CLINICS February 2012

I S S N - 1807-5932 printed version I S S N - 1980-5322 online version

Volume 67 Number 2 - February/2012

Scientific Journal of Hospital das ClĂ­nicas Faculdade de Medicina da Universidade de SĂŁo Paulo

CLINICS Editor Mauricio Rocha-e-Silva Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Area Editors Ana Maria de Ulhoa Escobar Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Anuar Ibrahim Mitre Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ashok Agarwal The Cleveland Clinic Foundation Cleveland, Ohio, USA Berenice Bilharinho Mendonc¸a Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Bruno Zilberstein Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Carlos Serrano Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Carmen Silvia Valente Barbas Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Claudia Regina Furquim de Andrade Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Edmund Chada Baracat Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Eliete Bouskela Universidade do Estado do Rio de Janeiro Rio de Janeiro, RJ, Brazil

Gustavo Franco Carvalhal Faculdade de Medicina da Pontifı´cia Universidade Cato´lica do Rio Grande do Sul Porto Alegre, Rio Grande do Sul, Brazil Heitor Franco de Andrade Jr. Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ivete Bedin Prado Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Joaquim Prado Moraes-Filho Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ludhmila Abrahao Hajjar Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Luı´z Eugeˆnio Garcez-Leme Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Lydia Masako Ferreira Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Maria Cecı´lia Solimene Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Nelson Wolosker Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Newton Kara-Junior Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Emilia Inoue Sato Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Olavo Pires de Camargo Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Fulvio Alexandre Scorza Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Geraldo Busatto Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Paulo Hoff Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Raul Coimbra University of California, San Diego La Jolla, CA, USA Renato Delascio Lopes Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Rubens Belfort Jr. Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ruth Guinsburg Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ruy Jorge Cruz Junior University of Pittsburgh Pittsburgh, PA, USA Sergio Paulo Bydlowski Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Silvia Vanessa Lourenc¸o Faculdade de Odontologia da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Simone Appenzeller Universidade Estadual de Campinas Campinas, SP, Brazil Sophie Franc¸oise Mauricette Derchain Faculdade de Cieˆncias Me´dicas, Universidade Estadual de Campinas Campinas, SP, Brazil Suely Kazue Nagahashi Marie Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Thelma Suely Okay Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Vale´ria Aoki Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Editorial Board Fa´bio Biscegli Jatene Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Marcelo Zugaib Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Francisco Laurindo Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Marco Martins Amatuzzi Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Cesar Gomes Victora Faculdade de Medicina da Universidade Federal de Pelotas Pelotas, RS, Brasil

Hiroyuki Hirasawa Chiba University School of Medicine Chiba, Japan

Daniel Romero Mun˜oz Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Irismar Reis de Oliveira Faculdade de Medicina da Universidade Federal da Bahia Salvador, BA, Brasil

Maria Aparecida Shikanai Yasuda Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Abhijit Chandra King George’s Medical College Lucknow, India

Artur Brum-Fernandes Universite´ de Sherbrooke Que´bec, Canada´

Adamastor Humberto Pereira Universidade Federal do Rio Grande do Sul Porto Alegre, RS, Brazil

Carmita Helena Najjar Abdo Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Adauto Castelo Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ademar Lopes Fundac¸a˜o Antoˆnio Prudente, Hospital do Caˆncer Sa˜o Paulo, SP, Brazil Alberto Azoubel Antunes Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Alexandre Roberto Precioso Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Edmund Neugebauer Witten/Herdecke University Witten, North Rhine - Westphalia, Germany Egberto Gaspar de Moura Jr. Universidade do Estado do Rio de Janeiro Rio de Janeiro, RJ, Brazil

Andrea Schmitt University of Goettingen Goettingen, Germany

Ernest Eugene Moore University of Colorado Denver Denver, CO, USA

Arnaldo Valdir Zumiotti Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Euclides Ayres Castilho Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Mauro Perretti William Harvey Research Institute London, UK

Irshad Chaudry University of Alabama Birmingham, AL, USA

Michael Gregory Sarr Mayo Clinic Rochester, MN, USA

Ivan Cecconello Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Milton de Arruda Martins Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Ke-Seng Zhao Southern Medical University Guangzhou, China

Mitchell C. Posner The University of Chicago Medical Center Chicago, IL, USA

Laura Cunha Rodrigues London School of Hygiene and Tropical Medicine - University of London London, UK

Moyses Szklo Johns Hopkins Bloomberg School of Public Health Baltimore, USA

Navantino Alves Faculdade de Cieˆncias Me´dicas de Minas Gerais Belo Horizonte, MG, Brazil Noedir Antonio Groppo Stolf Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Pedro Puech-Lea˜o Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Peter Libby Brigham and Women’s Hospital Boston, Boston, MA, USA Philip Cohen University of Houston Health Center

Houston, Texas, USA

Samir Rasslan Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Rafael Andrade-Alegre Santo Toma´s Hospital Republic of Panama´, Panama´ Ricardo Antonio Refinetti Faculdade de Medicina da Universidade Federal do Rio de Janeiro Rio de Janeiro, RJ, Brazil Roberto Chiesa San Raffaele Hospital Milan, Italy Ronald A. Asherson Netcare Rosebank Hospital Rosebank, Johannesburg, South A´frica

Tarcisio Eloy Pessoa de Barros Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Valentim Gentil Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Wagner Farid Gattaz Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Board of Governors Alberto Jose´ da Silva Duarte Ana Claudia Latronico Xavier Berenice Bilharinho de Mendonc¸a Clarice Tanaka Claudia Regina Furquim de Andrade Cyro Festa Neto Dalton de Alencar Fischer Chamone Daniel Romero Mun˜oz Edmund Chada Baracat Eduardo Massad Eloisa Silva Dutra de Oliveira Bonfa´ Euripedes Constantino Miguel Fa´bio Biscegli Jatene Flair Jose´ Carrilho Francisco Vargas Suso Gerson Chadi Gilberto Luis Camanho Irene de Lourdes Noronha Irineu Tadeu Velasco Ivan Cecconello Jorge Elias Kalil

Jose´ Antonio Franchini Ramires Jose´ Eduardo Krieger Jose´ Ota´vio Costa Auler Jose´ Ricardo de Carvalho Mesquita Ayres Lenine Garcia Branda˜o Luiz Augusto Carneiro D’Albuquerque Luiz Fernando Onuchic Magda Maria Sales Carneiro-Sampaio Manoel Jacobsen Teixeira Marcelo Zugaib Marcos Boulos Marcus Castro Ferreira Maria Aparecida Shikanai Yasuda Maria Irma Seixas Duarte Miguel Srougi Nelson de Luccia Noedir Antonio Groppo Stolf Olavo Pires de Camargo Paulo Andrade Lotufo Paulo Hila´rio Nascimento Saldiva Paulo Marcelo Gehm Hoff

Editorial Director Kavita Kirankumar Patel-Rolim Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Pedro Puech-Lea˜o Remo Susanna Ricardo Ferreira Bento Roberto Kalil Roberto Zatz Roger Chammas Samir Rasslan Sandra Josefina Ferraz Ellero Grisi Selma Lancman Tarcı´sio Eloy Pessoa de Barros Uenis Tannuri Umbertina Conti Reed Valentim Gentil Venaˆncio Avancini Ferreira Alves Vicente Odone Wagner Farid Gattaz Werther Brunow de Carvalho William Carlos Nahas Wilson Jacob

Editorial Assistant Nair Gomes Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Daniela Aquemi Higa Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ariane Maris Gomes Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Editorial Office: Rua Dr. Ovı´dio Pires de Campos, 225 - 6 ˚ Andar Email: clinics@hcnet.usp.br Website: www.scielo.br/clinics

CEP 05403-010 Sa˜o Paulo/SP Tel.: +55-11-2661-6235 Submission: http://www.editorialmanager.com/clinics

Indexations: LILACS; MEDLINE; PubMed; PubMed Central; SciELO; Science Citation Index Expanded (ISI Web of Knowledge); Scopus; Ulrich’s Periodical Directory; Qualis/Capes - Classified as an International Circulation Journal in Medicine.

Clinics. Sa˜o Paulo: Scientific Journal of Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, 2005Monthly Periodical: January to December ISSN 1807-5932 printed version ISSN 1980-5322 online version Formerly Revista do Hospital das Clı´nicas da FMUSP, 1946–2004.

1. Medicine-scientific production. 2. Medical Sciences I. Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo.

CDD 610

PUBLICATION INFORMATION AND EDITORIAL POLICIES CLINICS publishes peer-reviewed articles of interest to clinicians and researchers in the medical sciences. CLINICS is registered with PubMed Central and SciELO. This complies with the policies of funding agencies, such as, the Wellcome Trust, the Research Councils UK - RCUK, the National Institutes of Health(NIH), and the DFG, German Research Foundation, which request or require deposition of the published articles that they fund into such publicly available databases. CLINICS supports the position of the International Committee of Medical Journal Editors (http://www.icmje.org/) on trial registration.

All trials initiated after January 1, 2012 must be registered prospectively (before patient recruitment begins) in a publicly accessible registry. Trials initiated before January 1, 2012 must be registered before submission to our journals. See the ICMJE FAQ on trial registration for further details. Visit http://www. who.int/ictrp/network/list_registers/en/index.html for the WHO’s list of approved registries. CLINICS suggests: http://www. clinicaltrials.gov/, a user friendly site.

Publication Fees CLINICS uses a business model in which expenses are recovered in part by charging a publication fee to the authors or research sponsors for each article they publish. Our 2011 prices are: Original Articles: R$ 1.700,00 (US$ 1,000.00); Review Articles: R$ 1.700,00 (US$ 1,000.00); Case Reports, Technical Notes, and Rapid Communications: R$ 1.000,00 (US$ 600.00); Invited Reviews, Editorials and Letters to the Editors: No charge.

Manuscripts involving human subjects or the use of laboratory animals must clearly state adherence to appropriate guidelines and approval of protocols by their institutional review boards.

Photographs that may identify patients or other human participants of studies shall be acceptable only when a legally valid permission (form provided when necessary) is signed by the said patient or other human participant or by his/her legally constituted representative.

Manuscripts should be submitted online, in English, digitalized in a word.doc-compatible software program. Authors are strongly advised to submit the manuscript in its final form to a spell check for English (USA). Submissions with excessive spelling or syntax mistakes, as well as articles in which the meaning is not sufficiently clear shall be returned to authors for correction.

Authors are also very strongly advised to limit abbreviations to the minimum possible. Abbreviations tend to make text unreadable. All abbreviations must be defined when first used.

Only terms or expressions used at least 5 times throughput the text should be abbreviated. Never use abbreviations spelt as common English words such as, for instance FUN, PIN, SCORE, SUN, etc.

Please make sure to submit your manuscript in the exact format described below. Failure to do so will cause the submission to be returned to you during the preliminary examination by the Editorial Office.

Manuscripts are invited in the following categories: ORIGINAL STUDY: Complete original studies should be submitted in this category. Three sections are offered: Basic, Clinical, and Surgical Research. Original Studies must follow the following format:

Title page:

N N N N

Title (up to 250 characters); Running title (up to 40 characters, letters and spaces); Full address of corresponding author only; Authors’ names (without titles or graduations).

Authors should have participated sufficiently in the work to take public responsibility for appropriate portions of the content. Such participation must be declared in this section of the manuscript.

Manuscript:

N

N N

N

N

N

N

N N

Abstract: limited to 250 words and structured into: Objectives, Method, Results, Conclusions. Citations or abbreviations (except internationally recognized such as weights, measures, physical or chemical) are not allowed. Authors are strongly recommended not to display numerical statistical information, but merely to state what is significantly different (or not) between described parameters.

Keywords: 3–6 items from the Medical Subject Headings (Mesh) should be used.

Introduction: should set the purpose of the study, give a brief summary (not a review) of previous relevant works, and state what new advance has been made in the investigation. It should not include data or conclusions from the work being reported. A final sentence summarizing the novelty to be presented is permissible.

Materials and Methods: should briefly give clear and sufficient information to permit the study to be repeated by others. Standard techniques need only be referenced. Previously published methods may be briefly described following the reference.

Ethics: when reporting experiments on human subjects, indicate whether the procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional or regional) and with the Helsinki Declaration of 1975, as revised in 1983. When reporting experiments on animals, indicate whether the institution’s or a national research council’s guide for, or any national law on the care and use of laboratory animals was followed.

Results: should be a concise account of the new information discovered, with the least personal judgment. Do not repeat in text all the data in the tables and illustrations, but briefly describe what such material contains.

Discussion: should be limited to the significance of the new information and relate the new findings to existing knowledge. Only unavoidable citations should be included. Citation to review articles are not encouraged in this section.

Acknowledgements: should be short and concise, and restricted to those absolutely necessary.

References in text: CLINICS adopts the Vancouver format. Cite references in text with Arabic numerals, in order of appearance, within parentheses (1), after previous word, with spacing as in this example: ‘‘Diabetes (2), hypertension (3,4) and alcoholism (5–9)

N

N

N

are complex medical problems (10).’’ Under exceptional circumstances authors’ names may appear in text: Single author: ‘‘Einstein (11) proposed a new theory …’’ Two authors: ‘‘Watson & Crick (12) reported on the structure of …’’ Three or more authors: ‘‘Smith et al (13) described …’’

Reference List: Only citations that appear in the text should be referenced. Unpublished papers, unless accepted for publication, should not be cited. Work accepted for publication should be referred to as ‘‘in press’’ and a letter of acceptance of the journal must be provided. Unpublished data should only be cited in the text as ‘‘unpublished observations’’, and a letter of permission from the author must be provided.

Authors are responsible for the accuracy and completeness of their references and for correct text citation. CLINICS adopts the Vancouver format; references must be restricted to directly relevant published works, papers, or abstracts that have been accepted for publication. Usually the total number of references should not exceed 35. For up to six authors, list all authors; for more than 6 authors, list first six, followed by ‘‘et al’’.

Tables and Figures: The maximum number of tables plus figures is six. Tables: Do not be incorporate Tables into the Manuscript. Upload each table individually into the system. Tables should be constructed using the Table feature in your word processor or using a spreadsheet program such as Excel. Tables should be numbered in order of appearance in the text with Arabic numerals. Each table should have a title and, if necessary, an explanatory legend. All tables must be referred to and succinctly described in the text. Under no circumstances should a table repeat data presented in an illustration. Statistical measures of variation (i.e., standard deviation, standard error) should be identified, and decimal places in tabular data should be restricted to those with mathematical and statistical significance. Figures: Do not incorporate Figures into the Manuscript. Photographs, illustrations, charts, drawings, line graphs, etc are all defined as Figures. We do not publish Pictures Graphics, Photos, etc. Number your figures consecutively in Arabic numerals in order of appearance. Upload each Figure individually into the system. Legend(s) should be descriptive and allow examination of the figure without reference to text. Legends should be incorporated into the Main Document, after the Tables (if any) or after the references. Photographic illustrations should must be of professional quality and uploaded as *.tiff files, Typewritten or hand lettering is unacceptable, as are figures generated by dot matrix printers. Generally, figures will be reduced to fit one column of text. Actual magnification of all photomicrographs should be provided, preferably by placing a length scale on the print. Line graphs and charts must never be sent as jpeg illustrations. It is usually best to prepare these as ExcelH files. When ready copy the line graph or chart to a word.doc sheet.

Comments: Authors should use this space to describe the novelty contained in their original study. Only the

Editor of CLINICS has access to this section of the submission.

FAST TRACK ARTICLES: Fast track articles should follow the same format described above for original studies. Fast track articles must be complete original studies with justifiable urgency for publication. The Editorial Office undertakes to produce a first action response in the shortest possible time and to publish accepted Fast Track articles in the next available issue. Only one article may be submitted as Fast Track in any calendar year by any author or co-author. In Comments, authors must explain the reasons for the claim to Fast Track. Rejection by journals with a higher Impact Factor that ours is an acceptable reason for requesting FAST TRACK, provided that the reviewers’ reports from the previous submission be included in the present submission. Information contained in the comments is limited to the Editor and shall remain confidential. No publication fee discount is allowed for accepted Fast Track articles.

REVIEW ARTICLES: Such articles should cover themes relevant to medical practice or mammalian function. Spontaneously submitted reviews are welcome, but potential authors should bear in mind that they are expected to have expertise in the reviewed field. The sections should be arranged as follows:

N N

Title page: as in Original Study.

Manuscript: Abstract, keywords and text should be arranged as best suited to cover the subject being reviewed. If appropriate, the manner of reference collection should be described. The use of headings, subheadings, and paragraph titles is encouraged for sake of clarity. Abbreviations, acknowledgements, Tables and Figures as in original studies. The number of references is at the discretion of the authors. No publication fee discount is allowed for spontaneously submitted review articles accepted for publication.

RAPID COMMUNICATIONS:

N N

Title page: as in Original Study.

Manuscript: Rapid communications are limited to 1,500 words, except for the reference list. Authors should format such contributions as best suited to the subject at hand. No abstract or key words are required. Please copy an introductory sentence into the abstract box on Page 1 of the submission procedure.

CASE REPORTS: Case Reports will be published only if justified by the extreme rarity of the case and/or novelty of adopted procedures. Case reports should be arranged as follows:

N N

Title page: as in Original Study.

Manuscript: Case reports are limited to 1,500 words, except for the reference list. The text should be divided into Introduction, Case Description, and Discussion. No abstract or key words should be included, but please make sure to copy your Introduction into the abstract box of the submission form. Abbreviations, References, Tables and Figures (maximum of four, altogether) follow the format described for Original Studies. No publication fee discount is allowed for case reports accepted for publication.

LETTERS TO THE EDITOR: Letters to the Editor expressing comments or dissenting opinions concerning articles recently published in CLINICS are not submitted to peer review and are published at the discretion of the Editor. A letter is a single section untitled text, followed by references, to include the article under discussion. No publication fee is charged for this class of manuscripts.

EDITORIAL: Editorials should cover broad aspects of Medical or Biological sciences. Such manuscripts are not submitted to peer review and are published at the discretion of the Editor. A letter is a single section untitled text, followed by references, if applicable.

No publication fee is charged for this class of manuscripts.

COMMENTARY: A commentary is an invited text with respect to an article being published by Clinics. No publication fee is charged for this class of manuscripts.

INVITED REVIEW: Invited reviews follow the general pattern proposed for general reviews but are by invitation only. No publication fee is charged for this class of manuscripts.

SPECIAL ISSUE ARTICLE: Special issue articles are by invitation only and follow a specific pattern set out by the Editor in charge of the collection.

Peer Review: Manuscripts are reviewed by at least two expert consultants. Accepted manuscripts are edited to comply with the Journal’s format, to remove redundancies, and to improve clarity and understanding without altering meaning. The edited text will be presented to authors for approval.

Submission: A copyright transfer form, signed by all authors, must be submitted by fax (55-11-2661-7524) or by mail as soon as the manuscript is submitted. Any financial or other relationships that could lead to a conflict of interest must be disclosed in the copyright transfer form. If the Editor considers this conflict of interest relevant to the paper, a footnote will be added to show the equity interest in or affiliation with the identified commercial firm(s).

As soon as authors are satisfied that the manuscript complies with the Journal format, our site should be accessed through www. clinics.org.br. The system will guide authors through the manuscript submission process and prompt authors to input information into specific fields as they are submitting their manuscript. The Editorial Office will be automatically notified of the submission and will send an email confirming it as soon as the submission letter reaches the office. Progress of the manuscript through the Editorial Office procedures will be available to authors at all times.

ISSN-1807-5932

CLINICS CONTENTS Clinics 2012 67(2):93–203

EDITORIAL

Bioethicists must rethink the concept of death: the idea of brain death is not appropriate for cryopreservation Claudio Cohen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

CLINICAL SCIENCES

Genotype analysis of the human endostatin variant p.D104N in benign and malignant adrenocortical tumors Beatriz Marinho de Paula Mariani, Ericka Barbosa Trarbach, Tamaya Castro Ribeiro, Maria Adelaide Albergaria Pereira, Berenice Bilharinho Mendonca, Maria Candida Barisson Villares Fragoso . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

Characterization of human adipose-derived stem cells and expression of chondrogenic genes during induction of cartilage differentiation Adila A Hamid, Ruszymah Bt Hj Idrus, Aminuddin Bin Saim, Somasumdaram Sathappan, Kien-Hui Chua . . . . . . . . 99

Does administering albumin to postoperative gastroschisis patients improve outcome?

