER, S. STAHL, H. STAHL, K. STAMPER, L.W. STAPLETON, J.T. STARK, O. STARKE BUZETTI, W.A. STASHENKO, E. STAUCH, A. STEINDEL, M. STEINKAMP, H. STELL, F. STEVENS, Y. STEVERDING, D. STIENSTRA, S. STILLE, K. STOBBE, N.S.
P 507 P 310 P 793 P 885, P 888, P 889, P 891, P 894 P 886, P 890 P 897 P 731 P 507 P 832 P 297, P 623 O 257, P 491 P 626 P 549 P 489 P 842 O 069 P 509 P 939 O 091, P 067, P 072 P 590 O 082 P 189, P 307 P 765 P 411 P 010, P 205, P 220, P 265, P 566 P 440 O 181 P 524 P 080, P 718 P 550 P 713 P 183 P 005 P 668 O 205, O 284, O 337 P 232 P 421 P 186 P 602 P 108 O 371 O 064 O 064 O 121 P 278 O 137, O 140, O 141, P 364 P 539 P 497 P 382 P 357, P 515, P 666, P 866 O 308, O 339 P 680 O 194, O 289 P 482 O 338 O 262 P 854
STOBER, C.B. STOCKDALE, L. STOLF, B.S. STRASSER, R. STRAUS, A.H. STREATFIELD, P.K. STRUB WOURGAFT, N. STRUCHINER, C.J. STUHLMANN, F. SUAREZ, J. SUAREZ, M. SUAREZ, M.S. SUAU, H.A. SUCRE, A.P. SUCUPIRA, M.V. SUDARSHAN, M. SULEMAN, M. SUMAN, S.S. SUMI, D.V. SUMOVA, P.
P 011 O 360 P 004, P 031, P 033, P 044 O 131 O 335 P 796 P 627 P 708 O 188 O 380 P 038 P 036 O 029 O 144, P 017 P 522 P 454, P 478, P 752 O 190 P 599 O 168 P 029 O 244
SUNDAR, S. SUNYOTO, T. SURANGO, B.P.S. SUZANNE, G. SVOBODOVA, M. SZELAG, E.A. TABBABI, A. TACCHINI COTTIER, F. TAFURI, W. TAFURI, W.L. TAHERI, 1. TAHERI, T. TAIPALE, A. TAKAGI, H. TAKAHASHI, H.K. TAKELE, Y. TAKIYA, C.M. TALADRIZ, A. TALHARI, A. TALHARI, A.C. TALHARI, S. TALIGNANI, L. TALLY, J.D. TALVANI, A.
O 113 O 002, O 140, O 143, O 206, O 207, O 235, O 299, O 012, O 015, O 089, O 315, O 316, O 344, O 352, O 357, O 055, O 058, O 063, O 163, O 175, O 265, O 268, P 011, P 086, P 088, P 154, P 186, P 363, P 364, P 383, P 401, P 454, P 478, P 483, P 495, P 582, P 597, P 749, P 752 O 269 P 918 P 544 O 049, O 086, O 298 O 260 O 097 P 790 O 133, P 410 O 250 P 309, P 310, P 311, P 317, P 629 P 311, P 629 P 606, P 839 P 066, P 206, P 316 P 340 O 118, O 025, P 109 O 107 O 173 O 335 O 133 P 432 P 461 P 189 P 307 P 307 P 189 P 140 P 607 O 180
TAMAS, M.J. TANDON, R. TANDON,, R. TANIGUCHI, H.H. TANIWAKI, N.N. TAPADAR, J. TAPIA, F.J. TARALLO, V.D. TARTAROTTI, A.L. TASLIMI, Y. TATE, E.W. TAVARES, A.M. TAVARES, C.A. TAVARES, C.A.P. TAVARES, J. TAVARES, L. TAVARES, L.C. TAVARES, L.H.M. TAVARES, M.F.M. TAVARES, N. TAVARES, N.M. TAVARES, V.B. TAYLOR, M.J. TAYPE, C.A. TEESCH, L.M. TEIXEIRA NETO, R.G. TEIXEIRA CARVALHO, A. TEIXEIRA NETO, R.G. TEIXEIRA PINTO, L.A.L. TEIXEIRA, C. TEIXEIRA, D.E. TEIXEIRA, E.M. TEIXEIRA, F. TEIXEIRA, M.C. TEIXEIRA, M.J. TEIXEIRA, N.C. TEIXEIRA, P.C. TEIXEIRA, S. TEIXEIRA, S.C. TEIXEIRA, S.M.R. TEIXEIRA, S.R. TEIXEIRA, T.L. TEIXEIRA, V.N. TEKWANI, B.L. TELES RABELLO, A.L. TELES JUNIOR, E.P. TELES, C.B.G. TELES, M.