Ana Cristina A. Tannuri, Luanna M. Silva, Antonio Jose´ G. Leal, Augusto Ce´sar F. de Moraes, Uenis Tannuri . . . . 107

Establishment of the bacterial fecal community during the first month of life in Brazilian newborns Ka´tia Brandt, Carla R. Taddei, Elizabeth H. Takagi, Fernanda F. Oliveira, Rubens T. D. Duarte, Isabel Irino, Marina B. Martinez, Magda Carneiro-Sampaio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

Is the measurement of inferior thyroid artery blood flow velocity by color-flow Doppler ultrasonography useful for differential diagnosis between gestational transient thyrotoxicosis and Graves’ disease? A prospective study ¨ zel, Selvinaz Velet, Mehmet Sait Bug˘dacı, Esra C Sayid Shafi Zuhur, Alper O ¸ il, Yu¨ksel Altuntas. . . . . . . . . . . . . . . . 125

Liver enzymes in patients with chronic kidney disease undergoing peritoneal dialysis and hemodialysis Isabella Ramos de Oliveira Liberato, Edmundo Pessoa de Almeida Lopes, Maria Alina Gomes de Mattos Cavalcante, Tiago Costa Pinto, Izolda Fernades Moura, Luiz Loureiro Ju´nior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

Gamma-tocotrienol modulation of senescence-associated gene expression prevents cellular aging in human diploid fibroblasts Suzana Makpol, Azalina Zainuddin, Kien Hui Chua, Yasmin Anum Mohd Yusof, Wan Zurinah Wan Ngah . . . . . . 135

Visual acuity and retinal function in patients with Bardet-Biedl syndrome Adriana Berezovsky, Daniel Martins Rocha, Paula Yuri Sacai, Sung Song Watanabe, Nı´vea Nunes Cavascan, Solange Rios Saloma˜o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

Classification of the severity of diabetic neuropathy: a new approach taking uncertainties into account using fuzzy logic Andreja P. Picon, Neli R. S. Ortega, Ricky Watari, Cristina Sartor, Isabel C. N. Sacco . . . . . . . . . . . . . . . . . . . . . . 151

Clinical and serological manifestations associated with interferon-a levels in childhood-onset systemic lupus erythematosus Mariana Postal, Nailu Ange´lica Sinicato, Karina Oliveira Pelic¸ari, Roberto Marini, Lilian Tereza Lavras Costallat, Simone Appenzeller . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

BASIC RESEARCHES

Comparison of two peptide radiotracers for prostate carcinoma targeting Bluma Linkowski Faintuch, Erica A. Oliveira, Eutimio G. F. Nunez, Ana M. Moro, P. K. Nanda, Charles J. Smith. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

The protective effect of cilostazol on isolated rabbit femoral arteries under conditions of ischemia and reperfusion: the role of the nitric oxide pathway Mariana R.G.A. Santos, Andre´a C. Celotto, Verena K. Capellini, Paulo R. B. Evora, Carlos E. Piccinato, Edwaldo E. Joviliano . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171

READERS OPINION

The detection of cysto-biliary communications during surgery for liver hydatid cysts: let’s speak the unspoken Oktay irko¨ru¨cu¨, Enver Reyhan, Hasan Erdem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

RAPID COMMUNICATIONS

Prevalence of erectile dysfunction in chronic renal disease patients on conservative treatment Jose´ Fernando Pereira Mesquita, Thiago Faria Ramos, Felipe Pereira Mesquita, Jose´ Murillo Bastos Netto, Marcus Gomes Bastos, Andre´ Avarese de Figueiredo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

Interobserver concordance in the BI-RADS classification of breast ultrasound exams Maria Julia G. Calas, Renan M.V.R. Almeida, Bianca Gutfilen, Wagner C.A. Pereira . . . . . . . . . . . . . . . . . . . . . . . 185

CASE REPORTS

Paroxetine-induced severe sleep bruxism successfully treated with buspirone Aysel Milanlıoglu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

Acupuncture and exercise capacity: A case report Reginaldo Ceolin do Nascimento, Carlos Alberto Cordeiro Hossri, Otavio Berwanger, Vitor Oliveira Carvalho . . . . . 193

A combined minimally invasive approach for the treatment of aortoesophageal fistula caused by the ingestion of a chicken bone: case report and literature review Xiaodong Chen, Jianwen Li, Jie Chen, Yu Zhou, Yuanqi Zhang, Hongfei Ding, Shuichuan Huang, Zhi Zhang . . . . . 195

Laparoscopic repair of an incarcerated bochdalek hernia in an elderly man Toygar Toydemir, Hakan Akıncı, Mehmet Tekinel, Erdog˘an Su¨leyman, Bu¨lent Acunas¸, Mehmet Ali Yerdel . . . . . . . 199

ERRATA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203

CLINICS 2012;67(2):93-94

DOI:10.6061/clinics/2012(02)01

EDITORIAL

Bioethicists must rethink the concept of death: the idea of brain death is not appropriate for cryopreservation Claudio Cohen Universidade de Sa˜o Paulo, Legal Medicine, Medical Ethics and Social and Occupational Medicine.

Death has historically been believed to be an event that coincided with the onset of clinical death. It is now understood that death is a series of physical events, not a single event, and that the determination of permanent death is dependent on other factors beyond the simple cessation of breathing and heartbeat. We may need to accept the concept of the life cycle assessment introduced by nanotechnology or astrobiology, a process that is often called transhumanism (1).

Cryopreservation is the use of antifreezing solutions and proteins or cryoprotectants and cooling to very low temperatures for the long-term storage of human bodies, animals, organs or tissues, typically at liquid nitrogen temperatures (-196 ˚C). Cryopreservation is not freezing.

At these low temperatures, any biological activity, including biochemical reactions that would lead to cell death, is effectively stopped. However, when cryoprotectant solutions are not used, the cells being preserved are often damaged when approaching low temperatures in the freezing process or when being warmed to room temperature. To be biologically useful, cryoprotectants must easily penetrate the cells and not be toxic to them.

Cryopreservation is used for embryo storage, for example, when in vitro fertilization has produced more embryos than are needed at a given time. Semen can be successfully used almost indefinitely after cryopreservation, with successful storage reported for as long as 21 years. Oocyte cryopreservation is a new technology in which eggs are extracted, frozen, and stored. They may later be thawed, fertilized, and transferred to the uterus as embryos.

As of 2010, there were more than 200 people in cryogenic storage, all of them in the United States or Russia. Most are stored in the Alcor and the Cryonics Institute, but more than ten are in Russia, and two are in Trans Time.

All modern cryonics organizations require full payment for all future costs associated with storage ‘‘in perpetuity’’ before patient cryostorage. The costs of cryonics vary significantly, ranging from the basic fee of $10,000 for neuro(head or brain only) cryopreservation at the European cryonics company KrioRus to more than $200,000 for

whole-body cryopreservation by Alcor (this value includes overseas and last-minute fees). Alcor’s neuropreservation (head only) is priced at $80,000 (2).

Cryonicists do not believe that cryopreserved humans or animals are dead. They often refer to a person who has been declared legally dead and cryopreserved as being in a state of deanimation. However, the process of bringing a person or animal from a state of cryopreservation to life is called resuscitation, a term that is used among cryonicists. The term ‘‘revival’’ may be more appropriate than ‘‘resuscitation’’ (3).

At the present time, we should not define death exclusively as brain death. We cannot assume that a cryopreserved individual is either dead or still alive, as cryonicists believe. A cryopreserved individual is legally dead, but cryonicists say that he/she can be reanimated.

This reanimation should not be associated with resurrection.

To address these issues, we must reassess the concept of death. Such a reassessment occurred in 1988, when the scientific community reviewed and changed the definition of death from cardio-respiratory arrest to brain death.

Various religions and the legal system now accept this concept.

A central premise of cryonics is that long-term memory, identity stored in durable cell structures, and patterns within the brain do not require continuous brain activity to survive. This premise is generally accepted in medicine; it is known that, under certain conditions, the brain can stop functioning and can later recover, with the maintenance of long-term memory. Additional scientific premises of cryonics include the following: (a) the brain structures that encode personality and long-term memory persist for some time after clinical death, (b) these structures are preserved by cryopreservation, and (c) future technologies that could restore encoded memories to functional expression in a healed person are theoretically possible (4).

In simpler terms, we need to understand cryopreservation as a concept of life similar to the one used for frozen embryos. Some people view frozen embryos as living, but others do not. As soon as they are implanted in a uterus, however, they are ultimately given life, which is the equivalent of human reanimation.

The bioethical question posed by this issue is whether a cryopreserved human being is entitled to rights. We can accept the cryonics patient as legally dead, or he/she can be regarded as a ‘‘potential person’’. We need to respect his/ her living will when opting for ‘‘reanimation’’ as we accept a ‘‘do not resuscitate’’ (DNR) decision.

A moral premise of cryonics is that cryopreserving people is the right thing to do when there is no other

Email: ccohen@usp.br Tel.: 55 11 9222-4022 Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

93

Rethink the concept of death Cohen C

CLINICS 2012;67(2):93-94

hope, but the individual’s autonomy should be respected, as it is when a person agrees to participate in a genetic experiment with germ stem cells. Some cryonicists believe, as a matter of principle, that anyone who would ordinarily be regarded as dead should instead be made a ‘‘permanent patient’’, subject to whatever advances the future might bring (5).

We must differentiate between the concept of brain death and clinical death, which is the medical concept that refers to the cessation of blood circulation and breathing, the two criteria necessary to sustain life. Death occurs when the heart stops beating in a regular rhythm, a condition called cardiac arrest. During clinical death, all tissues and organs in the body steadily accumulate a type of injury called ischemic injury. The term is also sometimes used in resuscitation research.

The importance of this paradigm shift is that death is a metaphysical concept that may be changed in the future according to the needs of cryopreservation experimentation.

REFERENCES 1. Bostrom N. ‘‘A history of transhumanist thought’’. Journal of Evolution and Technology. 2005. Retrieved 2006-02-21. (Nobel Laureate in Medicine, Founding Director, NASA Astrobiology Institute).

2. ‘‘Prices and Funding’’ Membership info: costs". Alcor Life Extension Foundation. And Modern Marvels: Deep Freeze. The History Channel.

2008. Retrieved on 2008-04-01.

3. Whetstine L, Streat S, Darwin M, Crippen D. ‘‘Pro/con ethics debate: when is dead really dead?’’. Critical Care. 2005;9(6):538–42, http:// dx.doi.org/10.1186/cc3894.

4. Guyton, Arthur C. ‘‘The Cerebral Cortex and Intellectual Functions of the Brain’’. Textbook of Medical Physiology (7th ed.). W. B. Saunders Company. 1986;p.658.

5. Donaldson T. ‘‘Neural Archeology’’. Cryonics (Alcor Life Extension Foundation): 1987;24–33. Retrieved 2010-03-13.

94

CLINICS 2012;67(2):95-98

DOI:10.6061/clinics/2012(02)02

CLINICAL SCIENCE

Genotype analysis of the human endostatin variant p.D104N in benign and malignant adrenocortical tumors Beatriz Marinho de Paula Mariani,I Ericka Barbosa Trarbach,III Tamaya Castro Ribeiro,II Maria Adelaide Albergaria Pereira,IV Berenice Bilharinho Mendonca,V Maria Candida Barisson Villares FragosoI I Faculdade de Medicina da Universidade de Sa˜o Paulo, Unidade de Suprarrenal, Sa˜o Paulo/SP, Brazil. II Faculdade de Medicina da Universidade de Sa˜o Paulo, Laborato´rio de Hormoˆnios e Gene´tica Molecular LIM/42, Sa˜o Paulo/SP, Brazil. III Faculdade de Medicina da Universidade de Sa˜o Paulo, Laborato´rio de Gene´tica Me´dica LIM/25, Sa˜o Paulo/SP, Brazil. IV Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Clı´nica Me´dica, Sa˜o Paulo/SP, Brazil. V Faculdade de Medicina da Universidade de Sa˜o Paulo, Unidade de Endocrinologia do Desenvolvimento da Faculdade de Medicina da Universidade de Sa˜o Paulo, Sa˜o Paulo/SP, Brazil.

OBJECTIVE: Endostatin is a potent endogenous inhibitor of angiogenesis. It is derived from the proteolytic cleavage of collagen XVIII, which is encoded by the COL18A1 gene. A polymorphic COL18A1 allele encoding the functional polymorphism p.D104N impairs the activity of endostatin, resulting in a decreased ability to inhibit angiogenesis.

This polymorphism has been previously analyzed in many types of cancer and has been considered a phenotype modulator in some benign and malignant tumors. However, these data are controversial, and different results have been reported for the same tumor types, such as prostate and breast cancer. The purpose of this study was to genotype the p.D104N variant in a cohort of pediatric and adult patients with adrenocortical tumors and to determine its possible association with the biological behavior of adrenocortical tumors.

METHODS: DNA samples were obtained from 38 pediatric and 56 adult patients (0.6–75 yrs) with adrenocortical tumors. The DNA samples were obtained from peripheral blood, frozen tissue or paraffin-embedded tumor blocks when blood samples or fresh frozen tissue samples were unavailable. Restriction fragment length polymorphism analysis was used to genotype the patients and 150 controls. The potential associations of the p.D104N polymorphism with clinical and histopathological features and oncologic outcome (age of onset, tumor size, malignant tumor behavior, and clinical syndrome) were analyzed.

RESULTS: Both the patient group and the control group were in Hardy–Weinberg equilibrium. The frequencies of the p.D104N polymorphism in the patient group were 81.9% (DD), 15.9% (DN) and 2.2% (NN). In the controls, these frequencies were 80.6%, 17.3% and 2.0%, respectively. We did not observe any association of this variant with clinical or histopathological features or oncologic outcome in our cohort of pediatric and adult patients with adrenocortical tumors.

KEYWORDS: Endostatin; Angiogenesis; p.D104N polymorphism; Adrenocortical tumor.

Mariani BMP, Trarbach EB, Ribeiro TC, Pereira MAA, Mendonca BB, Fragoso MCBV. Genotype analysis of the human endostatin variant p.D104N in benign and malignant adrenocortical tumors. Clinics. 2012;67(2):95-98.

Received for publication on August 15, 2011; First review completed on September 14, 2011; Accepted for publication on September 14, 2011 E-mail: beatriz_galuzzi@hotmail.com Tel.: 55 11 2661-7512

by the cleavage of the C-terminal non-collagenous domain (NC1) is known as endostatin and has been shown to exhibit anti-angiogenic activity in vitro and in vivo (3). As an angiogenic inhibitor, endostatin prevents tumor growth and expansion by controlling the formation of new blood vessels (4). The observation that individuals with trisomy 21 (Down’s syndrome) rarely develop solid tumors suggests the presence of an endogenous angiogenesis inhibitor encoded by a gene on chromosome 21 (5). A single nucleotide polymorphism c.4309G.A (p.D104N) was identified in the endostatin domain of COL18A1 (6).

Lughetti et al. reported that, relative to control individuals, patients who are heterozygous for this polymorphism (DN) had a 2.5-fold greater chance of developing solid tumors, such as prostate cancer (7). This finding likely

INTRODUCTION Collagen XVIII/endostatin is a heparan sulfate proteoglycan component of nearly all epithelial and endothelial basement membranes (1). The gene COL18A1 is located on chromosome 21 at 21q22.3 and contains 10 collagenous domains that are interrupted and flanked by noncollagenous domains (2). A proteolytic fragment produced

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

95

COL18A1 polymorphism and adrenocortical tumors Mariani BMP et al.

CLINICS 2012;67(2):95-98

indicates that this polymorphism results in a less active protein and that individuals who carry one or more mutant alleles at this locus are more prone to developing aggressive prostate cancer (7). In contrast, Macpherson et al. observed no significant difference in the normal homozygous (DD), heterozygous (DN), and mutant homozygous (NN) frequencies between androgen-independent prostate cancer patients and control individuals (8).

During embryonic development and postnatal growth and repair, angiogenesis or neovascularization from the preexisting vasculature is crucial (9). The formation of new blood vessels is a complex multistage process that involves the proteolytic degradation of the basement membrane, the loss of endothelial cells into the surrounding stroma, and finally, the re-adhesion of endothelial cells to form new capillary tubes (9). The growth of tumors is associated with increased angiogenesis, during which the formation of new blood vessels is a fundamental step in tumor development and expansion. In addition, neovascularization is a critical component of tumor metastasis and disease progression (10). This connection between angiogenesis and tumor progression has been documented in clinical studies, which have demonstrated a correlation between unbalanced angiogenic factors and metastatic tumors with poor survival rates (11). The normal (i.e., non-tumorous) adrenal gland is a highly vascularized tissue that is centripetally irrigated by a network of fenestrated capillaries (12). Whereas adrenocortical tumors and the normal adrenal cortex have a similar vascular pattern (13), adrenocortical carcinomas display a disorganized vasculature, with large vessels interspersed with irregular networks of microcapillaries (13). As endostatin is involved in angiogenesis, we hypothesized that the presence of the D104N polymorphism may influence the outcome of adrenocortical cancer.

Table 1 - Clinical data from 94 patients with adrenocortical tumors.

Characteristic Number of patients Median age in years (range) Gender Hormone-secreting tumors Tumor size (cm) Weiss score 0–2 3–5 6–9 MacFarlane stage I–II III–IV Specific death Time to death, months (range) Follow-up, months (range) *

Pediatric group

Adult group

38 2.5 (0.8–18) 10M/28F 38 5.6 (1.5–20)

56 35.28 (18–74.8) 6M/50F 47 4.5 (2–20)

12 15 11

29 14 13

32 6 7 13.7 (7–16) 66 (1–167)

34 22 16 13.6 (8–35.4) 44.6 (1–203)

A Weiss score $3 and a MacFarlane stage $III were considered adrenocortical carcinoma in the pediatric group. A Weiss score $3 was considered adrenocortical carcinoma in the adult group.

DNA amplification and restricted fragment length polymorphism analysis A fragment from exon 42 of the COL18A1 gene was amplified using PCR with the following primers: 59-ACA AAC ACC CAC ACC CAT C-39 and 59-GGG CTC CTA TCT GCA GTT TC-39. For paraffin-embedded tumor blocks, the following primers were used: 59-GCT GGG AGG CTC TGT TCT CAG-39 59-TAG GTT CCC AGT GCC GTG TGA-39.

When the DNA was obtained from fresh tissue or leukocytes, we were able to amplify a 261 bp fragment, but when the DNA was obtained from paraffin-embedded tumor blocks, new primers were designed to amplify a 119 bp fragment. PCR amplification was performed using a previously described protocol (14).

Both of the fragments (261 and 119 bp) were subjected to RFLP analysis using the restriction enzyme MseI, and the fragments were separated by electrophoresis in a 2.0% agarose gel.

The 261 and 119 bp fragments were subjected to RFLP analysis using the restriction enzyme MseI, and the fragments were separated by electrophoresis in a 2.0% agarose gel (Figure 1).

OBJECTIVE The aim of this study was to analyze the frequency of the DD (normal homozygous), DN (heterozygous), and NN (mutant homozygous) genotypes of the p.D104N polymorphism in patients with adrenocortical tumors and to identify any associations with clinical and biological features. The cohort consisted of pediatric and adult patients with adrenocortical tumors (benign and malignant) who were seen at the Hospital das Clı´nicas, University of Sa˜o Paulo, Brazil, from 1990 to 2010.

PATIENTS AND METHODS Subjects DNA samples were obtained from different tissues depending on the viability of the samples. Peripheral blood (n = 17), fresh frozen tissue (n = 70), and paraffin embedded tissue samples (n = 7) were collected from 94 patients with adrenocortical tumors. Thirty-eight patients were pediatric (1 to 17 yrs of age; 10 carcinomas, 7 with a poor outcome), and fifty-six were adults (18 to 70 yrs of age; 25 carcinomas, 17 with a poor outcome).

The clinical data are presented in Table 1. All of the cases were diagnosed between 1990 and 2010. In addition, 300 alleles from 150 healthy volunteers were analyzed to determine the frequency of the D104N endostatin polymorphism.

Figure 1 - Electrophoresis of PCR and RFLP – The lines 2, 4 and 5 indicate the heterozygous pattern. The line 8 represents the homozygous pattern and the M corresponds to the molecular weight marker Øx.

96

CLINICS 2012;67(2):95-98

COL18A1 polymorphism and adrenocortical tumors Mariani BMP et al.

polymorphism has also been evaluated in other types of cancers, such as leukemia (18). However, there have been contradictory reports regarding the association between this polymorphism and prostate tumors and breast tumors when analyzed in different cohorts (7,18).

Adrenocortical tumors are highly vascularized tumors; we therefore hypothesized that a functional polymorphism located in the endostatin-coding region of COL18A1 may be associated with the poor behavior of the tumors, leading to early metastasis and death.

In the present study, we identified no association between the p.D104N endostatin polymorphism and tumor aggressiveness or other clinical features in pediatric and adult patients. The lack of an association observed in our adrenocortical cancer population has also been observed in other tumors types. Similar studies of patients with leukemia and multiple myeloma revealed no association between the endostatin p.D104N polymorphism and disease outcome (18,19). The discrepancy between these results may be due to the limited size of the studied population. For example, studies performed by Iughetti et al. and Lourenc¸o et al. (20) revealed a high risk of sporadic breast cancer in individuals homozygous (NN) for the p.D104N polymorphism. However, these studies examined a small number of patients, and the allelic frequencies were not in Hardy–Weinberg equilibrium (X2 = 22.87, p,0.001). Alternatively the different results could also be due to disease heterogeneity. Other candidate genes should be investigated with respect to their involvement in angiogenesis.

Grants: This work was supported by grants from the Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo FAPESP (07/51504-8) and from the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico - CNPq (307951/ 06-5 to B.B.M.).

Statistical analyses Descriptive analyses were performed to determine the frequencies and distributions of the demographic variables.

The genotype frequencies in the adrenocortical tumor patients and control individuals were compared using contingency table analyses and the chi-square or Fisher’s exact test, when necessary. The odds ratio and the corresponding confidence intervals were estimated using standard methods. Statistical analyses were performed using STATA 9.0 statistical software. The Hardy–Weinberg equilibrium was tested with the chi-square test to determine the goodness-to-fit (one degree of freedom).

RESULTS The frequencies of the alleles in both the patients and controls were in Hardy–Weinberg equilibrium.

Of the 94 patients, 15 were heterozygous for the p.D104N polymorphism (DN), and 2 were homozygous for the p.D104N polymorphism (NN). Of the 150 controls, 26 were heterozygous for the p.D104N polymorphism, and 3 were homozygous for the mutant allele. The genotype frequency of this polymorphism did not differ between the patients (17%) and controls (19%) (p = 0.82).

Of the 17 patients carrying the mutant allele, 14 were adults; 8 of these adult patients were diagnosed with adenoma, and 6 were diagnosed with carcinoma. All of these patients were heterozygous (DN) for the polymorphism (p = 0.50). The three remaining patients carrying the mutant allele were pediatric patients, and all were diagnosed with a benign adrenocortical tumor. Two of these pediatric patients were homozygous (NN) for the mutant allele, and one was heterozygous (DN) (p = 0.38). No statistical difference was observed among the genotypes frequencies (DD, DN, and NN) in this cohort with respect to the clinical data, including the presence of Cushing’s syndrome (p = 0.76), the presence of virilization (p = 0.07), tumor weight or size (p = 0.35), Weiss score (p = 0.3), metastasis occurrence (p = 0.43), disease recurrence (p = 0.51), and patient mortality (p = 0.8).