O 004 P 041, P 042, P 381 O 089 P 489, P 601, P 631, P 658, P 658, P 767 P 507 P 389, P 418 O 063 P 046, P 047 P 025, P 127, P 203, P 254 O 010 P 854 O 239, O 025 O 152 P 830 P 375 P 226 P 520 P 018, P 123, P 362, P 376 P 519 O 319 O 220, P 772, P 786 P 371 P 943 P 373 O 238 P 136 O 275 P 036 P 581 O 158 P 245 O 302 P 798 P 069, P 071 O 276, P 643 P 683 P 168 O 160, O 161, O 320, P 038 P 103 P 516 O 081, P 587, P 588 O 182, P 185 O 059 P 854 P 034, P 171, P 174 P 020 P 033 P 035 P 161, P 229, P 511 P 165 O 350 P 161, P 176, P 229, P 247, P 511 P 098 P 324 O 081 P 321 P 344 P 345 P 250
TELES, P.P.A. TELES, Y.C.F. TELLEVIK, M.G. TELLEZ, J. TELLO, A. TEMI SHIO, M. TEMPONE, A.G. TEMPONE, A.J. TENORIO, A.P.M. TEODORO, A.K.M. TEODORO, U. TERCEIRO, B.R.F. TERRAO, M. TERUYA, E.O. TERZIC, D. TESTASICA, M.S. TESTASICCA, M.C.S. TETIK, A.V. THAMMAPALO, S. THANGARAJ, K. THELLIER, M. THIES, S.F. THOMAZ SOCCOL, V.T. THOMAZ SOCCOL, V. THOMPSON, A. THOMSON, A. TIBURCIO, A.M.L. TIBURCIO, M.G.S. TILLEY, P. TIMMIS, J. TINOCO, N.A.B. TIVELLI, P. TIWARI, A. TIWARY, P. TODT, M. TOLEDO, C.R.S. TOLEDO, M.S. TOLEDO, R.S. TOLEZANO, J.E. TOLOUEI, S. TOMAS, A.M. TOMOKANE, T.Y. TONELLI, G. TONIN, L. TOPNO, R.K. TORALES, M. TORO LONDOﾃ前, M.
P 630, P 931, P 932 P 440 P 230 P 515 P 766 O 338 P 482 P 370, P 409, P 641 P 351, P 461, P 505 P 367, P 389, P 390, P 464, P 612 O 246 P 488, P 528, P 594 P 911, P 912 P 236, P 738, P 849, P 858, P 862 P 164 O 348 P 131 O 013, P 426 P 145 P 253 O 321 P 662 P 137 P 457 P 754, P 755 P 098, P 738 P 441, P 444, P 742, P 761, P 776 P 596, P 636, P 642 P 450 P 145 P 214 P 462, P 781 P 803, P 807 P 476 O 340 P 210 P 054, P 056 P 114 O 063 P 154, P 495, P 582, P 597 O 209 P 756 O 335 P 829 P 631, P 857 P 395, P 489, P 601, P 658, P 767 P 299, P 300 O 070, O 059, P 262 P 471 O 182, O 188, P 185 P 394 P 736 P 146, P 291, P 793 P 798 P 075 O 056, O 061, P 599 O 270 P 529
TORO, M.A. TORRACA, T.S.S. TORRELLAS, A.M. TORRENT, M. TORRES SANTOS, E.C. TORRES, A.R.A. TORRES, B. TORRES, C. TORRES, F.D. TORRES, M.S. TORRES, O.D.S. TORRES, T.M. TORRES, U.P.S. TORRICO, M.C. TOUMI, A. TOVAR, B. TOZ, S.Ö. TRAJANO, L.A.M. TRANNIN, M.A. TRAUB CSEKÖ, Y.M. TRAVI, B.L. TRENTIN, B. TRINCONI, C.M. TRIPATHI, C.D.P. TRIVEDI, V. TRONCO, C.T. TROVATTI, E. TRUJILLO, J.J. TRUPPEL, J.H. TSEGAYE, I. TSIGANKOV, P. TSUGE, C. TSUNASHIMA, R. TULADHAR, R. TUMA, K.S. TUMBAICO, J.W.T. TUNA, G.E. TUNON, G.I. TUON, F.F. TURCO, S.J. TURGAY, N. TURNER, J. TUTTERROW, Y. UBEDA, J. UCHOA, F.C. UEDA NAKAMURA, T. ULIANA, S. ULIANA, S.R.B.
O 307 P 090, P 164 O 259 P 475 P 120, P 408, P 421 P 266, P 267 P 222 O 196 P 659, P 850 P 479 P 657 P 285 P 830 P 438 O 125, P 039, P 305, P 306, P 916 P 254 O 135 P 585 O 136, P 073, P 119 O 246 P 447 O 345, P 059 P 419 O 089, O 120, P 043, P 388 O 104, P 130 P 473, P 477 P 433 P 904 P 738 P 750 O 346 O 179 P 313 P 094 P 952 P 918 O 321 P 740 O 168, P 435 O 075 P 117, P 287 O 158 O 092, O 027 P 336 P 489 P 625 O 282 P 405 P 404, P 418, P 419, P 473, P 477
ULLMAN, B. UMAÑA PEREZ, A. UNCOS, A. UNRAU, P. UNVER, A. UPEGUI, Y.A. URANW, S. URBINA, J.A. URIBE, S.I. UTGES, M.E. UZONNA, J.E. VALADARES, D.G. VALDERRAMA, A. VALDERRAMA, C.H. VALDES, A.C. VALDIVIA, H.O. VALDIVIA, H.V. VALDIVIESO, E. VALE, M.M. VALENCA, W.O. VALENCIA, A. VALENCIA, B. VALENCIA, B.M. VALENZUELA, J. VALENZUELA, J.G. VALERIO BOLAS, A. VALETE ROSALINO, C.M. VALLEJO, A. VALVERDE, J.G. VALVERDE, J.L.G. VAN DER AUWERA, G. VAN DER GEEST, S. VAN DER VEER, C. VAN GRIENSVEN, J. VAN LUENEN, H.G. VAN WYNSBEGHE, N. VANAERSCHOT, M. VANEGAS, O. VANERSCHOT, M. VANWALLEGHEM, G. VARGAS, D.A. VARGAS, E. VARGAS, M.L.F. VARIKUTI, S. VARJAO, J.R. VASCONCELOS, A.C. VASCONCELOS, M.F.
O 132 O 325 O 038, P 485 O 054 P 117 O 280, P 327, P 469 O 078, O 111, O 357, O 067 P 352 O 252, P 899 P 951 P 773 O 373 P 362 P 895 O 008 P 743, P 744 O 351 P 680 P 835 P 429, P 494 P 705 P 492 O 196 P 144, P 438 P 397, P 535 O 159, O 025, O 026, P 038, P 844 O 161, O 318, P 958 O 160, O 320, P 029 O 331, P 772 O 237, P 037 P 090, P 164 P 243 P 255, P 273, P 286 P 918 O 254, O 255, O 290, P 061, P 062, P 438 O 098 P 151 O 228, O 262, O 263, O 269 O 107 P 647 O 002, O 143, O 111, O 344, P 382, P 383, P 384, P 385, P 386, P 672, P 679 P 017 O 113 P 101 O 147, P 089, P 100 P 836 P 532 O 339 P 820 P 051 P 408 P 421
VASCONCELOS, R.O. VASCONCELOS, T. VASQUEZ, D. VASQUEZ, G.M. VASSALLO, M. VAUCHY, C. VAZQUEZ, M.A. VELAND, N. VELASQUEZ, E. VELASQUEZ, L.G. VELAZCO, E. VELAZQUEZ, S. VELDHUIS, S. VELET, M. VELEZ, A. VELEZ, I. VELEZ, I.D. VELO, E. VELOSO, V.M. VELURU, N.K. VENDRAME, C.M.V. VENDRAME, C.V. VERA, H. VERAS, L.M.C. VERAS, P.S. VERAS, P.S.T. VERCOSA, B.L.A. VERCOZA, B.R.F. VERDIANO, V.A. VERDONCK, K. VERGARA, D. VERMA, N. VERMELHO, A.B. VERMONT, M. VERONESI, F. VERSTEEG, I. VERWEIJ, J. VIANA, J.B.M. VIANA, K.F. VIANA, S.M. VIANNA, E.N. VICTOIR, K. VICTORIA, C. VIDAL, M.L. VIDAL, R.O. VIDOTTO, O. VIEGAS, R.P.