ACKNOWLEDGMENTS We would like to thank the lab staff members Mirian Yumie Nishi, Cristina Rossi and Mariana Ferreira de Assis Funari for their technical management; Maria Rita Passos Bueno for supportive discussions regarding the COL18A1 gene; and Rodrigo A. Toledo and Luciani Carvalho their support. We would also like to thank all of the individuals who took part in this study, including the patients and control subjects and especially Dr. Maria Claudia Zerbini. We would also like to thank FAPESP, which provided financial assistance for this study and Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo (HCFMUSP) for making this study possible.

DISCUSSION Following the observation that individuals with trisomy 21 (Down’s syndrome) rarely develop solid tumors, Folkman and Kalluri proposed that an endogenous angiogenesis inhibitor is likely encoded by a gene on chromosome 21 (5). Endostatin was first isolated from the conditioned media of a non-metastatic murine hemangioendothelioma cell line, EOMA. EOMA cell–conditioned media inhibited the proliferation of FGF-2-stimulated bovine capillary endothelial cells (15).

Preclinical experiments in which endostatin is exogenously administered to cancer patients have been used to evaluate its anti-tumor activity and to determine whether its endogenous anti-angiogenic effect controls the formation of new blood vessels, which is a necessary event for tumor metastasis (16).

A non-synonymous polymorphism (p.D104N) in exon 42 of COL18A1, which encodes endostatin, was described by Yugetti et al. The presence of this mutation leads to decreased activity of the protein, which may 1) interfere with the anti-angiogenic role of endostatin and 2) be associated with the progression of solid tumors, such as breast cancer and prostate cancer (7,17). This particular

AUTHOR CONTRIBUTIONS Mariani BMP was responsible for the PCR and RFLP statistical analyses.

Trarback EB was responsible for the primer design and optimization.

Ribeiro TC was responsible for the DNA extraction. Pereira MAA was responsible for the patient assistance. Mendonca BB supported the work, suggested the study and was also responsible for the patient assistance.

Fragoso MCBV was responsible for the patient assistance and work support.

REFERENCES 1. Halfter W, Dong S, Schurer B, Cole GJ. Collagen XVIII is a basement membrane heparan sulfate proteoglycan. J Biol Chem. 1998;273(39): 25404-12, http://dx.doi.org/10.1074/jbc.273.39.25404.

2. Rehn M, Pihlajaniemi T. Alpha 1(XVIII), a collagen chain with frequent interruptions in the collagenous sequence, a distinct tissue distribution, and homology with type XV collagen. Proc Natl Acad Sci U S A.1994; 91(10):4234-8, http://dx.doi.org/10.1073/pnas.91.10.4234.

97

COL18A1 polymorphism and adrenocortical tumors Mariani BMP et al.

CLINICS 2012;67(2):95-98

3. Boehm T, Folkman J, Browder T, O’Reilly MS. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature.

1997;390(6658):404-7, http://dx.doi.org/10.1038/37126.

4. O’Reilly MS, Holmgren L, Shing Y, Chen C, Rosenthal RA, Moses M, et al. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell. 1994.

1;79(2):315-28.

5. Folkman J, Kalluri R. Cancer without disease. Nature. 2004; 26;427(6977):787.

6. Visakorpi T. Molecular genetics of prostate cancer. Ann Chir Gynaecol.

1999;88(1):11-6.

7. Iughetti P, Suzuki O, Godoi PH, Alves VA, Sertie AL, Zorick T, et al. A polymorphism in endostatin, an angiogenesis inhibitor, predisposes for the development of prostatic adenocarcinoma. Cancer Res. 2001;61(20):7375-8.

8. Macpherson GR, Ng SS, Forbes SL, Melillo G, Karpova T, McNally J, et al.

Anti-angiogenic activity of human endostatin is HIF-1-independent in vitro and sensitive to timing of treatment in a human saphenous vein assay. Mol Cancer Ther. 2003;2(9):845-54.

9. Zatterstrom UK, Felbor U, Fukai N, Olsen BR. Collagen XVIII/endostatin structure and functional role in angiogenesis. Cell Struct Funct.

2000;25(2):97-101, http://dx.doi.org/10.1247/csf.25.97.

10. Folkman J. Angiogenesis inhibitors generated by tumors. Mol Med. 1995;1(2):120-2.

11. Zetter BR. Angiogenesis and tumor metastasis. Annu Rev Med. 1999;49:407-24, http://dx.doi.org/10.1146/annurev.med.49.1.407.

12. Murakami T, Oukouchi H, Uno Y, Ohtsuka A, Taguchi T. Blood vascular beds of rat adrenal and accessory adrenal glands, with special reference to the corticomedullary portal system: a further scanning electron microscopic study of corrosion casts and tissue specimens. Arch Histol Cytol.1989;52(5):461-76, http://dx.doi.org/10.1679/aohc.52.461.

13. Mage nnis DP, McNicol AM. Vascular patterns in the normal and pathological human adrenal cortex. Virchows Arch. 1998;433(1):69-73, http://dx.doi.org/10.1007/s004280050218.

14. Chang JG, Chen PH, Chiou SS, Lee LS, Perng LI, Liu TC. Rapid diagnosis of beta-thalassemia mutations in Chinese by naturally and amplified created restriction sites. Blood. 1992;80(8):2092-6.

15. O’Reilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, et al.

Endostatin: an endogenous inhibitor of angiogenesis and tumor growth.

Cell. 1997;88(2):277-85, http://dx.doi.org/10.1016/S0092-8674(00)818486.

16. Mundhenke C, Thomas JP, Wilding G, Lee FT, Kelzc F, Chappell R, et al.

Tissue examination to monitor antiangiogenic therapy: a phase I clinical trial with endostatin. Clin Cancer Res. 2001;7(11):3366-74.

17. Balasubramanian SP, Cross SS, Globe J, Cox A, Brown NJ, Reed MW. Endostatin gene variation and protein levels in breast cancer susceptibility and severity. BMC Cancer. 2007;7:107, http://dx.doi.org/10.1186/ 1471-2407-7-107.

18. Liu TC, Peng CT, Lin SF, Chang CS, Chen TP, Chang JG. Assocation of endostatin D104N with leukemia. Kaohsiung J Med Sci. 2003;19(1):1-5, http://dx.doi.org/10.1016/S1607-551X(09)70440-1.

19. Ortega MM, Nascimento H, Costa FF, Teori MT, Lima CS. A polymorphism in the angiogenesis inhibitor, endostatin, in multiple myeloma. Leuk Res. 2003;27(1):93-4, http://dx.doi.org/10.1016/S01452126(02)00218-7.

20. Lourenco GJ, Cardoso-Filho C, Goncales NS, Shinzato JY, Zeferino LC, Nascimento H, et al. A high risk of occurrence of sporadic breast cancer in individuals with the 104NN polymorphism of the COL18A1 gene. Breast Cancer Res Treat. 2006;100(3):335-8, http://dx.doi.org/10.1007/s10549006-9259-z.

98

CLINICS 2012;67(2):99-106

DOI:10.6061/clinics/2012(02)03

CLINICAL SCIENCE

Characterization of human adipose-derived stem cells and expression of chondrogenic genes during induction of cartilage differentiation Adila A Hamid,I,II Ruszymah Bt Hj Idrus,I,II Aminuddin Bin Saim,II,III Somasumdaram Sathappan,IV Kien-Hui ChuaI,II I Universiti Kebangsaan Malaysia, Department of Physiology, Faculty of Medicine, Jalan Raja Muda Abdul Aziz, Kuala Lumpur, Malaysia. II Universiti Kebangsaan Malaysia, Tissue Engineering Centre, Faculty of Medicine, Jalan Yaacob Latif, Bandar Tun Razak, Kuala Lumpur, Malaysia. III Ear, Nose & Throat Consultant Clinic, Ampang Puteri Specialist Hospital, Jalan Mamanda 9, Ampang Jaya, Selangor, Malaysia. IV Subang Jaya Medical Centre, Selangor, Malaysia.

OBJECTIVES: Understanding the changes in chondrogenic gene expression that are involved in the differentiation of human adipose-derived stem cells to chondrogenic cells is important prior to using this approach for cartilage repair. The aims of the study were to characterize human adipose-derived stem cells and to examine chondrogenic gene expression after one, two, and three weeks of induction.

MATERIALS AND METHODS: Human adipose-derived stem cells at passage 4 were evaluated by flow cytometry to examine the expression of surface markers. These adipose-derived stem cells were tested for adipogenic and osteogenic differentiation capacity. Ribonucleic acid was extracted from the cells for quantitative polymerase chain reaction analysis to determine the expression levels of chondrogenic genes after chondrogenic induction.

RESULTS: Human adipose-derived stem cells were strongly positive for the mesenchymal markers CD90, CD73, CD44, CD9, and histocompatibility antigen and successfully differentiated into adipogenic and osteogenic lineages. The human adipose-derived stem cells aggregated and formed a dense matrix after chondrogenic induction. The expression of chondrogenic genes (collagen type II, aggrecan core protein, collagen type XI, COMP, and ELASTIN) was significantly higher after the first week of induction. However, a significantly elevated expression of collagen type X was observed after three weeks of chondrogenic induction.

CONCLUSION: Human adipose-derived stem cells retain stem cell characteristics after expansion in culture to passage 4 and serve as a feasible source of cells for cartilage regeneration. Chondrogenesis in human adiposederived stem cells was most prominent after one week of chondrogenic induction.

KEYWORDS: Chondrogenesis; Cell-based therapy; Cartilage; Chondrocytes; Adipose; Stem cells; Differentiation.

Hamid AA, Idrus RBH, Saim AB, Sathappan S, Chua KH. Characterization of human adipose-derived stem cells and expression of chondrogenic genes during induction of cartilage differentiation. Clinics. 2012;67(2):99-106.

Received for publication on June 13, 2011; First review completed on July 7, 2011; Accepted for publication on September 21, 2011 E-mail: kienhui@medic.ukm.my / ckienhui@hotmail.com Tel.: 603 92897299

sufficient number of autologous chondrocytes for the treatment of large cartilage defects is difficult. Moreover, the procedure requires an invasive protocol for harvesting the autologous cartilage from a non-weight-bearing site of the patient’s knee cartilage and creates additional damage to normal cartilage. Studies have shown that the donor site for autologous chondrocyte harvesting can experience pathological changes to the articular cartilage in the joint (2).

Clearly, an alternative source of cells must be used to avoid the shortcomings of using autologous chondrocytes.

Previous studies have indicated that adult mesenchymal stem cells can be found in human adipose tissue (3).

Adipose tissue represents a good candidate tissue for obtaining adult stem cells for regenerative therapy because it is readily available in a large quantity and can be obtained through less invasive procedures. Furthermore, the stromal vascular portion of adipose tissue has been reported to

INTRODUCTION The repair of cartilaginous defects remains a significant clinical challenge, due to the poor spontaneous regeneration of articular cartilage. This poor regeneration is a result of an insufficient blood supply and the sparse number of chondrocytes embedded in the dense extracellular matrix of articular cartilage. Chondrocyte-based therapy for cartilage repair has demonstrated promising clinical outcomes (1). While this procedure is generally successful, obtaining a

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

99

Sequential chondrogenesis in adipose stem cells Hamid AA et al.

CLINICS 2012;67(2):99-106

contain up to 2% of cells that are able to differentiate into various cell types, such as osteoblasts, chondrocytes, adipocytes, and neurons, compared with only 0.002% of cells with this capability in bone marrow (4). Flow cytometry analysis has been extensively used to examine the surface immunophenotype of stem cells isolated from humans and other species. Previous studies have shown that human adipose derived stem cells (HADSCs) express a characteristic adhesion molecule (CD9), mesenchymal stem cell markers (CD90, CD44, and CD73), and the histocompatibility antigen HLA-ABC. Meanwhile, hematopoietic antigens (CD31, CD34 and CD45), a stem-cell factor (CD117), and the histocompatibility antigen HLA-DR-DP-DQ have been reported to be antigens that are absent from the surface of HADSCs (3). To assess whether HADSCs retain the immunophenotype of stem cells after expansion in culture, flow cytometry analysis was performed using the panel of antibodies described above. Due to the ease of their collection and their capacity for multipotential differentiation, HADSCs are worthy of consideration as a source of cells for treatment of cartilage lesions. Stem cells can be easily expanded in culture for several passages to obtain a sufficient and homogeneous population of cells before differentiating into cartilage-generating cells (chondrocytes). However, the characteristics and differentiation capacity of serially passaged HADSCs have not yet been reported in detail.

In the developing embryo, all bones of the axial skeleton are formed via cartilage in an intermediate step in the process of endochondral ossification. During this process, mesenchymal cells condense and begin to differentiate along a pathway that ultimately leads to chondrocyte hypertrophy and mineralized tissue. O’Driscoll et al. have proposed a model that includes four stages of chondrogenesis: (a) commitment of the cells to chondrocytes (048 hours), (b) cell proliferation (1-10 days), (c) differentiation (7-28 days), and (d) matrix production (10-42 days) (5).

Chondrogenesis begins with the aggregation and condensation of loose mesenchymal stem cells. During this early event, the condensing mesenchyme expresses various extracellular matrix (ECM) components, including collagen type II, aggrecan core protein, cartilage oligomeric matrix protein (COMP), and the important transcription factor SRY box 9 (Sox9) (6,7). As the mesenchymal stem cells differentiate into chondrocytes, the cells begin to produce ECM materials rich in aggrecan core protein and COMP. Chondrogenesis by HADSCs may involve a process similar to that described above. However, to date, the detailed molecular changes during the chondrogenic induction of HADSCs have not been examined, although this information is crucial to determine if a cell-based therapy protocol is useful for repairing cartilage lesions. To understand the molecular events that are involved in the differentiation of uncommitted HADSCs to chondrocytes and the formation of cartilage tissue, we investigated the sequential expression of chondrogenic genes in HADSCs during chondrogenic induction after the first, second, and third week of culture. Knowledge of the rapid changes in chondrogenic gene expression during chondrogenic induction in HADSCs will provide a better understanding of the potential of these stem cells for cartilage repair. Knowledge of genes that are expressed during chondrogenic induction in culture is of fundamental importance for the use of stem cell-based approaches for the treatment of cartilage lesions.

MATERIALS AND METHODS Human adipose-derived stem cell isolation and culture This study was approved by the Universiti Kebangsaan Malaysia Research and Ethical Committee (Project code FF083-2007). Lipoaspirate was obtained from consented patients (n = 6) after liposuction procedures that were performed at the Subang Jaya Medical Center in Malaysia.

The specimens were placed in sterile containers and brought to the Biotechnology Laboratory, Department of Physiology, Faculty of Medicine, Universiti Kebangsaan, Malaysia to be processed within 24 hours. Each specimen was digested with 0.3% collagenase type I (Invitrogen, Carlsbad, CA, USA) in an orbital incubator at 37 ˚C for four hours. Next, the cell suspension was centrifuged, and the cell pellet was washed with phosphate-buffered saline (PBS, Invitrogen) to remove red blood cells and other impurities.

After a final centrifugation, the cell pellet was suspended in the control medium Ham’s F12:Dulbecco’s Modified Eagle Medium (151) supplemented with 10% fetal bovine serum (FBS), 1% antibiotic and antimycotic, 1% glutamax (Invitrogen), and 1% Vitamin C (Sigma, St. Louis, MO, USA). All cultures were maintained in a 5% CO2 incubator at 37 ˚C. The medium was changed every three days. After reaching 70% confluence, the primary culture (P0) was trypsinized using 0.05% trypsin-EDTA (Invitrogen) and passaged at a culture expansion ratio of 154 until passage 4 (P4).

Flow cytometry analysis Flow cytometry was performed on HADSCs at P4. The HADSCs were trypsinized using Accutase (Innovative Cell Technologies, San Diego, CA, USA). The cell suspension was subsequently centrifuged at 1200 rpm for 10 minutes and washed with DPBS containing 0.5% Bovine Serum Album (Sigma). HADSCs were filtered through a 70-mm nylon membrane, and the number of cells was determined by hemocytometer. A total of 26105 cells were incubated with either fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated antibodies for 30 minutes. The following CD surface markers were tested: CD9/PE, CD34/FITC, CD117/PE, CD31/PE, CD44/FITC, CD45/ FITC, CD73/FITC, CD90/FITC, HLA-ABC/FITC and HLA-DR-DP-DQ/FITC (BD Biosciences, NJ, USA). Ten thousand events were acquired for each CD surface marker on a Becton Dickinson FACSCalibur flow cytometer. The data analysis was performed using CELLQuestPro acquisition software (BD Bioscience, Franklin Lakes, NJ, USA).

Adipogenic induction of human adipose-derived stem cells (HADSCs) Isolated HADSCs were expanded in culture until passage 4 (P4) in an equal volume mix of Ham’s F-12 medium and Dulbecco’s Modified Eagle Medium supplemented with 10% FBS (control medium). The HADSCs were induced in an adipogenic induction medium that contained 200 mM indomethacin, 10 mM insulin, 0.5 mM 3-isobutyl-1-methylxanthine, and 1 mM dexamethasone (Sigma) at a seeding density of 400 cells/mm2. Media were replaced every three days, and the cells were maintained in culture for up to three weeks. After three weeks, the cultures were fixed in 10% formalin, and adipogenic differentiation was determined by Oil Red O staining.

100

CLINICS 2012;67(2):99-106

Sequential chondrogenesis in adipose stem cells Hamid AA et al.

Superscript III reverse transcriptase (Invitrogen). The reaction mix and protocol were performed following the manufacturer’s recommendations. The reaction cycle was 10 min at 23 ˚C, 60 min at 42˚C, and 10 min at 94 ˚C. To quantify the expression level of chondrogenic genes after the first, second, and third week of culture in the chondrogenic induction medium or the control medium, quantitative polymerase chain reaction (qPCR) was performed using cDNA as the template. The genes of interest were collagen type II, aggrecan core protein, SOX9, COMP, ELASTIN, collagen type XI, collagen type I (dedifferentiation marker), and collagen type X (hypertrophy marker). The specific primer sequences were designed using Primer 3 software (http://frodo.wi.mit.edu/primer3/) based on the published GenBank database sequences (Table 1). QPCR was performed in a Bio-Rad iCycler (Bio-Rad, Hercules, CA, USA), and the data were analyzed using the Bio-Rad iCycler software. Each qPCR mixture consisted of iQ SYBR Supermix (Bio-Rad), forward and reverse primers (5 mM of each) and 1 ml of cDNA template. The following PCR conditions were used: cycle 1: 95˚C for 3 minutes (1X) and cycle 2: Step 1, 95˚C for 10 second and Step 2, 61˚C for 30 second (40X). The PCR cycles were followed by a meltingcurve analysis to determine the specificity of the PCR products. Next, the data were normalized to the expression of the housekeeping gene GAPDH. The formula for calculating the relative mRNA expression from the data are as follows: Relative mRNA expression: 2DDCT DDC T = CT housekeeping gene – CT gene of interest CT = Threshold cycle (relative measure of the concentration of the target in the PCR reaction)

Osteogenic induction of human adipose-derived stem cells (HADSCs) HADSCs at passage 4 (P4) were induced in an osteogenic induction medium that contained 100 nM dexamethasone, 10 mM b-glycerophosphate, and 5 mg/ml ascorbate-2phosphate (Sigma) at a seeding density of 200 cells/mm2.

The cultures were fed with the osteogenic medium every three days for up to three weeks. After three weeks, the cultures were fixed in 10% formalin, and osteogenic differentiation was determined by Alizarin Red staining.

Chondrogenic induction of human adipose-derived stem cells (HADSCs) HADSCs at P4 were cultured in T25 flasks at a cell density of 9.66104 cells/cm2 in either the chondrogenic induction medium or the control medium. The chondrogenic induction medium contained F12:DMEM (1:1) supplemented with 1% FBS, 1% antibiotic and antimycotic, 1% glutamax, 1% vitamin c, 1% insulin-transferrin-selenium-X (ITS), 50 ng/ mL insulin-like growth factor 1 (IGF-1), 50 mg/mL ascorbate-2-phosphate, 100 nM dexamethasone, 40 mg/ml Lproline, and 5 ng/mL transforming growth factor-b3 (TGF-b3) (Sigma). The morphological features of cultured cells were monitored every day using an inverted light microscope (Olympus, Shinjuku-ku, Tokyo, Japan). HADSCs were harvested after the first, second and third week of culture for the quantification of sequential chondrogenic gene expression.

Total RNA isolation and quantitative PCR analysis Total RNA was extracted from cultured HADSCs after the first, second, and third week of culture in either the control medium or the chondrogenic induction medium using TRI Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s protocol. The HADSCs were homogenized in TRI reagent and centrifuged at 12,000 x g for 15 minutes at 4 ˚C to separate the cell debris. Total RNA was precipitated with 10 mL Polyacryl Carrier (Molecular Research Center). The total RNA pellet was then washed with 75% ethanol, dissolved in RNase- and DNase-free water (Invitrogen) and stored at -80˚C until use. Complementary DNA (cDNA) was synthesized from total RNA using

Statistical Analysis The data are presented as the mean ¡ the standard error of the mean (SEM). The gene expressions of collagen type II, aggrecan core protein, SOX9, COMP, ELASTIN, collagen type I and collagen type X in the control and chondrogenic induction groups after induction for one, two and three weeks were subjected to one-way analysis of variance with a Tukey post-hoc test and were analyzed using SPSS 15.0 (SPSS Inc., Chicago, IL). A p-value,0.05 was considered to be significant.