P 177, P 190 P 268 P 794 O 164 P 680 P 221 O 118 P 766 O 254 P 040, P 380 P 031 P 259 O 307, P 529 O 023 P 272 O 011, O 196, O 380, P 904 O 202 O 364 O 011, O 280, O 283, O 361, O 365, O 196, O 252, O 164, O 380, P 327, P 328, P 469, P 589, P 899, P 904, P 910 O 254 P 843 P 533, P 816 O 104 P 590 P 294 P 680 P 561 O 305 P 074, P 326, P 522, P 524, P 526 O 036, O 181, O 044, P 106, P 391 P 051 P 399, P 414 P 813, P 815 O 267, O 269, P 144 O 011 O 061 O 108, O 057, O 261 P 445 P 460 P 604 O 060 O 255 P 293, P 296, P 608, P 609, P 614, P 617 P 197, P 199, P 292 P 034, P 171, P 174 P 816, P 954 P 663 P 863 P 293 O 030 P 829 P 788
VIEIRA BRANQUINHO, H.C. VIEIRA FILHO, J.N. VIEIRA GONCALVES, R. VIEIRA, A.T.P. VIEIRA, E. VIEIRA, E.L.M. VIEIRA, P.M.A. VIEIRA, P.S. VIEIRA, R.F.C. VIEIRA, S. VIEIRA, T.S.S. VILA NOVA, N.S. VILACA, R. VILAS, F. VILELA, M.L. VILLANUEVA LIZAMA, L.E. VILLELA REZENDE, G. VILLELA, E.V. VIOL, M.A. VISHWAKARMA, P. VITAL, W.C. VITALE, F. VITALE, M. VIVANCO, B.C. VIVARINE, A.C. VIVARINI, A.C. VIVERO, R. VIVERO, R.J. VIVEROS, R.J. VLKOVA, M. VOGAS, G.P. VOJTISKOVA, J. VOLF, P. VOLFOVA, V. VOLKOVA, V. VON STEBUT, E. VONATSOU, P. VOTYPKA, J. VOTÝPKA, J. VOUGOGIANNOPOULOU, K. VOULDOUKIS, I. WACKWELLA, H. WAGHABI, M.C. WAGNER, G.K. WALLACE, J. WARBURG, A. WARD, R.J.
P 321 P 319 P 551 O 233 P 883 P 250 O 050 O 091, P 067, P 072, P 714 P 557 P 395, P 852 P 786 O 301, O 304 P 622 P 786 P 766, P 769 P 933 P 647 O 276 P 217 P 402 P 596, P 642 P 522, P 714, P 900, P 903 O 187, P 064, P 400, P 693, P 837 P 050 P 156, P 172, P 175 P 222 P 183 O 037 O 196, O 380 O 252 P 951 P 910 P 029 P 393 O 294 O 086 O 049 O 216, O 244, O 245, O 248, O 249, O 251, O 298, O 318, P 029, P 717 O 248, P 029 O 049, O 086, O 260 O 333 P 901 O 216, O 248, O 249 P 717 P 406 O 214, P 068 P 670 P 065 P 482 P 450 O 216, O 248, O 249, P 029, P 751 P 557
WARN, P. WARNASOORIYA, W.A. WARREN, W. WASUNNA, M. WATOWICH, S. WEI, L. WEINA, P.J. WEINKOPFF, T. WEINSTOCK, G. WEISE, M.H. WELDEGEBREAL, T. WENCESLAU, A.A. WENDLANDT, E.B. WENG, J.L.H. WENTKER, P. WERKHAUSER, R.P. WERNECK, G.L. WESTROP, G. WEYENBERGH, J. WEYENBERGH, J.V. WHEELER, L. WICKREMASINGHE, R. WIESE, M. WILKINSON, A.J. WILLIAMS, G.P. WILLIAMS, R. WILLIAMS, R.A. WILLIAMS, R.M. WILSON, M. WILSON, M.E. WILSON, R. WINSTANLEY, H. WINTER, L.M.F. WIRTH, T. WOLDEYES, M. WONDIMU, H. WONG RAMIREZ, C. WRIGHT, M.H. WU, M. WURTELE, M. WUTTKE, J. WYLLIE, S. WYREPKOWSKI, C.C. XANDER, P. XAVIER, C.C. XAVIER, E.M.