Table 1 - List of primers used in qPCR for chondrogenic genes (www.ncbi.nlm.nih.gov/nucleotide).

Chondrogenic Gene

Gene Bank Accession No.

Primer sequences

Human Collagen Type I, Col I

NM_000088

Human Collagen Type II, Col II

NM_001844

Human Collagen Type X, Col X

NM_000493

Human Collagen Type XI, Col XI

NM_080681

Human Aggrecan Core Protein, ACP

NM_001135

Human Sox9, SOX9

NM_000346

Cartilage Oligomeric Protein, COMP

NM_000095

ELASTIN

NM_000501

GAPDH

NM_002046

F: 59-agg gct cca acg aga tcg aga-39 R: 59-tac agg aag cag aca ggg cca-39 F: 59-cta tct gga cga agc agc tgg ca-39 R: 59-atg ggt gca atg tca atg atg g-39 F: 59-gct aag ggt gaa agg ggt tc-39 R: 59-ctc cag gat cac ctt ttg ga-39 F: 59-agc agg ggg tga gac ctg tg -39 R: 59-tgc tcc aga gca ggg gta gg-39 F: 59-cac tgt tac cgc cac ttc cc-39 R: 59-acc agc gga agt ccc ctt cg-39 F: 59-gcg gag gaa gtc ggt gaa ga-39 R: 59-ccc tct cgc ttc agg tca gc-39 F: 59-aac tca ggg cag gag gat gt-39 R: 59-tgt cct ttt ggt cgt cgt tc-3 F: 59-ggc ctg gag gca aac ctc tt-39 R: 59-cca cca act cct ggg aca cc-39 F: 59-tcc ctg agc tga acg gga ag-39 R: 59-gga gga gtg ggt gtc gct gt-39

101

Sequential chondrogenesis in adipose stem cells Hamid AA et al.

CLINICS 2012;67(2):99-106

confirmed by (Figure 1D).

Table 2 - Immunophenotyping of human adipose-derived stem cells (HADSCs) at passage 4. Data are presented as the mean ¡ standard error of the mean (SEM). n = 6.

Surface Markers

HADSCs at P4

CD90 CD9 CD73 CD44 HLA ABC CD45 CD117 CD31 CD34 HLA DP, DQ, DR

99.9¡0.1 91.2¡2.5 99.8¡0.1 99.2¡0.2 99.3¡0.4 6.2¡0.7 5.8¡0.8 1.9¡0.3 2.4¡0.5 3.0¡0.3

positive

staining

with

Alizarin

Red

Morphological features of HADSCs during chondrogenic induction After HADSCs were incubated in the chondrogenic medium for 24 hours, the cells began to move closer to one another and form cell aggregates. This aggregation demonstrated that cellular condensation activity started shortly after chondrogenic induction. The cell aggregates became larger after a week in culture (Figure 2A). After 14 days in the chondrogenic induction medium, most of the cells in the culture were involved in aggregate formation, and empty areas were present in the culture flasks (Figure 2B). On day 21 of chondrogenic induction, the cell aggregates became darker in color, which denoted the formation of dense cell aggregates with increased extracellular matrix production (Figure 2C). HADSCs in the control medium did not form any cell aggregates during the three-week culture period. HADSCs in the control medium continued to grow and form multilayers at the end of the third week of culture (Figure 2D).

RESULTS Immunophenotype of HADSCs HADSCs displayed positive staining for the mesenchymal surface markers CD90, CD73, CD44, CD9, and HLA-ABC (Table 2). HADSCs at passage 4 exhibited high levels of expression of CD90, CD73, CD9 and CD44, which stained more than 90% of the total cell population. More than 99% of HADSCs expressed the histocompatibility antigen molecule HLA-ABC. In contrast, only a small proportion (less than 7%) of HADSCs expressed the hematopoietic markers CD117, CD34, CD31, CD45, and the histocompatibility antigen class II HLA-DR-DP-DQ.

Sequential expression of chondrogenic genes in HADSCs The HADSCs cultured in the chondrogenic induction medium displayed a significantly higher expression of chondrogenic genes (collagen type II, SOX9, aggrecan core protein, COMP, collagen type XI, and ELASTIN) than the control group (Figure 3). Collagen type II mRNA was expressed only in the chondrogenic induction groups. The HADSCs expressed significantly higher levels of collagen type II mRNA after the first week (3.09E-04¡1.89E-04) in the chondrogenic induction medium compared with both

Adipogenic and osteogenic differentiation of HADSCs After 21 days of culture in the adipogenic induction medium, lipid droplets had formed in the induced HADSCs (Figure 1A). Positive Oil Red O staining further confirmed the formation of lipid droplets in the culture (Figure 1B).

Under osteogenic induction conditions, dark extracellular matrix material was detected after the induction period (Figure 1C). Deposition of calcified matrix by the cells was

Figure 2 - Human adipose-derived stem cells (HADSCs) cultured in a chondrogenic induction medium. (A) After one week of chondrogenic induction, HADSCs showed early cell condensation (arrow). (B) Cell aggregates (arrow) became larger in size and started to store matrices during the second week of chondrogenic induction. (C) Cell aggregates (arrow) became dense and dark in color, which denoted increased matrix deposition at the third week of culture in a chondrogenic induction medium. (D) HADSCs grown in a control medium did not show any cell aggregation after three weeks in culture. Magnification 200X.

Figure 1 - Human adipose-derived stem cells (HADSCs) cultured in adipogenic and osteogenic induction media. (A) Lipid droplet production in HADSCs after 21 days of adipogenic induction. (B) Positive Oil Red O staining of the lipid droplets. (C) Calcium deposition in HADSCs after 21 days of osteogenic induction. (D) Positive Alizarin Red staining. Magnification 400X.

102

CLINICS 2012;67(2):99-106

Sequential chondrogenesis in adipose stem cells Hamid AA et al.

Figure 3 - Chondrogenic gene expression in human adipose-derived stem cells cultured for one, two, and three weeks in a chondrogenic induction medium compared with a control medium. A significantly higher expression of the following chondrogenic genes was observed in the chondrogenic induction medium compared with the control medium in the first, second, and third weeks of culture: Collagen type II (a), Collagen type X (b), Collagen type XI (c), Aggrecan core protein (d), SOX9 (e), COMP (f) and ELASTIN (g). n = 6, p,0.05.

103

Sequential chondrogenesis in adipose stem cells Hamid AA et al.

CLINICS 2012;67(2):99-106

the second week (2.72E-09ยก2.72E-09) and the third week (1.28E-07ยก1.28E-07) of culture. Aggrecan core protein (ACP) was also expressed at the highest level after the first week of chondrogenic induction. The induced cells displayed a fourfold higher ACP expression after the first week (1.46E-03ยก0.56E-03) compared with the same culture duration of the control group. Subsequent culturing of the cells in the chondrogenic medium caused a reduction in ACP expression to 2.50E-04ยก1.46E-04 at the second week and 3.13E-04ยก1.02E-04 at the third week of culture.

Similarly, COMP expression was significantly higher in the HADSCs after the first week of culture in the chondrogenic induction medium compared with the other groups. The HADSCs expressed 2.5-fold higher COMP mRNA expression after the first week of culture compared with the control group, 10-fold higher COMP expression after the second week and 8-fold higher COMP expression after the third week. Both ELASTIN and collagen type XI were also expressed at the highest level after the first week of chondrogenic induction compared with the other groups. However, SOX9 expression reached the highest level at the third week of culture in the chondrogenic induction medium. SOX9 expression at the third week was significantly higher than at the first and second week in the chondrogenic induction group and all of the control groups.

In contrast, collagen type I (a dedifferentiation marker) was expressed at significantly higher levels at the first week of HADSC culture in the control medium. HADSCs in the chondrogenic induction medium expressed a much lower level of collagen type I expression; this expression was maintained at a steady state throughout the three weeks of culture compared with the control groups.

Finally, HADSCs expressed significantly higher levels of collagen type X (a hypertrophy marker) mRNA expression at the third week of culture in the chondrogenic induction medium compared with the other groups. HADSCs expressed 8.5-fold higher collagen type X mRNA expression at the third week of culture compared with cells cultured in the control medium for the same culture period and 4-fold higher collagen type X expression compared with the first and second week of culture of HADSCs in the chondrogenic medium.

osteocytes (10). High expression of CD44 and CD73 in HADSCs has also been reported in previous studies (11-13).

A small population of HADSCs at P4 expressed the hematopoietic surface markers CD34, CD45, and CD31.

This small percentage of cells is negligible and does not interfere with the multipotent ability of HADSCs (12,14).

The culture of HADSCs at P4 was also free of fibroblasts, which are positive for CD34 (15). The low expression of molecular MHC class II (HLA-DP-PQ-DR) suggests the potential of HADSCs for allogenic cell transplantation, due to a lower risk of rejection (16). CD117 is known to play a role in the survival, proliferation and migration of bone marrow stem cells, but it is poorly expressed in HADSCs, as shown by previous work (3). Thus, our results show that after expansion in culture, HADSCs retained stem cell surface marker expression and were unlikely to be contaminated with hematopoietic cells and other differentiated cells (13). Second, after expansion in culture to passage 4, the HADSCs demonstrated the capacity for multilineage differentiation into adipogenic and osteogenic cells. These results further support the data suggesting that the HADSCs expanded in culture in this study retained stem cell characteristics and could be used for cartilage regeneration.

The development of cartilage tissue involves the process of mesenchymal stem cell condensation, aggregation, and differentiation into chondroblasts followed by cell maturation, hypertrophy, calcification, and ultimately cell death (17). Cartilage formation during embryonic development starts with the formation of aggregates of mesenchymal progenitor cells and condensation, which play an important role in creating the microenvironment for chondrogenesis (18). Although the successful chondrogenic differentiation of human mesenchymal stem cells has been reported, the sequential mRNA expression profile has not been examined during the induction stage. In this study, after 24 hours of culture in a chondrogenic induction medium, HADSCs began to attract one another and form cell aggregates. This aggregation denoted the condensation process of the early stage of chondrogenesis. Following cell condensation, HADSCs began to express significantly higher levels of collagen type II, aggrecan core protein, COMP, ELASTIN, and collagen type XI in the first week of induction. In addition, the expression of ELASTIN mRNA in this study showed the feasibility of using differentiated HADSCs for elastic cartilage regeneration, such as in ear (19) and trachea reconstruction (20). The transcription factor SOX9 is an early marker that is expressed during cell condensation to regulate collagen type II and cartilage-specific matrix synthesis (21). Our results showed that continued culture in the chondrogenic induction medium further increased SOX9 expression in HADSCs (compared with the control group), and SOX9 expression reached its highest level at the third week of culture, which could further promote the production of cartilage matrix. Other studies have also shown successful chondrogenic induction upon transfection of SOX9 into human bone-marrow stem cells for cartilage formation (22).

Our study showed that the expression of most of the chondrogenic genes in induced HADSCs (collagen type II, aggrecan core protein, COMP, ELASTIN, and collagen type XI) was reduced at the second and third week of culture.

This reduction may have been due to the extracellular matrix mRNA expression returning to the baseline level

DISCUSSION The characterization of any source of stem cells after expansion in culture is crucial for determining if the cultured cells retain the stemness criteria required for cellbased therapy and testing purposes. HADSCs were cultured to passage 4 to obtain sufficient cells for analysis in this study. Standard characterization methods assess the expression of mesenchymal markers and the multilineage differentiation capacity of the stem cells. Based on flow cytometry analyses, HADSCs at P4 showed high expression of the mesenchymal adhesion molecule CD9 and surface marker CD44. CD9 plays an important role in cell adhesion during in vitro culture and acts as a useful marker for evaluating the characteristics and potential of stem cells (8). HADSCs at P4 expressed high levels of CD44, a receptor for hyaluronic acid that aids HADSC migration into the injured tissue during the tissue regeneration process (9). Another specific surface marker for mesenchymal stem cells is CD73, which is not expressed in hematopoietic stem cells, osteoblasts, or

104

CLINICS 2012;67(2):99-106

Sequential chondrogenesis in adipose stem cells Hamid AA et al. were co-supervisors of the project, involved in data analysis and manuscript drafting. Sathappan S was co-researcher in the project, provided specimens and was involved in data analysis. Chua KH was the corresponding author, principal investigator, responsible for the planning of the experiment, supervision of the progress of the experiment, data analysis, and final approval of the manuscript.

after the condensation and aggregation process during the first week of induction. Thus, continuous culture of the cells in the chondrogenic induction medium did not promote further extracellular matrix production to increase the size of cell aggregates. These results suggest that chondrogenic induction of stem cells for one week may be sufficient for cartilage regeneration. In addition, chondrogenesis is hypothesized to be regulated by the sequential exposure of the chondrogenic induction factors in a specific timeframe.

Therefore, further studies should evaluate the sequential combination of various growth factors to achieve more complete chondrogenesis in HADSCs. The high expression of a hypertrophy marker, collagen type X, at the third week of chondrogenic induction suggests that the continuous induction of HADSCs could lead the cells toward a hypertrophic state and increased bone matrix synthesis. Several factors have been shown to be related to chondrocyte hypertrophy in vitro, such as the use of the TGF family and dexamethasone (23-24).

Therefore, the removal of these two factors in the later period of induction could decrease the hypertrophy process in HADSCs. Serum-derived growth factors may also limit the continuation of chondrogenesis in stem cells. The ideal chondrogenic induction medium should be totally serumfree such that interference from the serum-derived growth factors can be avoided. However, cells also need proteins such as fibronectin for adherence and other factors from serum to reduce apoptosis (25). Our previous report has shown that supplementation with ITS, IGF-1, bFGF, and TGF-ß2 in the culture medium can promote human chondrocytes to maintain differentiated phenotypes and increased proliferation (26). Furthermore, the formation of good trachea cartilage also requires serum supplementation (20). Human serum from either an autologous or allogenic source is also known to promote articular chondrocyte growth (27). In addition, supplementation with a combination of human serum and bFGF can enhance human nasal septum chondrocyte proliferation and can promote cartilage regeneration (28). Perhaps a serum-free chondrogenic induction medium should also include factors used in chondrocyte culture to reduce cell death and increase chondrogenesis.

HADSCs expanded in culture to passage 4 retained stem cell characteristics and served as a feasible source of cells for cartilage regeneration. Chondrogenesis in HADSCs was most prominent after the first week of chondrogenic induction, but a longer induction period can provoke hypertrophy. Thus, the findings of this study suggest that future studies should investigate the use of HADSCs induced for one week in chondrogenic conditions for cartilage regeneration. This approach could be used in the near future to reduce the time and costs associated with preparing stem cells for the treatment of cartilage lesions.

REFERENCES 1. Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med. 1994;331:889-95.

2. Lee CR, Grodzinsky AJ, Hsu HP, Martin SD, Spector M. Effects of harvest and selected cartilage repair procedures on the physical and biochemical properties of articular cartilage in the canine knee. J Orthop Res. 2000;18:790-9, http://dx.doi.org/10.1002/jor.1100180517.

3. Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, et al. 2002.

Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell.

2002;13:4279-95, http://dx.doi.org/10.1091/mbc.E02-02-0105.

4. Strem BM, Hedrick MH. The growing importance of fat in regenerative medicine. Trends Biotechnol. 2005;23:64-6, http://dx.doi.org/10.1016/ j.tibtech.2004.12.003.

5. O’Driscoll SW, Fitzsimmons JS. The role of periosteum in cartilage repair. Clin Orthop Relat Res. 2001;391:S190-207, http://dx.doi.org/ 10.1097/00003086-200110001-00019.

6. Ng LJ, Tam PP, Cheah KS. Preferential expression of alternatively spliced mRNAs encoding type II procollagen with a cysteine-rich aminopropeptide in differentiating cartilage and nonchondrogenic tissues during early mouse development. Dev Biol. 1993;159:403-17, http:// dx.doi.org/10.1006/dbio.1993.1251.

7. Akiyama H, Chaboissier MC, Martin JF, Schedl A, de Crombrugghe B. The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev. 2002;16:2813-28, http://dx.doi.org/10.1101/ gad.1017802.

8. Kim YJ, Yu JM, Joo HJ, Kim HK, Cho HH, Bae YC, et al. Role of CD9 in proliferation and proangiogenic action of human adipose-derived mesenchymal stem cells. Pflugers Arch. 2007;455:283-96, http:// dx.doi.org/10.1007/s00424-007-0285-4.

9. Zhu H, Mitsuhashi N, Klein A, Barsky LW, Weinberg K, Barr ML, et al.

The role of the hyaluronan receptor CD44 in mesenchymal stem cell migration in the extracellular matrix. Stem Cells. 2006;24:928-35, http:// dx.doi.org/10.1634/stemcells.2005-0186.

10. Haynesworth SE, Baber MA, Caplan AI. Cell surface antigens on human marrow-derived mesenchymal cells are detected by monoclonal antibodies. Bone. 1992;13:69-80, http://dx.doi.org/10.1016/8756-3282(92) 90363-2.

11. Mitchell JB, McIntosh K, Zvonic S, Garrett S, Floyd ZE, Kloster A, et al.

Immunophenotype of human adipose-derived cells: temporal changes in stromal-associated and stem cell-associated markers. Stem Cells.

2006;24:376-85, http://dx.doi.org/10.1634/stemcells.2005-0234.

12. Katz AJ, Tholpady A, Tholpady SS, Shang H, Ogle RC. Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hADAS) cells. Stem Cells. 2005;23:412-23, http://dx.doi.org/ 10.1634/stemcells.2004-0021.

13. Kolf CM, Cho E, Tuan RS. Mesenchymal stromal cells. Biology of adult mesenchymal stem cells: regulation of niche, self-renewal and differentiation. Arthritis Res Ther. 2007;9:204, http://dx.doi.org/10.1186/ ar2116.

14. Charrie`re G, Cousin B, Arnaud E, Andre´ M, Bacou F, Penicaud L, et al.

Preadipocyte conversion to macrophage: Evidence of plasticity. J Biol Chem. 2003;278:9850-5, http://dx.doi.org/10.1074/jbc.M210811200.

15. D’Andrea F, De Francesco F, Ferraro GA, Desiderio V, Tirino V, De Rosa A, et al. Large-scale production of human adipose tissue from stem cells: a new tool for regenerative medicine and tissue banking. Tissue Eng. Part C Methods. 2008;14:233-42, http://dx.doi.org/10.1089/ ten.tec.2008.0108.

16. McIntosh K, Zvonic S, Garrett S, Mitchell JB, Floyd ZE, Hammill L, et al.

The immunogenicity of human adipose-derived cells: temporal changes in vitro. Stem Cells. 2006;24:1246-53, http://dx.doi.org/10.1634/ stemcells.2005-0235.

17. Izzo MW, Pucci B, Tuan RS, Hall DJ. Gene expression profiling following BMP-2 induction of mesenchymal chondrogenesis in vitro. Osteoarthritis Cartilage. 2002;10:23-33, http://dx.doi.org/10.1053/joca.2001.0478.

18. Stott NS, Jiang TX, Chuong CM. Successive formative stages of precartilaginous mesenchymal condensations in vitro: modulation of cell adhesion by Wnt-7A and BMP-2. J Cell Physiol. 1999;180:314-24, http://dx.doi.org/10.1002/(SICI)1097-4652(199909)180:3,314::AIDJCP2.3.0.CO;2-Y. 19. Ruszymah BH, Lokman BS, Asma A, Munirah S, Chua K, Mazlyzam AL, Isa MR, Fuzina NH, Aminuddin BS. Pediatric auricular chondrocytes gene expression analysis in monolayer culture and engineered elastic

ACKNOWLEDGMENTS This study was made possible by a grant from the Ministry of Science, Technology and Innovation of Malaysia: eScienceFund 02-01-02-SF0290.

AUTHOR CONTRIBUTIONS Hamid AA was the first author, responsible for performing the experiment, analyzing the data and drafting the manuscript. Idrus RBH and Saim AB

105

Sequential chondrogenesis in adipose stem cells Hamid AA et al.

20.

21.

22.

23.

CLINICS 2012;67(2):99-106

24. Leboy PS, Sullivan TA, Nooreyazdan M, Venezian RA. Rapid chondrocyte maturation by serum-free culture with BMP-2 and ascorbic acid.

J Cell Biochem. 1997;66:394-03.

25. Wong M, Tuan RS. Nuserum, a synthetic serum replacement, supports chondrogenesis of embryonic chick limb bud mesenchymal cells in micromass culture. In Vitro Cell Dev Biol Anim. 1993;29A:917-22, http:// dx.doi.org/10.1007/BF02634229.

26. Chua KH, Aminuddin BS, Fuzina NH, Ruszymah BHI. InsulinTransferrin-Selenium prevent human chondrocyte dedifferentiation and promote the formation of high quality tissue engineered human hyaline cartilage. Eur Cell Mater. 2005;9:58-67.

27. Munirah S, Ruszymah BH, Samsudin OC, Badrul AH, Azmi B, Aminuddin BS. Autologous versus pooled human serum for articular chondrocyte growth. J Orthop Surg. (Hong Kong) 2008;16:220-9.

28. Chua KH, Aminuddin BS, Fuzina NH, Ruszymah BHI. Basic fibroblast growth factor with human serum supplementation: enhancement of human chondrocyte proliferation and promotion of cartilage regeneration. Singapore Med J. 2007;48:324-32.

cartilage. Int J Pediatr Otorhinolaryngol. 2007;71:1225-34, http:// dx.doi.org/10.1016/j.ijporl.2007.04.014.

Ruszymah BH, Chua K, Latif MA, Hussein FN, Saim AB. Formation of in vivo tissue engineered human hyaline cartilage in the shape of a trachea with internal support. Int J Pediatr Otorhinolaryngol. 2005;69:1489-95, http://dx.doi.org/10.1016/j.ijporl.2005.04.026.

Ng LJ, Wheatley S, Muscat GE, Conway-Campbell J, Bowles J, Wright E, et al. SOX9 binds DNA, activates transcription and coexpresses with type II collagen during chondrogenesis in the mouse. Dev Biol. 1997;183:10821, http://dx.doi.org/10.1006/dbio.1996.8487.

Babister JC, Tare RS, Green DW, Inglis S, Mann S, Oreffo RO. Genetic manipulation of human mesenchymal progenitors to promote chondrogenesis using ‘‘bead-in-bead’’ polysaccharide capsules. Biomaterials.

2008;29:58-65.

Cancedda R, Castagnola P, Cancedda FD, Dozin B, Quarto R. Developmental control of chondrogenesis and osteogenesis. Int J Dev Biol. 2000;44:707-14.

106

CLINICS 2012;67(2):107-111

DOI:10.6061/clinics/2012(02)04

CLINICAL SCIENCE

Does administering albumin to postoperative gastroschisis patients improve outcome?

Ana Cristina A. Tannuri,I Luanna M. Silva,I Antonio Jose´ G. Leal,I Augusto Ce´sar F. de Moraes,II Uenis TannuriI I

Faculdade de Medicina da Universidade de Sa˜o Paulo, Pediatric Surgery Division, Pediatric Liver Transplantation Unit and Laboratory of Research in Pediatric Surgery (LIM 30), Sa˜o Paulo/SP, Brazil. II Faculdade de Medicina da Universidade de Sa˜o Paulo, Child Institute, Post-Graduate Program in Science, Sa˜o Paulo, Brazil. GEPECIN – Science of Nutrition Research Group. OLFIS – Latin American Epidemiological Research Group.

OBJECTIVES: Newborns who undergo surgery for gastroschisis correction may present with oliguria, anasarca, prolonged postoperative ileus, and infection. New postoperative therapeutic procedures were tested with the objective of improving postoperative outcome.

PATIENTS AND METHODS: One hundred thirty-six newborns participated in one of two phases. Newborns in the first phase received infusions of large volumes of crystalloid solution and integral enteral formula, and newborns in the second phase received crystalloid solutions in smaller volumes, with albumin solution infusion when necessary and the late introduction of a semi-elemental diet. The studied variables were serum sodium and albumin levels, the need for albumin solution expansion, the occurrence of anasarca, the length of time on parenteral nutrition, the length of time before initiating an enteral diet and reaching a full enteral diet, orotracheal intubation time, length of hospitalization, and survival rates.

RESULTS: Serum sodium levels were higher in newborns in the second phase. There was a correlation between low serum sodium levels and orotracheal intubation time; additionally, low serum albumin levels correlated with the length of time before the initiation of an oral diet and the time until a full enteral diet was reached. However, the discharge weights of newborns in the second phase were higher than in the first phase. The other studied variables, including survival rates (83.4% and 92.0%, respectively), were similar for both phases.

CONCLUSIONS: The administration of an albumin solution to newborns in the early postoperative period following gastroschisis repair increased their low serum sodium levels but did not improve the final outcome. The introduction of a semi-elemental diet promoted an increase in body weight at the time of discharge.

KEYWORDS: Gastroschisis; Gastroschisis repair; Hyponatremia; Morbidities; Neonates; Body wall defects.

Tannuri ACA, Silva LM, Leal AJG, Moraes AGF, Tannuri U. Does administering albumin to postoperative gastroschisis patients improve outcome? Clinics. 2012;67(2):107-111.

Received for publication on August 9, 2011; First review completed on August 19, 2011; Accepted for publication on September 21, 2011 E-mail: uenist@usp.br Tel.: 55 11 30812943

and fluid losses into the traumatic edema zone. These problems occur in association with increased intra-abdominal pressure due to reduced peritoneal cavity capacity and a consequent decrease in diuresis (8).

The classic treatment for NBs in the first two days after gastroschisis correction includes the administration of large volumes of hydration solutions and, if necessary, repeated expansion of the extracellular compartment with crystalloid solutions, which invariably leads to anasarca and diluted low serum levels. These results have deleterious consequences, specifically interstitial pulmonary edema and respiratory dysfunction (9). In a previous investigation involving a large number of patients with surgically repaired gastroschisis at three tertiary university centers, we demonstrated that the presence of hyponatremia and hypoalbuminemia in the postoperative period correlated with the number of days that the patient required a ventilator (10).

To avoid this vicious cycle, we proposed a different treatment in the current study. Instead of administering

INTRODUCTION In recent decades, the worldwide incidence of gastroschisis has increased (1-3). Despite survival rates higher than 90% (4,5), newborns (NBs) with gastroschisis demonstrate significant postoperative morbidity associated with long periods of hospitalization (6).

Surgical treatment for gastroschisis includes viscera reduction inside the cavity and abdominal wall closure in one- or two-stage procedures, always with at least a slight intra-abdominal tension (7). Patients in the early postoperative period tend to present with hydrosaline retention

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

107

Albumin for gastroschisis patients Tannuri ACA et al.

CLINICS 2012;67(2):107-111

and albumin levels in the first 72 hours of life. Furthermore, the occurrence of anasarca and the length of orotracheal intubation (OTI) were verified.

Because we usually opted for a mild sedation, the ventilator support method of choice for both groups was synchronized intermittent mandatory ventilation.

Hyponatremia was defined as sodium serum levels ,135 mEq/L, and hypoalbuminemia was defined as albumin serum levels ,3 g/dL. No patient received loop diuretics during the study period. No severity score was used.

To examine the nutritional support, we evaluated the necessary parenteral nutrition (PN) time, time until the initiation of the enteral diet, and time until reaching the full enteral diet. We also recorded the occurrence of enterocolitis, the length of hospitalization, and mortality.

Initially, we used the Shapiro-Wilk test to investigate the framing of the numeric variables in the Gaussian model of distribution. Mann-Whitney’s U test was used to compare the median values for colloid solution administration with enteral diet, PN time, length of hospitalization, serum sodium levels, and serum albumin levels. The chi-squared test was used to compare the percentages of newborns who presented anasarca. Simple linear regression tests were used to analyze the relationship between length of hospitalization, gestational age, birth weight, low serum sodium levels, low serum albumin levels, PN time, and OTI time. A multiple linear regression model was fitted to assess the association between each outcome (serum albumin levels, serum sodium levels, and the need for albumin solution infusion) and the independent variables. The adjusted analysis was conducted following a previously formulated hierarchical model in three levels: 1) gestational age; 2) birth weight; and 3) hospitalization time, OTI time, time to first enteral feeding, and time of full enteral feeding. In this model, the variables were controlled for factors in the same level and in the superior levels. P-values #0.20 were adopted in the univariate analysis as necessary to determine which variables to include in the multiple analyses or when there was a ß modification of more than 10% for any variable already in the model. The variables were then entered using the forward method. The significance level was set at p,0.05. Moreover, homoscedasticity was assessed graphically in all regression models. The Stata statistical software package, Version 10.0 (Stata Corp., College Station, TX, USA) was used for all statistical calculations.

large volumes of crystalloid solution, a basal maintenance solution was administered in standard volumes, even if diuresis rates were low. In cases with more intense oliguria, albumin solution was administered in an attempt to maintain the intravascular compartment volume.

Another problem frequently found in NBs who have undergone gastroschisis repair is the prolonged postoperative ileus (11,12). The introduction of an enteral diet can be difficult because of repeated vomiting, abdominal distention, intestinal malabsorption of nutrients, and enterocolitis episodes. Special diets may reduce the risk of these complications (13), but this practice has not yet been proven in the medical literature.

The objectives of the present study were as follows: 1) to evaluate whether albumin solution administration improves the outcomes for NBs in the early postoperative period by decreasing the occurrence of hyponatremia and 2) to verify whether the introduction of a semi-elemental diet presents some advantage over natural diets.

MATERIALS AND METHODS The data for this study were obtained by reviewing the records for all NBs with gastroschisis who were treated between January 2000 and June 2010 at the Neonatal Intensive Care Unit of the Child Institute of the University of Sao Paulo School of Medicine. This prospective study protocol was approved by the institution’s ethical committee. The study was divided into two phases.

Phase 1 (January 2000 to December 2003): Postoperative treatment consisted of an infusion of 120 to 150 mL/kg/day of crystalloid solutions (containing 37.5 mEq Na+/L, 20 mEq K+/L and 9 mEq Ca++/L) followed by 20 mL/kg/ 30 min of normal saline, repeated up to three times whenever the NBs presented with oliguria (defined as urinary output ,0.8 mL/kg/h) in the first two days of the postoperative period. Colloid solution (containing normal saline and 10% albumin) in volumes of 20 mL/kg/30 min was administered only if there was no increased diuresis.

Enteral nutrition was introduced as soon as the flow of fluids through the orogastric tube indicated restoration of intestinal transit. A whole-milk formula appropriate for each NB’s gestational age was used.

Phase 2 (January 2004 to June 2010): Postoperative treatment consisted of an infusion of 80 to 100 mL/kg/ day of crystalloid solutions and 20 mL/kg of albumin solution when oliguria occurred (i.e., diuresis less than 0.8 mL/kg/h). The orogastric tube was removed when appropriate, and after one or two days without vomiting or abdominal distension, a semi-elemental diet (PregominH) was introduced followed by a gradual transition to a full enteral formula.

In both phases, the aim of the selected surgical treatment was always primary closure of the defect. A staged reduction with silo was used only if the total reduction of visceral contents would impair hemodynamic and respiratory function.

The following data were evaluated: gestational age, weight (at birth and at hospital discharge), APGAR score (at 1 and 10 minutes), type of gastroschisis (simple or complicated; associated with atresia, necrosis and/or intestinal perforation), and type of surgical treatment. We evaluated whether the NBs received a colloid infusion in the early postoperative period and the average serum sodium

RESULTS One hundred thirty-six NBs were included in this study: 36 NBs in the first phase and 100 NBs in the second phase.

The comparisons of perinatal data and surgical treatment between the two phases are presented in Table 1.

Comparisons and correlations of serum sodium levels, serum albumin levels, anasarca and the need for albumin solution infusion Table 2 shows the data for serum sodium levels, serum albumin levels, and OTI time in the two study phases and the results of the statistical comparisons.

Albumin solution administration was necessary in 47.2% (17/36) and 56.0% (56/100) of NBs in the study’s first and second phases, respectively (p = 0.47).

108

CLINICS 2012;67(2):107-111

Albumin for gastroschisis patients Tannuri ACA et al.

Table 3 - Results of the multiple linear regression analysis evaluating the association between independent variables and patient outcomes.

Table 1 - Gestational age (GA), APGAR scores at 1 and 10 minutes, birth weight (mean¡sd), occurrence of complicated gastroschisis and silo usage in both phases of the study.

Variable GA (weeks) APGAR 1 min.

APGAR 10 min.

Birth weight (g) Silo Complicated gastroschisis

Phase 1

Phase 2

P-value

36 1/7¡2 7.8 ¡1.7 9.3¡0.7 2334.8¡478.3 16.6% 23.3%

36 6/7¡1 7.0¡2.3 9.0¡0.7 2415.8¡ 463.2 13.6% 10.0%

0.22 0.16 0.53 0.30 0.97 0.13

Serum albumin level Gestational age (per 1-week increase) Birth weight (per 100-g increase) OTI time (per 1-day increase) Time to the initiation of an enteral diet (per 1-day increase) Time to full enteral diet (per 1-day increase) Hospitalization time (per 1-day increase) Serum sodium level Gestational age (per 1-week increase) Birth weight (per 100-g increase) OTI time (per 1-day increase) Time to the initiation of an enteral diet (per 1-day increase) Time to full enteral diet (per 1-day increase) Hospitalization time (per 1-day increase) Need for colloid infusion Gestational age (per 1-week increase) Birth weight (per 100-g increase) OTI time (per 1-day increase) Time to the initiation of an enteral diet (per 1-day increase) Time to full enteral diet (per 1-day increase) Hospitalization time (per 1-day increase)

In the first phase, four newborns responded well to treatment with normal saline boluses, increasing their urinary output with no need for colloid solution administration. Their mean OTI time was 2.5 days, and their mean natremia level was 123.5 mEq/L. The percentage of newborns who presented with anasarca was 19.4% in the first phase and 8% in the second phrase (p = 0.117).

In all cases, there was no statistical correlation between serum albumin levels and gestational age, birth weight, and OTI time or hospitalization time. However, there was a strong correlation between low serum albumin level and the time to both the initiation of enteral feeding and a full enteral diet (p,0.001) (Table 3).

There were no correlations between serum sodium levels and gestational age, hospitalization time, birth weight, or time to first and full enteral feedings. There was, however, a correlation between low serum sodium levels and increased OTI time (p = 0.04). The need for colloid solution infusion did not correlate with any of the studied variables (Table 3).

*

variables with p.0.2 were excluded from the model.

Table 4 - Initiation time for an enteral diet or a full diet, length of time for parenteral nutrition, weight at discharge, and hospitalization time in the two phases of the study (mean¡sd).

Variable

Serum sodium levels (mEq/L) Serum albumin levels (g/dL) OTI (days)

p-value

123.4¡8.6

128.6¡6.4

0.006

2.5¡0.5

2.0¡0.5

0.02

4.3¡6.5

5.5¡9.4

0.98

0.312 0.928 0.300 0.630 0.613 0.439

This study aimed to clarify the epidemiological, surgical, and postsurgical data for newborns with gastroschisis.

Initially, we investigated the therapeutic changes related to extracellular volume expansion and the use of colloid solutions and their impact on fluid and electrolyte balance and the final outcomes for NBs with gastroschisis. The NBs in the two phases of the study (i.e., from 2000 to 2003 and from 2004 to 2010) had similar gestational ages and birth weights, which allowed statistical comparisons between the two groups.

However, although the serum albumin levels were higher in the patients from Phase 1, the serum sodium levels were significantly lower in this phase, which may reflect the excessive use of crystalloid solution infusion during this phase. Such extreme crystalloid administration was also ineffective because 47.2% of the NBs in this group remained oliguric and required albumin solution expansion to improve diuresis. A similar proportion of patients from Phase 2 received colloid solution infusion without previous crystalloid administration.

Table 2 - Serum sodium levels, serum albumin levels, and OTI time in the two phases of the study (mean¡sd).

Phase 2

0.533 0.514 0.041 0.392 0.319 0.389

DISCUSSION

The nutritional data, including the fasting time before beginning the enteral diet, the length of time before the patient received a full enteral diet, the duration of PN administration, the length of hospitalization, and weight at discharge, and the results of the statistical comparisons are summarized in Table 4.

The time until the first and full enteral feeding, the length of time for PN and the length of hospitalization were similar during both phases. However, the discharge weights of the NBs in the second phase were higher than those of the NBs in the first phase (p = 0.03). It is important to note that no patient presented with peripheral edema at the time of discharge.

Finally, episodes of necrotizing enteritis occurred in two NBs in the first phase and in none of the NBs in the second phase (p = 0.11).

Phase 1

0.382 0.812 0.278 ,0.001 ,0.001 0.382

The survival rate in Phase 1 was 83.4%; in Phase 2, it was 92% (p = 0.25).

Comparisons regarding nutritional support

Variable

p-value*

Outcomes

Initiation of an enteral diet (days) Full enteral diet (days) PN time (days) Weight at discharge (g) Hospitalization time (days)

109

Phase 1

Phase 2

p-value

17.9¡12.8

21.6¡12.9

0.06

29.9¡27.1 30.8¡17.2 27.3¡28.6 29.2¡17.6 2561.3¡510.7 2814.5¡515.8 35.2¡34.8 34.9¡19.5

0.18 0.06 0.03 0.16

Albumin for gastroschisis patients Tannuri ACA et al.

CLINICS 2012;67(2):107-111

The joint analysis of the data on serum sodium and albumin levels from the two time periods did not show any correlation between these data and the NBs’ initial birth weights. However, in both phases of the study, low serum sodium and albumin levels were observed. In experimental studies of chicken embryos and rabbit fetuses with iatrogenic gastroschisis, low sodium and chloride serum levels, low amino acid uptake and glucose deficits were observed along with the minor expression of a series of genes involved in enterocyte nutrient absorption (14,15). Finally, Carroll et al. observed that human fetuses with gastroschisis present with lower total serum protein levels and higher quantities of protein in the amniotic fluid, which should reflect protein losses and intrauterine growth deficits (16).

Thus, after the surgical correction of gastroschisis, the NBs should present with large exudative losses (e.g., water, sodium, and proteins) to the so-called ‘‘third space’’ or the inflamed bowel walls. The low serum albumin levels lead to decreased coloidosmotic plasma pressure, with fluid leakage to the extravascular areas, decreased intravascular volume, and decreased renal perfusion. In addition, the increased intra-abdominal pressure from the closed-wall defect contributes to decreased renal perfusion, which is responsible for oliguria. Thus, NBs with gastroschisis would have increased antidiuretic hormone secretion to promote free water retention and blood-volume reestablishment. The infusion of crystalloid solutions, however, cannot increase the blood volume because the infused liquid continues to leak into the interstitial space due to the previous low albumin levels; as a result, the serum sodium concentration remains low. Consequently, edema in the pulmonary interstice occurs, with defects in gaseous exchange and ventilatory mechanics. This effect explains the correlation between low serum levels and increased OTI time because both of these values are probably related to the excessive infusion of crystalloid solutions (p = 0.04). Furthermore, newborns in the first phase received sodium at levels much higher than normal maintenance, which increased their tendency to present with anasarca. Finally, despite the therapeutic administration of albumin solutions in the second phase of the study, the serum albumin levels decreased (p = 0.02), although the serum sodium levels may have increased (p = 0.006). The albumin levels may have initially decreased when albumin was administered, probably because the intravascular space had been expanded. The Phase 2 patients did indeed have a tendency to be less edematous than the newborns in Phase 1, which explains why the low sodium levels in the second phase did not correlate with longer OTI times.

The low serum albumin levels did not correlate with longer OTI times, although a correlation with the time to first and full enteral feeding was observed. Additionally, the need for albumin solution expansion did not correlate with any of the studied variables. These facts suggest that the low albumin levels combined with hemodynamic alterations and oliguria (requiring the administration of exogenous albumin), which are significant morbidity factors, did not influence the time of postoperative ileus. In fact, the mechanisms of the intestinal dysfunction observed in gastroschisis patients are not fully known. Intestinal dysfunction can be explained by changes and immaturity of the enteric nervous plexus and by exposure of the intestine to the amniotic fluid, as described in experimental investigations in rabbits (12, 17).

The results of the present study agree with several studies of adult patients undergoing abdominal surgery or after trauma. These studies found that maintaining albumin concentrations above 3.5 g/dL with exogenous albumin did not alter the length of postoperative ileus or hospital stay and did not improve pulmonary function or final mortality (18).

There are few studies of albumin treatment in pediatric surgical patients. In an interesting study by Kenny et al. (19), hypoalbuminemia occurred in 27% of postoperative neonates, and the mortality of the hypoalbuminemic infants was greater but was unrelated to the albumin level or albumin treatment. Finally, two randomized controlled studies comparing isotonic saline to 5% albumin in hypotensive term and preterm neonates showed that these two solutions are equally effective in treating hypotension and that patients treated with albumin had greater fluid retention (20,21). It is possible that the increased fluid retention was responsible for the decreased serum albumin levels observed in our Phase 2 patients. Another explanation would be the extravasation of albumin into the interstitial space.

Another interesting difference between the two study periods is the higher body weights found in the second phase, even though no weight differences were observed between the two phases at the beginning of the enteral diet.

Moreover, there was a tendency toward more episodes of necrotizing enterocolitis in the first group than in the second group. Such data suggest a possible benefit of initially introducing a semi-elemental diet and then gradually transitioning to integral formulas. This result agrees with a previous report suggesting that the early introduction of minimal enteral feeding and the controlled increase of nutritional elements after bowel reintegration significantly improves the NBs’ outcomes after the surgical repair of gastroschisis (13).

The comparisons regarding nutritional support demonstrated that no differences occurred between the two phases of the study, even though the albumin solution administration in the second phase of the study may have exacerbated the low serum sodium levels and decreased the serum albumin levels (Table 2). In both phases of the study, the time to initiation of the enteral diet, time to reach the full enteral diet, time on PN, and length of hospitalization did not vary. These results are in accord with the conclusion of a previous study of 163 gastroschisis patients that demonstrated that hyponatremia and hypoalbuminemia could not have influenced intestinal function because these alterations were not correlated with the number of days on PN (10).

In conclusion, the present study suggests that the administration of albumin solution to NBs in the early postoperative period of gastroschisis repair improved the decreased serum sodium levels but did not improve the final outcomes. In addition, the introduction of a semielemental diet was responsible for increased body weight at hospital discharge.

ACKNOWLEDGMENTS This study was funded by a grant from FAPESP (number 09/1807-0).

AUTHOR CONTRIBUTIONS Tannuri ACA, Tannuri U, and Leal AJG performed medical and surgical care of the patients and wrote the manuscript. Silva LM examined the medical records and statistical analyses. Moraes ACF performed the final revision of the statistical analyses.

110

CLINICS 2012;67(2):107-111

Albumin for gastroschisis patients Tannuri ACA et al.

11. Phillipps JD, Raval MV, Redden C, Weiner TM. Gastroschisis, atresia, dysmotility: surgical treatment strategies for a distinct clinical entity.

J Pediatr Surg. 2008;43:2208-12, http://dx.doi.org/10.1016/j.jpedsurg.

2008.08.065.

12. Oyachi N, Lakshmanan J, Ross MG, Atkinson JB. Fetal gastrointestinal motility in a rabbit model of gastroschisis. J Pediatr Surg. 2004;39:366-70, http://dx.doi.org/10.1016/j.jpedsurg.2003.11.044.