P 450 O 217 O 142 O 356, P 627 P 531 O 171 P 607 P 518 O 326, O 250 O 142 P 450 O 133 P 655 O 073 O 192 O 064 P 293, P 296, P 608, P 609, P 614, P 617 P 468, P 682, P 689, P 700, P 703, P 708, P 720, P 723, P 770, P 936, P 948 P 356 P 618 O 238, O 291, P 022 P 450 P 670 P 670 O 115, P 356 O 001, O 004 O 152 O 152 P 356 O 033 P 450 P 264 P 278 O 352 O 073, O 302, O 087, P 013, P 285 O 166, P 087, P 289, P 423, P 785 O 015 O 142 O 100 O 048, P 789 O 137, O 140, O 141, P 711 P 410 O 216 P 595 P 882 O 152 P 055 O 030 P 671 O 153 P 549 P 156, P 172, P 175 O 103 P 850
XAVIER, I.M. XAVIER, V. XIMENES, M.F.F.M. XU, B. YACOUB, A. YADAV, A. YAGHOOBI ERSHADI, M.R. YAMAGISHI, J. YAMASHIRO KANASHIRO, E. YAMASHIRO KANASHIRO, E.H. YAMAZHAN, T. YAN, S. YAO, C. YARDLEY, V. YASHIGUCHI, Y. YATES, P. YAZIDI, R. YEO, M. YEPEZ, J.Y. YLI KAUHALUOMA, J. YOKOYAMA YASUNAKA, J.K. YOKOYAMA YASUNAKA, J.K.U. YOUNIS, B.M. YUNA, A.G.S. Z, H. ZAATOUR, A. ZAATOUR, W. ZACARIAS, D.A. ZACKAY, A. ZAHEDIFARD, F. ZAIDI, A. ZAMBONI, D. ZAMBRANO, C.D.P. ZAMBRANO, P. ZAMITH MIRANDA, D. ZAMPIERI, R.A. ZANGGER, H. ZANNA, R.L.D. ZAULI, D.A.G. ZEN RUFFINEN, C. ZERPA, O. ZGHIDI ABOUZID, O. ZHANG, W. ZHANG, W.W. ZHANG, Y. ZHIOUA, E. ZIJLSTRA, E.E. ZILBERSTEIN, D.
P 788 P 175 P 917 P 285, P 915 P 055, P 697 P 501 P 063 O 195, O 127, O 318 O 090 P 472 P 637 P 117, P 287 P 055, P 697 O 302 P 437, P 596, P 642 P 485 O 348 P 922 P 308 P 836 P 338 P 477 P 419 P 692 P 918 O 035 O 289 P 942 O 198, O 322, O 069, P 052, P 060, P 275, P 290 P 078 O 239, O 025 P 603 O 210 P 702, P 706, P 710 P 709 P 170 O 048, O 129, P 789 O 035, O 074, O 351, P 112, P 116 P 911 O 276, P 069 O 203 O 375, P 127, P 203, P 298, P 302, P 910 O 101 O 155 O 348, O 349 P 055 P 039, P 654 O 019, O 024, O 225 O 072, O 147, O 204, O 346, O 348, O 131
ZINI, R.M. ZORRILLA, V.O. ZULETA, M. ZULUAGA, J.J. ZUﾃ選GA, C. ZUQUE, F.R.S. ZUQUE, F.T.S. ZUQUE, M.A.S. ZWETSCH, A.
P 857 P 680 O 196 O 365 O 380 P 745, P 799 P 745, P 799 P 745, P 799, P 873 P 870, P 933
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