13. Walter-Nicolet E, Rousseau V, Kieffer F, Fusaro F, Bourdaud N, Oucherif S, et al. Neonatal outcome of gastroschisis is mainly influenced by nutritional management. J Pediatr Gastroenterol Nutr 2009;48:612-7.

14. Shaw K, Buchmiller TL, Curr M, Lam MM, Habib R, Chopourian HL, et al. Impairment of nutrient uptake in a rabbit model of gastroschisis.

J Pediatr Surg. 1994;29:376-8, http://dx.doi.org/10.1016/0022-3468 (94)90570-3.

15. Srinathan SK, Langer JC, Wang JL, Rubin DC. Enterocytic gene expression is altered in experimental gastroschisis. J Surg Res. 1997; 68:1-6, http://dx.doi.org/10.1006/jsre.1996.4986.

16. Carroll SG, Kuo PY, Kyle PM, Soothill PW. Fetal protein loss in gastroschisis as an explanation of associated morbidity. Am J Obstet Gynecol. 2001;184:1297-301, http://dx.doi.org/10.1067/mob.2001.

114031.

17. Santos MM, Tannuri U. Alterations of enteric nerve plexus in experimental gastroschisis: is there a delay in the maturation? J Pediatr Surg. 2003;38:1506-11, http://dx.doi.org/10.1016/S0022-3468(03)005049.

18. Uhing MR. The albumin controversy. Clin Perinatol. 2004;31:475-488, http://dx.doi.org/10.1016/j.clp.2004.03.018.

19. Kenny SE, Pierro A, Isherwood D, Donnell SC, Van Saene HK, Lloyd DA. Hypoalbuminaemia in surgical neonates receiving parenteral nutrition.

J Pediatr Surg. 1995;30:454-7, http://dx.doi.org/10.1016/00223468(95)90054-3.

20. So KW, Fok TF, Ng PC, Wong WW, Cheung KL. Randomised controlled trial of colloid or crystalloid in hypotensive preterm infants. Arch Dis Child Fetal Neonatal. 1997;Ed 76:F43-6, http://dx.doi.org/10.1136/ fn.76.1.F43.

21. Oca MJ, Nelson M, Donn SM. Randomized trial of normal saline versus 5% albumin for the treatment of neonatal hypotension. J Perinatol. 2003; 23:473-6, http://dx.doi.org/10.1038/sj.jp.7210971.

REFERENCES 1. Suita S, Okamatsu T, Yamamoto T, Handa N, Nirasawa Y, Watanabe Y, et al.

Changing profile of abdominal wall defects in Japan: results of a national survey. J Pediatr Surg. 2000;35:66-72, http://dx.doi.org/10.1016/S00223468(00)80016-0.

2. Penman DG, Fisher RM, Noblett HR, Soothill PW. Increase in incidence of gastroschisis in the South West of England in 1995. Br J Obstet Gynecol. 1998;105:328-31, http://dx.doi.org/10.1111/j.1471-0528.1998. tb10095.x.

3. Nichols CR, Dickinson JE, Pemberton PJ. Rising incidence of gastroschisis in teenage pregnancies. J Matern Fetal Med. 1997;6:225-9, http:// dx.doi.org/10.1002/(SICI)1520-6661(199707/08)6:4,225::AIDMFM8.3.0.CO;2-L. 4. Fillingham A, Rankin J. Prevalence, prenatal diagnosis and survival of gastroschisis. Prenat Diagn. 2008;28:1232-7, http://dx.doi.org/10.1002/ pd.2153 5. Eggink BH, Richardson CJ, Malloy MH, Angel CA. Outcome of gastroschisis: a 20-year case review of infants with gastroschisis born in Galverston, Texas. J Pediatr Surg. 2006;41:1103-8, http://dx.doi.org/ 10.1016/j.jpedsurg.2006.02.008.

6. Maramreddy H, Fisher J, Slim M, Lagamma EF, Parvez B. Delivery of gastroschisis patients before 37 weeks of gestation is associated with increased morbidities. J Pediatr Surg. 2009;44:1360-6, http://dx.doi.org/ 10.1016/j.jpedsurg.2009.02.006.

7. Marven S, Owen A. Contemporary postnatal surgical management strategies for congenital abdominal wall defects. Semin Pediatr Surg.

2008;17:222-35, http://dx.doi.org/10.1053/j.sempedsurg.2008.07.002.

8. Banieghbal B, Gouws M, Davies MR. Respiratory pressure monitoring as an indirect method of intra-abdominal pressure measurement in gastroschisis closure. J Pediatr Surg. 2006;16:79-83, http://dx.doi.org/10.1055/s2006-924051.

9. Drewett M, Michailidis GD, Burge D. The perinatal management of gastroschisis. Early Hum Dev. 2006;82:305-12, http://dx.doi.org/ 10.1016/j.earlhumdev.2006.02.003.

10. Tannuri ACA, Sbragia L, Tannuri U, Silva LM, Leal AJ, Schmidt AF, et al.

Evolution of critically ill patients with gastroschisis from three tertiary centers. Clinics. 2011;66:17-20, http://dx.doi.org/10.1590/S180759322011000100004.

111

CLINICS 2012;67(2):113-123

DOI:10.6061/clinics/2012(02)05

CLINICAL SCIENCE

Establishment of the bacterial fecal community during the first month of life in Brazilian newborns Ka´tia Brandt,I,II Carla R. Taddei,III,IV Elizabeth H. Takagi,III Fernanda F. Oliveira,III Rubens T. D. Duarte,V Isabel Irino,III Marina B. Martinez,III Magda Carneiro-SampaioI I

Universidade de Sa˜o Paulo, School of Medicine, Hospital das Clı´nicas, Children’s Hospital, Sa˜o Paulo/SP, Brazil. II Instituto de Medicina Integral Prof.

Fernando Figueira, Recife/PE, Brazil. III Universidade de Sa˜o Paulo, School of Pharmaceutical Sciences, Department of Clinical and Toxicological Analyses, Sa˜o Paulo/SP, Brazil. IV Universidade de Sa˜o Paulo, School of Arts, Science and Humanities, Sa˜o Paulo/SP, Brazil. V Universidade de Sa˜o Paulo, Laboratory of Microbial Ecology, Oceanographic Institute, Sa˜o Paulo/SP, Brazil.

OBJECTIVE: The establishment of the intestinal microbiota in newborns is a critical period with possible long-term consequences for human health. In this research, the development of the fecal microbiota of a group of exclusively breastfed neonates living in low socio-economic conditions in the city of Sa˜o Paulo, Brazil, during the first month of life, was studied.

METHODS: Fecal samples were collected from ten neonates on the second, seventh, and 30th days after birth. One of the neonates underwent antibiotic therapy. Molecular techniques were used for analysis; DNA was extracted from the samples, and 16S rRNA libraries were sequenced and phylogenetically analyzed after construction. A real-time polymerase chain reaction (PCR) was performed on the samples taken from the 30th day to amplify DNA from Bifidobacterium sp.

RESULTS: The primary phylogenetic groups identified in the samples were Escherichia and Clostridium.

Staphylococcus was identified at a low rate. Bifidobacterium sp. was detected in all of the samples collected on the 30th day. In the child who received antibiotics, a reduction in anaerobes and Escherichia, which was associated with an overgrowth of Klebsiella, was observed throughout the experimental period.

CONCLUSION: The observed pattern of Escherichia predominance and reduced Staphylococcus colonization is in contrast with the patterns observed in neonates living in developed countries.

KEYWORDS: Microbiota; Breastfeeding; 16S rRNA; Escherichia; Clostridium.

Brandt K, Taddei CR, Takagi EH, Oliveira FF, Duarte RTD, Irino I, et al. Establishment of the bacterial fecal community during the first month of life in Brazilian newborns. Clinics. 2012;67(2):113-123.

Received for publication on July 19, 2011; First review completed on August 12, 2011; Accepted for publication on September 26, 2011 E-mail: crtaddei@usp.br Tel.: 55 11 3091-3636

first facultative anaerobic colonizers were from the family Enterobacteriaceae, derived from the mother and the hospital environment (2,3). Recently, a shift from this pattern was observed and described as a diminished initial colonization by enterobacteria and high rates of colonization by Staphylococcus (4-6), which was probably derived from the skin of the parents (6). It has been suggested that this change correlates with the stricter hygiene measures applied during and after labor in developed countries (4,5,7-9). In developing countries, however, ideal hygiene control has not yet been achieved, and repercussions in early gut colonization are expected. Initial studies conducted in developing countries observed that neonates were soon colonized by Escherichia coli and other Enterobacteriaceae. Researchers observed that babies harbored a large number of bacterial species, and they had a rapid turnover of bacterial strains in their microbiota resulting from high bacterial exposure in the environment (7,8). A clear understanding of the colonization process of neonates guts in developing countries at the present time is somewhat difficult, as the few available studies were mostly performed more than a decade ago using culture techniques (7,8).

INTRODUCTION Microbial colonization of the gastrointestinal tract begins immediately after birth and is carried out by microbes that are derived from the mother and the environment. The gut, during colonization, has classically been described as being initially dominated by a range of facultative bacteria, such as Enterobacteriaceae, Streptococcus, and Staphylococcus. As the available oxygen is consumed, strict anaerobes, including Bifidobacterium, Bacteroides, and Clostridium (1), proliferate.

The course of microbial colonization of the newborn gut is determined by a complex interaction of factors, including the mode of delivery, feeding practice, and the bacterial load in the environment (1). In developed countries decades ago, the

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

113

Fecal microbiota in Brazilian neonates Brandt K et al.

CLINICS 2012;67(2):113-123

and running water, although the access to water in each home varied. The sewer system was inadequate. Only families who lived in communities located in the neighborhoods near the HU-USP and who were referred to this center for medical follow-up were enrolled in the study.

Three fecal samples were collected from each newborn.

The first was collected on the 2nd day of life, while the newborn was still in the hospital. The second and the third samples were collected at home (by the mother) on the 7th and 30th days after birth, respectively. A medical appointment was scheduled for these time periods (the 7th and 30th days). The mothers were instructed to collect the fecal sample immediately after elimination with a standardized spoon, to place it in a sterile plastic container, and to keep it in a freezer (-4oC) until the appointment, which occurred later on the same day. Samples were transported inside a polystyrene container filled with ice. Despite their low socio-economic status, all of the families possessed a freezer coupled to a refrigerator and were capable of producing ice for this transport. Because of their geographic proximity, they took less than one hour to deliver the samples to the hospital.

One of the ten children who participated in the study required the use of a 10-day oral cephalexin treatment because of a skin infection that began on the 7th day of life.

No 7th-day fecal sample was collected from this child. The 2nd- and 30th-day samples were collected, and the microbial profile of this child is described separately from those of the other children in the group.

Studies on the microbial colonization of the gut performed using culture techniques may be limited because some bacteria are believed to be unculturable (10).

Molecular techniques are able to overcome this limitation because they permit microbes to be identified from the genetic material present in the sample, with no dependence on cultivation. The techniques are mostly based on the study of 16S rRNA, the bacterial small ribosomal subunit RNA. The 16S rRNA contains regions of nucleotide sequences that are highly conserved across the bacterial world, interspersed by variable and hypervariable regions that allow for phylogenetic analyses of the microorganisms (10).

Colonization studies performed in developed countries using molecular methods have permitted more detailed and dynamic analyses of the newborn gut colonization process.

Some molecular studies showed colonization patterns similar to those that have been classically described, with the earliest colonizers identified as aerobes, including Staphylococcus, Streptococcus, and Enterobacteriaceae, and later colonizers tending to be strict anaerobes, such as Eubacterium and Clostridia (11). Others observed divergent initial bacterial colonization events, such as the predominance of the anaerobic species Clostridium in the first days of life (12) and the important participation of previously unidentified bacterial groups, such as the anaerobe Ruminococcus (12,13).

Different molecular techniques can be applied to the study of intestinal microbiota, depending on the question being addressed. When the objective is to explore the biodiversity of a sample, construction of a 16S rRNA library can be used (10). Unfortunately, this process may suffer limitations, especially related to the PCR stage (14). The valuable information obtained by the construction of a 16S rRNA library fully justifies its potential pitfalls, but it is important to analyze the results in a judicious manner (15).

This study is the first to use a molecular approach to analyze the gut colonization process of a group of exclusively breastfed neonates born in Sa˜o Paulo, Brazil, and living in a low socio-economic environment. In one child, the microbial events associated with the use of an antibiotic were also assessed.

DNA extraction and 16S rRNA amplification DNA was extracted from stools using the QIAamp DNA Stool Mini-Kit (Qiagen, Canada) according to the manufacturer’s instructions. Two bacteria-specific primers, 27F (59AGA GTT TGA TCC TGG CTC AG 39) and 1492R (59 GGT TAC CTT GTT ACG ACT T 39), were used to amplify the 16S rRNA gene coding region (16). Amplification reactions were performed in a total volume of 100 mL containing 180 ng of DNA that was extracted from each fecal sample. The PCR amplifications were performed using the following procedure: 94 ˚C for 5 min, followed by 35 cycles of 94 ˚C for 45 sec, 62 ˚C for 1 min, 68 ˚C for 2 min, and a final extension period of 68 ˚C for 10 min. The amplified 16S rRNA was purified using the UltraClean PCR kit (Invitrogen, USA).

MATERIALS AND METHODS Study group and sample collection The study was carried out from June through November of 2007, in the city of Sa˜o Paulo, Brazil. The study group consisted of ten newborns. All of the mothers underwent prenatal care at a health center at the University of Sa˜o Paulo. They were well nourished and healthy, and they had not taken any antibiotics during the last month of gestation.

The babies were vaginally delivered at the University Hospital of the University of Sa˜o Paulo (HU-USP). The newborns had uneventful births and had no associated morbidities; they were full-term and had adequate birth weights. After delivery, they remained in the hospital for 48 hours without incident. During the hospital stay, they did not receive any medication or supplemental fluids.

Exclusive breastfeeding was established from birth and continued throughout the research period.

The families belonged to low socio-economic communities. In general, the parents had little schooling and low incomes; families lived in multi-habitation homes, and some of them lived in slums. These communities had electricity

Cloning of the 16S rRNA library The purified PCR products were ligated into a pCRH2.1 TOPO plasmid vector and then transformed into competent E. coli cells using an Original TA Cloning Kit (Invitrogen, USA). 16S rRNA libraries were constructed for each time point (2nd, 7th, and 30th days). After the bacterial transformation, approximately 90 clones from each child at each time point were selected, and their lysates were used as the template in an amplification reaction using the M13 primers supplied by the cloning kit.

DNA sequencing and phylogenetic analysis The amplicons obtained as described above were purified using the UltraClean PCR kit. The products were analyzed using an automated MegaBACE 1000 DNA Analysis System (GE Healthcare, USA) and Cimarron 3.12 Base Caller (Cimarron Software Inc., USA), and the sequencing reactions were performed using the T7 primer supplied by the

114

CLINICS 2012;67(2):113-123

Fecal microbiota in Brazilian neonates Brandt K et al.

To determine the rrn copy number in the fecal samples, standard curves were calculated by correlating the rrn gene copy number and the threshold cycle (Ct) values in a tenfold serial dilution of Bifidobacterium animalis subsp. lactis HN019. The data quantification was expressed as the range of the values encountered in the group and the mean.

cloning kit. Sequencing quality was analyzed using the PRED17 online software, and nucleotides with a Phred score of less than 10 were discarded. Qualified sequences were manually edited using the BioEdit software, v7.0.5.3 (18) to remove the vector and primer sequences. Chimeric artifacts were checked by the Chimera Slayer algorithm in the Mothur program, v1.13.0 (19) using a Silva aligned database as a reference. The phylogenetic affiliations of the resulting sequences (approximately 450 bp and larger) were determined using the Classifier program of the Ribosomal Database Project (RDP) (20) and were confirmed by BLASTn comparison using the NCBI GenBank database.

The Mothur program was used to perform the rarefaction analysis and estimation of the microbial diversity using operational taxonomic unit (OTU)-based approaches (ACE and Chao1 richness estimators, Shannon and Simpson diversity indices) and hypothesis-based approaches (Libshuff statistics and UniFrac distance comparisons) (19). The sequences were aligned using the Silva 102 SSU reference database as a template (21), and a Phylip distance matrix file was generated to cluster the sequences into OTUs. The diversity analyses at the family and genera levels were based on definitions of OTUs with at least 90 and 95% sequence similarity, respectively. A Venn diagram was also drawn to show the number of shared and unique OTUs among each sample. Phylogenetic trees were constructed using the ARB software (22) with the neighbor-joining method and Felsenstein correction. A phylogenetic tree with only the non-chimeric clone sequences was subjected to the weighted and unweighted UniFrac tests, which tested the hypothesis that a group of sequences (that is, branches of a phylogenetic tree) may be unique to any community.

Nucleotide sequence accession number The nucleotide sequences reported in this paper will appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases with the following accession numbers: 2nd day - GU145617 to GU146046, 7th day - GU131996 to GU132306, and 30th day - GU229292 to GU229644.

Ethical consideration This study was approved by the Ethics Committee of the HU-USP (under registration number 574/05). The mothers of all of the infants enrolled in the research signed an informed consent form.

RESULTS Data related to the individuals The studied neonates had a mean gestational age of 39.6¡1.6 weeks (range 37.3 to 41.6 weeks) and a mean birth weight of 3,328¡388 g (range 2,725 to 3,560 g). Table 1 displays the characteristics of the neonates and their antibiotic use.

16S rRNA libraries Approximately 2,610 clones were obtained to construct the 16S rRNA gene library. A total of 2,419 clones were sequenced. After excluding the low-quality or chimeric sequences, 1,765 sequences, each encompassing approximately 450 bp from the 59 end of the 16S rRNA gene (Table 2), were gathered and used to analyze the prevalence and relative abundance of microbes at each time point (Table 3, Figures 1 and 2). Rarefaction curves and the diversity estimation were constructed using only the forward-aligned sequences (1,077 sequences, Table 2). The rarefaction curves (Figure 3) illustrate the number of sequences that were acquired and the number of OTUs that these sequences represent at a species-level cutoff ($97% similarity). Although the clone library coverage was high for all of the samples (.96%, Table 2), which indicates

Data presentation The data from nine of the ten neonates in the study, who progressed without incidents throughout the research period, were analyzed as a group (Group data). The data from the other neonate, who had received an antibiotic, were analyzed separately (Antibiotic exposure data).

The relative abundance of bacterial genus encountered in the fecal molecular analysis was calculated as the percentage of sequences obtained for a specific bacterial genus in relation to the total number of sequences recovered at each time point. The distribution of the abundances of each genus was graphically presented. The prevalence was calculated as the percentage of neonates in the group that harbored a specific bacterial genus.

Table 1 - Data showing the general characteristics and antibiotic use of the neonates.

Real-time PCR for Bifidobacteria Bifidobacterium was not detected in the fecal samples using the 16S rRNA library technique. Therefore, a real-time PCR reaction for Bifidobacterium spp. was conducted on the 30thday fecal samples for further investigation. The primers and probe were the same as those validated by Penders et al.

(23). The amplification reaction was conducted in a total volume of 50 mL, containing 1x TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA), 300 nmol/L of both primers, 150 nmol/L TaqMan probe, and 0.2 ng of the purified target DNA. The amplification reactions were performed using the following procedure: 50 ˚C for 2 min, 95 ˚C for 10 min, and 42 cycles each of 95 ˚C for 15 seconds and 60 ˚C for 1 min. PCR products were detected with an Applied Biosystems Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA).

Gestational age (weeks) 1 2 3 4 5 6 7 8 9 10

40.5 40.1 41.1 41.6 37.6 39.3 37.3 40 40.6 38.1

* Cephalexin for 10 days.

M = male.

F = female.

115

Birth weight 3.560 2.870 2.765 3.725 3.030 3.300 3.210 3.290 3.520 4.015

g g g g g g g g g g

Gender

Antibiotic use

F F F M M F M M M M

No No Yes* No No No No No No No

Fecal microbiota in Brazilian neonates Brandt K et al.

CLINICS 2012;67(2):113-123

Table 2 - Richness estimators, diversity indices, and coverage of bacterial 16S rRNA gene sequences from 2-, 7-, and 30day-old neonates (the OTU was defined as a $95% similarity cutoff).

Sample

No. of sequencesa

2-day-old 7-day-old 30-day-old

Richness estimators

OTUs

421 304 352

28 17 27

Diversity indices

Coverage (%)

ACE

Chao1

Simpson

Shannon

67 18 31

48 17 29

0.440 0.246 0.132

1.516 1.891 2.437

96.9 99.9 98.5

a – the number of forward-oriented clone inserts (59-39) after excluding those of weak quality and suspected chimeras.

0.05/6 = 0.00833), which indicates that each neonate microbial community had a different structure. In agreement with these results, both the unweighted and weighted UniFrac tests indicated that each sample had a different community structure (significance values ,0.001 for each sample and a critical threshold of 0.05/3 = 0.01666 for each test).

that the sampling effort was sufficient to cover the community diversity, only the 7th-day sample rarefaction curve showed a slight plateau (Figure 3). Nevertheless, the slope of the 7th-day curve was shallower than the slope of the curves obtained for the 2nd and 30th days, which suggests that greater numbers of OTUs are expected in these two libraries.

Richness estimates obtained by both ACE and Chao1 were in agreement, demonstrating that the microbiota of the 2nd-day group had a greater richness than those of the other time points (Table 2). Interestingly, both the Simpson and Shannon indices showed the lowest diversity in the 2nd-day group and the highest diversity in the 30th-day group (Table 2). In fact, the large number of clones similar to Escherichia found in the 2nd-day group of fecal material accounted for the dominance measured by the diversity indices, as shown in the compositional analysis. On the other hand, the microbiota of the 30-day-old neonates had a different community structure, composed of a smaller number of bacteria distributed more equally among communities; that is, these communities appeared less dominant. A detailed analysis of the OTUs found in each sample group (Table 2) is shown in a Venn diagram (Figure 4). The 2nd-day sample group had the greatest number of unique sequences (14 OTUs), followed by the 30th-day (10 OTUs), and 7th-day (4 OTUs) sample groups. Nevertheless, approximately 12.7% of the gut species (6 OTUs) were shared among all time points, which suggests that these genera were present throughout the first month of life.

Analysis by hypothesis-based approaches, which tests if the microbial communities have the same structure by chance, revealed significant differences among each group.

The Libshuff test resulted in significance values (,0.0001 for each sample comparison) below the critical threshold (i.e.,

Community composition analysis A detailed, genus-level analysis of the OTU affiliations, as determined by the RDP Classifier and BLASTn matches, is given in Figure 1. All groups of OTUs revealed members of the phyla Firmicutes, Bacteroidetes, and Proteobacteria (Figure 5).

Group data 2nd-day analysis. A total of 606 sequences were obtained from nine neonate fecal samples (Tables 2 and 3). In the initial 16S rRNA library profile, Escherichia predominated (Figure 1), with a relative abundance of 61% and a prevalence of 100% (Table 3). Other facultative anaerobes were observed, including gram-negative enterobacteria, namely, Enterobacter (44% prevalence, 9% relative abundance) and Klebsiella (22% prevalence, 1% relative abundance), and gram-positive cocci, namely, Streptococcus (56% prevalence, 7% relative abundance) and Staphylococcus (22% prevalence, 2% relative abundance). The strict anaerobes, Bacteroides (33% prevalence, 10% relative abundance) and Clostridium (44% prevalence, 3% relative abundance), were already present in these early days of life.

7th-day analysis. A total of 478 sequences were obtained from nine neonate fecal samples (Tables 2 and 3). By this time point, Escherichia had become less predominant, but it still accounted for the highest number of sequences as it was responsible for 47% of the sequences obtained and occurred in

Table 3 - Relative abundances and prevalences of bacterial genera identified in fecal samples of nine neonates throughout the first month of life.

2nd day (n = 9)

Bacterial genus P*

Bacteroides Clostridium Escherichia Enterobacter Klebsiella Lactobacillus Staphylococcus Streptococcus Uncultured Bacteria Veillonella Others

RA**

7th day (n = 9) P*

30th day (n = 9) RA**

P*

RA**

%

%

%

%

%

%

33 44 100 44 22 11 22 56 60 22 25

10 3 61 9 1 1 2 7 3 1 2

22 22 78 0 22 22 33 44 78 44 44

4 1 47 0 2 3 10 13 10 7 3

33 56 67 33 44 33 0 44 80 44 44

10 33 31 1 2 5 0 6 5 5 2

116

CLINICS 2012;67(2):113-123

Fecal microbiota in Brazilian neonates Brandt K et al.

Figure 1 - Bacterial profile of the 16S rRNA library constructed with fecal samples from a group of nine neonates who were not given antibiotics. The sequences were grouped into genera. The data shown are pooled per time point.

Figure 2 - Bacterial profile of the 16S rRNA library constructed with fecal samples from one child who used an oral antibiotic for 10 days. The sequences were grouped into genera. The data are shown per time point.

117

Fecal microbiota in Brazilian neonates Brandt K et al.

CLINICS 2012;67(2):113-123

Figure 3 - Rarefaction curves from the 16S rRNA libraries constructed at each time point, including sequences obtained from the neonate who received antibiotic treatment. An OTU is defined as having $95% similarity.

78% of the neonates. Other enterobacteria (Klebsiella and Enterobacter) were observed in individual neonates at a low abundance. By the 7th day, the facultative anaerobe Streptococcus was still identified in a larger proportion of neonates than Staphylococcus (prevalence of 44% versus 33%), but the two species were similarly abundant (13% versus 10%).

The anaerobic components, Bacteroides and Clostridium, were

infrequently detected; both anaerobes were identified in 22% of the neonates, with relative abundances of 4% and 1%, respectively. Veillonella was more frequently identified in this analysis (prevalence of 44% and relative abundance of 7%). The anaerobic lactic acid bacterium Lactobacillus was detected in 22% of the samples at a low relative abundance (3%). Other microorganisms that were less frequently identified included Acinetobacter and Parabacteroides, which were detected in different neonates. At this time point, a number of OTUs with genetic similarity to the clones designated ‘‘uncultured bacterium’’ in the data bank were detected in 78% of the samples, and this prevalence was higher than those at other time points.

30th-day analysis. A total of 506 sequences were obtained from nine neonate fecal samples (Tables 2 and 3). Overall, a more uniform distribution of abundances was observed (Figure 1). Clostridium joined Escherichia as the main genera encountered at this time point. Both were of similar relative abundance (Clostridium 33% and Escherichia 31%), and both were detected in a significant number of neonates (the prevalence of Clostridium was 56% and that of Escherichia was 67%). Other enterobacteria were found at a low abundance: Klebsiella was identified in 44% of the neonates with a relative abundance of 2%, and Enterobacter was detected in 33% of the neonates with a relative abundance of 1%. Streptococcus could be identified in 44% of the neonates with a relative abundance of 6%, but sequences related to Staphylococcus were not identified at this time point. The anaerobic components dominated this library for this time point (in total, they were responsible for 58% of the sequences). Apart from Clostridium, Bacteroides could be detected in 33% of the neonates with a relative abundance of 10%; Veillonella was more predominant, being detected in 44% of the neonates with a relative abundance of 5%; and Lactobacillus was moderately better represented in this 30thday library than in the libraries from earlier time points, being observed in 33% of the neonates with a relative abundance of 5%. Other microorganisms that could be identified were Acinetobacter baumanii, Citrobacter, Enterococcus, and Prevotella.

Figure 4 - Venn diagram showing the number of unique and shared OTUs among the neonates. A genus-based OTU definition was used ($95% similarity cutoff).

118

CLINICS 2012;67(2):113-123

Fecal microbiota in Brazilian neonates Brandt K et al.

Figure 5 - Phylogenetic relationships among the OTUs detected in newborn fecal samples. Black branches represent the 2-day-old samples, white branches represent 7-day-old samples and gray branches represent 30-day-old samples. Reference sequences are indicated by their taxonomic names, and uncultured clones are followed by their GenBank accession numbers.

main phylogenetic group identified (Figure 2), responsible for 67% of the sequences (versus 64% encountered in the nonantibiotic group). As for the anaerobic components, Bacteroides was also the major anaerobe identified, but Clostridium was not detected (Figure 2). A small percentage of sequences (6%) were attributed to uncultured bacteria.

Antibiotic exposure data

2nd-day analysis. A total of 106 sequences were obtained from the second-day fecal sample of the child who later underwent antibiotic treatment. The bacterial profile of the 16S rRNA library at this initial time point was similar to those observed for the remainder of the group. Escherichia was the

119

Fecal microbiota in Brazilian neonates Brandt K et al.

CLINICS 2012;67(2):113-123

Richness estimates obtained by both the ACE and Chao1 indices and diversity estimates obtained by both the Simpson and Shannon indices showed the lowest diversity in the second-day group and the highest diversity in the 30th-day group. These results demonstrate that the neonates’ microbiota shifted from a simple to a more complex bacterial profile. It has previously been demonstrated that the microbiota of infants grow in complexity of microbial communities until a stable, adult-type microbiota is achieved (24). Each neonate microbial community had a different structure and was composed of members of the phyla Firmicutes, Bacteroidetes, and Proteobacteria. This characteristic of an individual microbiota pattern, or inter-individual variability, has been described for the first days of life (24,25).

This variability must reflect the consequences of the selective forces that act inside each individual (e.g. number of bacterial receptors in the gut and each ones particular gastrointestinal physiology, gastric acid, peristalsis, bile flow), the impact of the environment and the inter-bacterial arrangements (1). The importance of each individual microbe to the host must be better understood. Although primordial microbial communities are vulnerable to the influences of external environment, ecological stability is achieved by approximately two years of age. Furthermore, although individuals are still exposed to environmental microbes after this age, further attempts to interfere with the composition of the microbial community will produce only transient effects (26).

Although individual characteristics were noted, a tendency toward a pattern of microbial colonization existed, and the findings were further considered in a group manner. When the bacterial profiles of the neonates’ fecal microbiota were analyzed as a group, Escherichia stood out as a major bacterial group at all time points. In the second-day samples, clones derived from Escherichia could be identified in all of the neonates; this microorganism accounted for 61% of the clones obtained. In subsequent samples, its relative abundance declined somewhat. However, even though other bacterial groups had become more abundant in the 30th-day samples, Escherichia was one of the major phylogenetic groups in the library. This high degree of enterobacteria colonization observed throughout the first month of life in these Brazilian neonates was observed in other studies conducted in newborns living in developing countries (7,9,27) and is possibly related to a high degree of environmental exposure and contamination. In a study that analyzed the colonization pattern of Pakistani babies, a massive colonization by enterobacteria was found to have already occurred in the first days of life, and this colonization persisted throughout the first few months (7). The environment was identified as the main source of contamination because even though all of the infants had been vaginally delivered at home, fewer than 50% of the Escherichia strains matched maternal strains, and most of the strains could be found in the immediate home environment (7).

This high rate of enterobacteria colonization, particularly in the first days of life, was also described in initial studies that analyzed the microbiota colonization pattern of neonates living in developed countries (2,28). However, in later years, some authors have asserted that this initial enterobacteria predominance would have greatly declined in neonates living in developed countries (4,5,29,30). Nowrouzian et al.

(30) observed that in a group of Swedish neonates, fewer than 50% were colonized by Escherichia by one week of age. This change in colonization may have occurred as a result of

Table 4 - Bifidobacterium counts obtained through realtime PCR in the 30-day-old samples.

Infant#

rrn copy number (ÂĄSD)

1 2 3 4 5 6 7 8 9 10

7.3861011 3.3761011 2.9561011 8.4461011 1.4661012 1.0261012 8.2161011 6.7361011 9.4961011 5.6161010

(1.01) (4.98) (7.51) (4.56) (9.67) (3.37) (1.95) (8,55) (1.45) (2.68)

SD: standard deviation of the means of experimental results.

7th-day analysis. The 7th-day fecal sample was not collected as the child could not attend the appointment on this day.

30th-day analysis. Sixty-nine sequences were obtained from the 30th-day fecal sample. The microbial profile after antibiotic use showed a significant decline in the relative abundance of Escherichia, which accounted for only 4% of the sequences. Instead, Klebsiella was responsible for a greater proportion of sequences obtained (28%). Anaerobic components were responsible for 35% of the clones, but they did not represent the major phylogenetic group of the library, as was observed in the remainder of the group at this time point. Bacteroides was the main anaerobe identified, and Clostridium was detected at low rates. The proportion of uncultured microorganisms increased; they were detected at a relative abundance of 23%.

Real-time PCR of Bifidobacteria Real-time PCR of Bifidobacterium detected this microorganism in all neonate stools from the 30th day, even though it was absent from the 16S rRNA library. Quantification revealed counts that ranged from 5.6161010 to 1.4661012 copies per mg of DNA (median value of 7.20x1011 copies per mg of DNA) in the group of children that did not receive antibiotics and 2.9561011 copies per mg of DNA in the feces of the child who underwent antibiotic treatment (Table 4).

DISCUSSION The study population represented a healthy group of neonates who were not submitted to any medical intervention and who were exposed to the average environmental conditions present in poor communities. These characteristics must be emphasized because some published data are from heterogeneous groups of neonates, which complicates interpretation of the observed outcomes. Data from the one baby who received an antibiotic were analyzed separately because this intervention had the potential to affect normal microbial establishment. All babies were vaginally delivered and exclusively breastfed, two factors that strongly influence the colonization process.

Taking into account this scenario and the need to fully describe the microbial characteristics of this group of Brazilian neonates, the 16S rRNA clone library methodology was applied, rather than high-throughput sequencing methods (24,25). This method allowed the characterization of the most abundant organisms, despite the fact that this approach may result in a lower assessment of the bacterial diversity.

120

CLINICS 2012;67(2):113-123

Fecal microbiota in Brazilian neonates Brandt K et al.

An intriguing feature of the constructed libraries was that no Bifidobacterium could be identified at any of the analyzed time points. Although some studies (37) have reported low rates of Bifidobacterium even in breastfed neonates, high Bifidobacterium levels are expected in exclusively breastfed infants owing to particular features (acid profile, iron content, and bifidogenic factors) of human milk that favor Bifidobacterium growth to the detriment of other microorganisms (34), and many studies have provided evidence for the differential high levels of Bifidobacterium colonization in breastfed neonates (23,25,29). Other studies have concluded that the finding of appropriate levels of Bifidobacterium might be related to methodological aspects of each particular study and that the particular approach used can alter whether high or low levels of Bifidobacterium are detected (25).

In view of this unexpected result, another molecular approach was used to investigate, in a more sensitive and specific manner, the occurrence of Bifidobacterium in the 30thday samples. A specific probe and primers for Bifidobacterium sp. were used in a real-time PCR reaction, which allowed the detection and quantification of such bacteria.

This technique allowed detection of Bifidobacterium in the samples from all of the children at one month of age.

Quantification of the samples revealed average counts of 7.20x1011 copies per mg of DNA. Thus, frequent colonization and high population levels of Bifidobacterium were in fact detected in these breastfed neonates, in accordance with other studies (23,29).

In a study that used culture techniques to analyze the difference in Bifidobacterium and Lactobacilli populations among Brazilian students from two different socioeconomic groups, de Mello et al. (38) observed lower numbers of Bifidobacterium and Lactobacilli in the children living in slums compared with those who attended private schools. The authors suggested that these microbial populations might have been affected by the unfavorable slum environment. A possible explanation for the divergent finding of low Bifidobacterium levels in children exposed to poor environment in Brazil observed by de Mello GQ et al. (38) in Brazilian students living in slums could be the fact that those children were older and had thus been subjected to environmental aggressions over a longer period of time, in the absence of the protective and bifidogenic effect of breastfeeding.

The inability of our molecular approach to amplify Bifidobacterium DNA from the samples can be explained in view of the inevitable bias involved in PCR-dependent methodologies (14). Bifidobacterium was also not identified in some other studies that used the 16S rRNA library methodology to analyze intestinal microbiota (16,24); therefore, the ability of this molecular technique to adequately detect the Bifidobacterium genus must be questioned.

The ecological structure that forms inside the gut can be disrupted by the use of antibiotics, which can suppress susceptible bacterial groups and favor the overgrowth of others (8). The future impact of this initial microbial imbalance is not clear, but it is possible that the use of antibiotics early in life may affect the immune regulation process (39). The impact of antibiotic use was investigated through the follow-up of one child who required the use of cephalosporin during the research period. It is known that different antibiotics affect microbiota components in different ways. Approximately ten days after antibiotic use, the microbiota profile of the antibiotic-treated child revealed

development and improved hygiene practices that alter the diversity and abundance of the microbes to which the neonates are exposed.

Moreover, in association with this phenomenon, an increased colonization rate by Staphylococcus was also detected (4-6). The source of the Staphylococcus found among infants’ intestinal microbiota was predominantly the parents’ skin and not the environment (6). It has been suggested that in the absence of enterobacterial competition, Staphylococcus proliferation would be favored (4). Interestingly, in the present study, clones derived from Staphylococcus were identified in few individuals and at low abundance. It is possible that the opposite to the Staphylococcus favoring phenomena described before has occurred in the microbiota of these Brazilian neonates, whereby the maintenance of a large enterobacterial population inhibited Staphylococcus proliferation. The high prevalence and relative abundance of clones attributed to Clostridium in the 30th-day analysis were also of interest. Clostridium clones could be identified in 60% of the neonates. This finding is not usually reported in exclusively breastfed neonates, in whom low rates of colonization have been described (23,31).

In 1,032 fecal samples from one-month-old Dutch infants, Clostridium was detected in only 21% of those who were breastfed and only at low levels (4.5 log10 CFU) (23).

Newborn colonization by Clostridium is attributed to a lesser extent to mother-to-child transmission and is mainly caused by environmental contamination and transfer via contaminated hands (32). The Clostridium colonization frequency has been correlated with the degree of contamination by this microorganism in the environment to which the child is exposed (33). It is possible that the neonates enrolled in this investigation were exposed to a high load of Clostridium in their domestic surroundings and that poor hygiene conditions favored contamination. Considering the fact that these neonates were exclusively breastfed, and knowing that being fed by human milk creates an intestinal milieu that is not favorable for Clostridium proliferation (34), higher colonization would be expected if they had not been breastfed.

This microbial pattern of persistently high levels of Escherichia without over-proliferation of Staphylococcus and relatively high rates of Clostridium colonization, unlike those reported in developed countries (4), might reflect the impact of environmental contamination on intestinal colonization.

The effect of the environmental microbial load to which an individual is exposed, and the microbial composition of this load, on the development of the immune system is debated in the ‘‘hygiene hypothesis’’ (35). According to the hygiene hypothesis, the stricter hygiene practices adopted in developed countries may modify the initial microbial exposure, with a negative impact upon immune regulation, possibly leading to the greater incidences of allergic and autoimmune diseases observed in these countries (35).

Studies have provided evidence for the role of the initial intestinal colonizers in the development of the naı¨ve immune system and shown that the sequence of bacterial events that occurs during colonization of the gastrointestinal tract may affect the host’s future health (36). The challenge is to fully understand which microbial events (their timing and composition) are able to modify the host’s future health and whether interventions in events that occur in the beginnings of life, could favor the establishment of a more beneficial microbiota.

121

Fecal microbiota in Brazilian neonates Brandt K et al.

CLINICS 2012;67(2):113-123

Takagi EH contributed to the study design, execution of experiments, data analysis and bioinformatics results. Oliveira FF contributed to the execution of experiments and data analysis. Irino I contributed to the execution of experiments. Duarte RTD contributed to the data analysis and bioinformatics results. Martinez MB designed the study, performed the data analysis and prepared the manuscript. Carneiro-Sampaio M conceived the original study, contributed to the data analysis and supervised the preparation of the manuscript.

changes in the bacterial communities compared with the remainder of the group. These differences could represent an effect of the antibiotic use. A significant reduction in the relative abundance of Escherichia was observed. This microorganism was found at levels 7.5 times lower than those encountered in the remaining children. In turn, Klebsiella was found at 14-fold higher levels. This change in the enterobacterial profile, with a replacement of Escherichia by an overgrowth of Klebsiella, has been highlighted by other researchers who analyzed the impact of similar antibiotic groups on the composition of children’s microbiota (8). The prevalence of anaerobic microorganisms may also have been reduced because of the antibiotic exposure. In contrast with the findings for the non-antibiotic user group, anaerobic bacteria were not detected as a predominant group over the aerobic components. This effect of antibiotics on the anaerobic bacteria components was also described by previous researchers, who reported a significant impact of antibiotics in reducing Bifidobacterium levels (8). The effects of alterations in Bifidobacterium levels could not be clearly established, as Bifidobacterium were encountered in the 30th-day samples at a level somewhat lower than that of the group average, although still within the group range, possibly indicating a quick reestablishment favored by breast milk.

Although described in only one child, a disruption of the initial microbial colonization process by the use of an antibiotic is suggested. The long-term impact of this intervention should be taken into account when analyzing the indication for antibiotics early in life because the microbiota composition may change definitively after antibiotic use.

Some studies have shown a tendency for microbiota composition to be restored after antibiotic use, but with some permanent changes (40). The microbiota profile of the antibiotic-treated child revealed changes in the bacterial communities during the first month of life. In the following months, this profile showed a turnover of Escherichia (resulting in 30% of the microbial population) but also became more diverse than the profiles of the children who were not given antibiotics (data not shown), suggesting that the microbial profile was not completely restored.

This molecular approach permitted the analysis of fecal microbiota establishment in a group of neonates from Sa˜o Paulo, Brazil. The observed bacterial predominance of Escherichia and Clostridium may be attributable to a highly contaminated environment, and neonate contamination may have been favored by hygiene habits. The observed pattern of predominance by Escherichia in the first days of life and the low rates of colonization by Staphylococcus differ from the colonization patterns described in developed countries. It is possible that a greater knowledge of these differences might help us to understand the ways in which intestinal microbes can favor adequate regulation of the immune system.

REFERENCES 1. Adlerberth I, Wold AE. Establishment of the gut microbiota in Western infants. Acta Pediatri. 2009;98(2):229-38, http://dx.doi.org/10.1111/ j.1651-2227.2008.01060.x.

2. Gothefors L, Carlsson B, Ahlstedt S, Hanson LA, Winberg J. Influence of maternal gut flora and colostral and cord serum antibodies on presence of Escherichia coli in faeces of the newborn infant. Acta Paediatr Scand.

1976;65(2):225-32, http://dx.doi.org/10.1111/j.1651-2227.1976.tb16542.x.

3. Murono K, Fijita K, Yoshiokawa M, Saijo M, Inyaku F, Kakehashi H, et al.

Acquisition of nonmaternal Enterobacteriaceae by infants delivered in hospitals. J Pediatr. 1993;122(1):120-5.

4. Adlerberth I, Lindberg E, Aberg N, Hesselmar B, Saalman R, Strannega˚rd IL, et al. Reduced enterobacterial and increased staphylococcal colonization of infantile bowel: an effect of hygiene lifestyle?

Pediatr Res 2006;59(1):96-101.

5. Adlerberth I, Strachan DP, Matricardi PM, Ahrne´ S, Orfei L, Aberg N, et al. Gut microbiota and development of atopic eczema in 3 European birth cohorts. J Allergy Clin Immunol. 2006;120(2):343-50.

6. Lindberg E, Adlerberth I, Hesselmar B, Saalman R, Strannega˚rd IL, Aberg N, et al. High rate of transfer of Staphylococcus aureus from parental skin to infant gut flora. J Clin Microbiol. 2004;42(2):530-4, http://dx.doi.org/10.1128/JCM.42.2.530-534.2004.

7. Adlerberth I, Jalil F, Carlsson B, Mellander L, Hanson LA, Larsson P, et al. High turnover rate of Escherichia coli strains in the intestinal flora of infants in Pakistan. Epidemiol Infect. 1998;121(3):587-98, http:// dx.doi.org/10.1017/S0950268898001484.

8. Bennet R, Eriksson M, Nord CE, Zetterstron R. Suppression of aerobic and anaerobic faecal flora in newborns receiving parental gentamicin and ampicillin. Acta Paediatr Scand. 1982;71(4):559-62, http:// dx.doi.org/10.1111/j.1651-2227.1982.tb09474.x.

9. Bennet R, Eriksson M, Tafari N, Nord CE. Intestinal bacteria of newborn Ethiopian infants in relation to antibiotic treatment and colonization by potentially pathogenic gram-negative bacteria. Scand J Infect Dis. 1991;23(1):63-9, http://dx.doi.org/10.3109/00365549109023376.

10. Zoetendal EG, Collier CT, Koike S, Mackie RI, Gaskins HR. Molecular ecological analysis of the gastrointestinal microbiota: a review. J Nutr.

2004;134(2):465-472.

11. Palmer C, Bik EM, DiGiulio DB, Relman DA, Brown PO. Development of the human intestinal microbiota. PLoS Biol. 2007;5(7):1556-72.

12. Favier CF, de Vos WM, Akkermans ADL. Development of bacterial communities in feces of newborn babies. Clin Microbiol. 2003;9(5): 219-29.

13. Wang M, Ahrne´ S, Antonsson M, Molinl G. T-RFLP combined with principal component analysis and 16S rRNA sequencing: an effective strategy for comparison of fecal microbiota in infants of different ages.

J Microbiol Method. 2004;59(1):53-69, http://dx.doi.org/10.1016/ j.mimet.2004.06.002.

14. Wintzingerode F, Go¨bel UB, Stackebrandt E. Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol Rev. 1997;21(3):213-29, http://dx.doi.org/ 10.1111/j.1574-6976.1997.tb00351.x.

15. Drancort M, Bollet C, Carlioz A, Martelin R, Gayral JP, Raoult D. 16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. J Clin Microbiol.

2000;38(10):3623-30.

16. Hayashi H, Sakamoto M, Benno Y. Phylogenetic analysis of the human gut microbiota using 16S rDNA clone libraries and strictly anaerobic culture methods. Microbiol Immunol. 2002;46(8):535-48.

17. Ewing B, Hillier L, Wendl M, Green P. Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 1998;8(3):175-85.

18. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser. 1999;41:95-8.

19. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, et al. Introducing mothur: Open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol. 2009;75(23):7537-41, http:// dx.doi.org/10.1128/AEM.01541-09.

20. Wang Q, Garrity GM, Tiedje JM, Cole JR. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy.

ACKNOWLEDGMENTS We thank Professor Luiz R. Trabulsi (in memorian) for having idealized the original study with Carneiro-Sampaio M.

AUTHOR CONTRIBUTIONS Brandt K conceived the study, performed children selection, sample collection and prepared the manuscript. Taddei CR designed the study, performed the experiments and data analysis and prepared the manuscript.

122

CLINICS 2012;67(2):113-123

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

Fecal microbiota in Brazilian neonates Brandt K et al.

31. Harmsen HJM, Wideboer-Veloo ACM, Raangs GC, Wagendorp AA, Klijn N, Bindels JG, et al. Analysis of intestinal flora development in breastfed and formula-fed infants by using molecular identification on detection methods. J Pediatr Gastroenterol Nutr. 2000;30(1):61-7, http:// dx.doi.org/10.1097/00005176-200001000-00019.

32. Bacon AE, Fekety R, Scaberg DR, Faix RG. Epidemiology of Clostridium difficile colonization in newborns: results using a bacteriophage typing system. J Infect Dis. 1988;158(2):349-54, http://dx.doi.org/10.1093/infdis/ 158.2.349.

33. Matsuki S, Ozaki E, Shozu M, Inoue M, Shimizu S, Yamaguchi N, et al.

Colonization by Clostridium difficille of neonates in hospital, and infants and children in three day-care facilities of Kanazawa, Japan. Int Microbiol. 2005;8(1):43-8.

34. Newburg DS. Oligosaccharides in human milk and bacterial colonization. J Pediatr Gastroenterol Nutr. 2000;30(2):S8-S17.

35. Rautava S, Ruuskanen O, Ouwehand A, Salminen S, Isolauri E. The hygiene hypothesis of atopic disease — an extended version. J Pediatr Gastroenterol Nutr. 2004;38(4):378–88.

36. Kelly D, King T, Aminov R. Importance of microbial colonization of the gut in early life to the development of immunity. Mutat Res. 2007;622(12):58-69.

37. Lundequist B, Nord CE, Winbeg J. The composition of the faecal microflora in breastfed and bottle fed infants from birth to eight weeks. Acta Paediatr Scand. 1985;74(1):45-51, http://dx.doi.org/10.1111/j.1651-2227.1985. tb10919.x.

38. de Mello RMP, de Morais MB, Tahan S, Melli LC, Rodrigues MS, Mello CS, et al. Lactobacilli and bifidobacteria in the feces of schoolchildren of two different socioeconomic groups: children from favela and children from private school. J Pediatr. 2009;85(4):307-14.

39. Kummeling I, Thijs C. Reverse causation and confounding-by-indication: do they or do they not explain the association between childhood antibiotic treatment and subsequent development of respiratory illness?

Clin Exp Allergy. 2008;38(8):1249-51, http://dx.doi.org/10.1111/j.13652222.2008.03047.x.

40. Tanaka S, Kobayashi T, Songjinda P, Tateyama A, Tsubouchi M, Kiyohara C, et al. Infuence of antibiotic exposure in the early postnatal period on the development of intestinal microbiota. FEMS Immunol Med Microbiol.

2009;56:80–87, http://dx.doi.org/10.1111/j.1574-695X.2009.00553.x.

Appl Environ Microbiol. 2007;73(16):5261–7, http://dx.doi.org/10.1128/ AEM.00062-07.

Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig W, Peplies J, et al.

SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res. 2007;35(21):7188-96, http://dx.doi.org/10.1093/nar/gkm864.

Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar, et al.

ARB: a software environment for sequence data. Nucleic Acids Research.

2004;32(4):1363-71, http://dx.doi.org/10.1093/nar/gkh293.

Penders J, Thijs C, Vink C, Stelma FF, Snijders B, Kummeling I, et al.

Factors influencing the composition of the intestinal microbiota in early infancy. Pediatrics.2006;118(2):511-21, http://dx.doi.org/10.1542/ peds.2005-2824.

Palmer C, Bik EM, DiGiulio DB, Helman DA, Brown PO. Development of the human intestinal microbiota. PLoS Biol. 2007;5(7):1556-72.

Roger LC, McCartney AL. Longitudinal investigation of the faecal microbiota of healthy full-term infants using fluorescence in situ hybridization and denaturing gradient gel electrophoresis. Microbiology.

2010;156(Pt 11):3317–28, http://dx.doi.org/10.1099/mic.0.041913-0.

Thompson-Chagoya´n OC, Maldonado J, Gil A. Colonization and impact of disease and other factors on intestinal microbiota. Dig Dis Sci. 2007;52(9):2069-77, http://dx.doi.org/10.1007/s10620-006-9285-z.

Mata LJ, Mejicanos ML, Jimenez F. Studies on the indigenous gastrointestinal flora of Guatemalan children. Am J Clin Nutr.

1972;25(12):1380-90.

Murono K, Fijita K, Yoshiokawa M, Saijo M, Inyaku F, Kakehashi H, et al.

Acquisition of nonmaternal Enterobacteriaceae by infants delivered in hospitals. J Pediatr. 1993;122(1):120-5.

Fallani M, Young D, Scott J, Norin E, Amarri S, Adam R, et al. Intestinal Microbiota of 6-week-old Infants Across Europe: Geographic Influence Beyond Delivery Mode, Breast-feeding, and Antibiotics. J Pediatr Gastroenterol Nutr. 2010;51(11):77-84, http://dx.doi.org/10.1097/ MPG.0b013e3181d1b11e.

Nowrouzian F, Hesselmar B, Saalman R, Strannegard IL, Aberg N, Wold AE, et al. Escherichia coli in infants’ intestinal microflora: colonization rate, strain turnover, and virulence gene carriage. Pediatr Res. 2003;54(1):8-14, http://dx.doi.org/10.1203/01.PDR.0000069843.20655.EE.

123

CLINICS 2012;67(2):125-129

DOI:10.6061/clinics/2012(02)06

CLINICAL SCIENCE

Is the measurement of inferior thyroid artery blood flow velocity by color-flow Doppler ultrasonography useful for differential diagnosis between gestational transient thyrotoxicosis and Graves’ disease? A prospective study ¨ zel,II Selvinaz Velet,I Mehmet Sait Bug˘dacı,III Esra C¸il,I Yu¨ksel AltuntasI Sayid Shafi Zuhur,I Alper O I

Sisli Etfal Training and Research Hospital, Endocrinology and Metabolism Clinic, Istanbul, Turkey. II Sisli Etfal Training and Research Hospital, Radiology Clinic, Istanbul, Turkey. III Sisli Etfal Training and Research Hospital, First Internal Medicine Clinic, Istanbul, Turkey.

OBJECTIVE: To determine the role of peak systolic velocity, end-diastolic velocity and resistance indices of both the right and left inferior thyroid arteries measured by color-flow Doppler ultrasonography for a differential diagnosis between gestational transient thyrotoxicosis and Graves’ disease during pregnancy.

METHODS: The right and left inferior thyroid artery-peak systolic velocity, end-diastolic velocity and resistance indices of 96 patients with thyrotoxicosis (41 with gestational transient thyrotoxicosis, 31 age-matched pregnant patients with Graves’ disease and 24 age- and sex-matched non-pregnant patients with Graves’ disease) and 25 ageand sex-matched healthy euthyroid subjects were assessed with color-flow Doppler ultrasonography.

RESULTS: The right and left inferior thyroid artery-peak systolic and end-diastolic velocities in patients with gestational transient thyrotoxicosis were found to be significantly lower than those of pregnant patients with Graves’ disease and higher than those of healthy euthyroid subjects. However, the right and left inferior thyroid artery peak systolic and end-diastolic velocities in pregnant patients with Graves’ disease were significantly lower than those of non-pregnant patients with Graves’ disease. The right and left inferior thyroid artery peak systolic and end-diastolic velocities were positively correlated with TSH-receptor antibody levels. We found an overlap between the inferior thyroid artery-blood flow velocities in a considerable number of patients with gestational transient thyrotoxicosis and pregnant patients with Graves’ disease.

CONCLUSIONS: This study suggests that the measurement of inferior thyroid artery-blood flow velocities with colorflow Doppler ultrasonography does not have sufficient sensitivity and specificity to be recommended as an initial diagnostic test for a differential diagnosis between gestational transient thyrotoxicosis and Graves’ disease during pregnancy.

KEYWORDS: Gestational transient thyrotoxicosis; Graves’ disease; Inferior thyroid artery; Color-flow Doppler ultrasonography; Pregnancy. ¨ zel A, Velet S, Bug˘dacı MS, C¸il E, Altuntas Y. Is the measurement of inferior thyroid artery blood flow velocity by color-flow Doppler Zuhur SS, O ultrasonography useful for differential diagnosis between gestational transient thyrotoxicosis and Graves’ disease? A prospective study. Clinics.

2012;67(2):125-129.

Received for publication on September 10, 2011; First review completed on September 13, 2011; Accepted for publication on September 30, 2011 E-mail: zuhur744@gmail.com Tel.: 90 212 373 50 00

treated, thyrotoxicosis may result in maternal and fetal complications (1-3). In contrast, gestational transient thyrotoxicosis (GTT) is a non-autoimmune cause of thyrotoxicosis of variable severity that is often associated with hyperemesis gravidarum (HG) (2-4). GTT is defined as transient thyrotoxicosis during early pregnancy with a lack of clinical features of GD, no evidence of hyperthyroidism before pregnancy and an absence of thyroid autoantibodies (2). The etiology of GTT is thought to be related to stimulation of the thyroid gland by human chorionic gonadotropin (hCG) or related molecular variants (2-6).

Apart from rare cases in which persistent and severe clinical

INTRODUCTION Thyrotoxicosis affects up to 0.1% to 0.4% of pregnancies.

Graves’ disease (GD) is the overwhelming autoimmune cause of thyrotoxicosis during pregnancy. Unless it is

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

125

Doppler for thyrotoxicosis in pregnancy Zuhur SS et al.

CLINICS 2012;67(2):125-129

symptoms might require treatment with low doses of antithyroid drugs, usually for a short period of time, GTT often resolves spontaneously before 18 weeks of gestation (1-7).

Other causes of thyrotoxicosis such as toxic multinodular goiter, single toxic adenoma and different forms of destructive thyroiditis are rare during pregnancy. Because nonspecific symptoms of hyperthyroidism may occur normally in pregnancy, the evaluation of thyrotoxicosis in a pregnant woman is difficult. In the presence of thyrotoxicosis with clinical features of GD, the diagnosis is straightforward. However, the differential diagnosis between GD and GTT is difficult in the absence of clinical features of GD. The assessment of TSH-receptor antibody (TRAb) levels is generally useful when investigating thyrotoxicosis of uncertain etiology, including thyrotoxicosis associated with HG (8). Although the new generation of TSH-receptor antibody (TRAb) assays is highly sensitive and specific for discriminating GD from various causes of thyrotoxicosis (9), these assays are not widely available, and cost remains an important factor when considering their use in routine clinical practice. Recently, the measurement of the peak systolic velocity (PSV) of the inferior thyroid artery (ITA) with color-flow Doppler ultrasonography (CFDUSG) was suggested as a sensitive diagnostic tool for the differential diagnosis of thyrotoxicosis (10,11). Previously, Kumar et al. indicated the utility of CFDUSG for the differential diagnosis of thyrotoxicosis during pregnancy (12). However, because of the small number of patients included, Kumar et al. were not able to determine the exact role of CFDUSG for a differential diagnosis between GTT and GD. Therefore, we conducted the present study to evaluate the role of PSV, end-diastolic velocity (EDV) and resistance indices (RIs) of the right and left ITA, measured with CFDUSG, for a differential diagnosis between GTT and GD during pregnancy.

thyroid hormone levels presenting within the first trimester of pregnancy and associated with HG, but self-limiting on follow-up; absence of clinical findings of GD; a normal TRAb level; and absence of a history of hyperthyroidism before pregnancy (13). GD in pregnant patients was defined based on clinical findings, high TRAb and free thyroid hormone levels, decreased TSH levels and presence of a history of hyperthyroidism before pregnancy. GD in nonpregnant patients was defined based on the clinical findings, decreased TSH level, high free thyroid hormone and TRAb levels and high uptake values for 99mTcpertechnetate. Destructive thyroiditis in pregnant women was defined as the presence of thyrotoxicosis lasting for fewer than three months and/or later development of transient hypothyroidism associated with a positive result for thyroid peroxidase and/or thyroglobulin antibodies, normal TRAb levels, decreased echogenicity and heterogeneous appearance of the thyroid parenchyma on USG and low uptake values for 99mTc-pertechnetate in non-pregnant patients with thyrotoxicosis. HG was defined as electrolyte disturbances, ketosis and weight loss of .%5 of the nonpregnant weight (13).

PATIENTS AND METHODS

Doppler ultrasonography

Hormone assays Serum samples to determine TSH, fT3 and fT4 levels for all subjects; TRAb level in all patients with thyrotoxicosis; and b hCG level in all pregnant patients were collected after an overnight fast and sent immediately for laboratory assessment on the day of ultrasonography examination.

Serum TSH, fT3, fT4 and b HCG levels were assessed using a direct chemiluminescence immunoassay (Siemens, ADVIA Centaur XP Immunoassay System, Tarrytown, NY, USA). Serum TRAb levels were assessed with TRAb-FastELISA (Euroimmun AG Seekamp 31, D-23560 Lu¨beck.

Germany) (14).

All thyroid ultrasound examinations were performed by the same radiologist (A.O.), who was blinded to the clinical status of the subjects. A color Doppler ultrasound scanner (Toshiba, Aplio XV, Tokyo, Japan) equipped with a 714 MHz broadband linear array transducer was used. The studies of the right and left ITAs were performed with transverse scanning, in which the vessels crossed the common carotid arteries posteriorly, or with longitudinal scanning of the ascending parts of the arteries, in which the vessels lay parallel to the common carotid arteries. The angle correction cursor was parallel to the direction of flow, and the Doppler angle was kept at or below 60 Ëš. The PSV, EDV and RI values were obtained from the right and left ITA. Maternal thyroid function was monitored with respect to fT3, fT4 and TSH levels at monthly intervals. All pregnant patients diagnosed with GTT were followed up until the normalization of the TSH level without treatment with antithyroid drugs. Antithyroid therapy with propiltiouracil (PTU) was administered after the assessment of thyroid blood flows to all pregnant patients diagnosed with GD, and the patients were followed up until delivery. The aim of the treatment was to maintain the FT3/FT4 in the upper quartile of the normal, non-pregnant range. Non-pregnant patients with GD were treated appropriately. Informed consent was obtained from all participants, and the study was approved by the local ethics committee.

Patients Seventy-eight pregnant women in their first trimester of pregnancy (between 8-12 weeks gestation) who were referred to our center for evaluation of thyrotoxicosis, 24 age-matched non-pregnant patients with GD and 25 ageand sex-matched healthy euthyroid subjects were enrolled in this study. All of the cases were newly diagnosed and had not received antithyroid therapy before inclusion in the study. A detailed history was taken, and all patients with thyrotoxicosis were examined physically for clinical features of GD (ophthalmopathy, a significant goiter, pretibial myxedema and nail changes). Patients with a history of previous thyroid surgery, radioactive iodine ablation therapy and radiation exposure to the neck; patients with single or multinodular goiter on gray-scale ultrasonography (USG); patients with destructive thyroiditis; and patients under treatment with thionamides or L-thyroxine were excluded from the study. Accordingly, six pregnant patients with thyrotoxicosis (two with multiple nodules and one with a single nodule on USG, one with factitious thyrotoxicosis, one with painless thyroiditis and one with a previous history of radiation therapy to the neck for the treatment of childhood lymphoma) were excluded from the study.

Eventually, 72 pregnant patients with thyrotoxicosis (41 with GTT and 31 with GD) were found to be eligible for the study. GTT was defined as suppressed TSH and high free

126

CLINICS 2012;67(2):125-129

Doppler for thyrotoxicosis in pregnancy Zuhur SS et al.

different among the three groups of patients with thyrotoxicosis (p,0.001 for all groups with thyrotoxicosis). The canonical r coefficient of the correlation demonstrated a positive correlation of the right and left ITA-PSV and EDV with the TRAb levels (r = 0.515, p = 0.440 for the right ITA PSV, r = 0.463, p = 0.461 for the left ITA-PSV, r = 0.615, p = 0.356 for the right ITA-EDV and r = 0.526, p = 0.382 for the left ITA-EDV, respectively). However, of the patients with GTT, nine (22%) had right ITA-PSV, 13 (31.7%) had left ITA-PSV, 12 (29.3%) had right ITA-EDV, and 13 (31.7%) had left ITA-EDV above the cutoff values. Similarly, of the pregnant patients with GD, seven (22.6%) had right ITAPSV, eight (25.8%) had left ITA-PSV, eight (25.8%) had right ITA-EDV, and eight (25.8%) had left ITA-EDV below the cutoff values. Hence, an overlap was found among ITA blood flow velocities in a considerable number of patients with GTT and pregnant patients with GD. The most appropriate cutoff values for the right and left ITA-PSV and EDV for differentiating GTT from GD during pregnancy are presented in Table 3.

The TSH, fT3 and fT4 levels returned to normal prepregnancy levels before the 20th week of pregnancy in patients with GTT. However, despite treatment with different doses of PTU, the TSH levels in the 20th week of pregnancy were significantly lower in pregnant patients with GD than in those with GTT (p,0.001). The fT3 and fT4 levels in the 20th week of pregnancy were not different between patients with GTT and pregnant patients with GD (p.0.05) (Table 1). Because the thyroid function returned to normal before the 20th week of pregnancy in patients with GTT, the thyroid function was only followed in pregnant patients with GD beyond the 20th week of pregnancy. The TSH levels also remained reduced after the 30th week of pregnancy in the majority of pregnant patients with GD, irrespective of treatment with PTU.

Statistical analysis Data were analyzed using SPSS version 12.0.0 for Windows (SPSS Inc., Chicago, IL). Scale variables are presented as the mean ¡ standard deviation (mean ¡ SD). Categorical data were evaluated using Chi-square analysis or with Pearson’s correlation and Fisher’s exact tests as appropriate. Student’s t-test was used for a comparison of parametric quantitative data. A one-way analysis of variance (ANOVA) was performed to compare quantitative variables within different groups followed by post-hoc analyses for multiple comparisons (Tamhane’s test). A canonical correlation analysis was used to determine the correlation between the ITA flow velocity and the TRAb level. Patients with GTT were tested to determine a diagnostic cutoff for the right and left ITA-PSV and EDV using receiver-operating-characteristic (ROC) analysis. p,0.05 was considered to be statistically significant.

RESULTS The demographic and laboratory features of the study participants are summarized in Table 1. In the present study, the right and left ITA-PSV and EDV in patients with GTT were lower than those of pregnant patients with GD (p,0.001) and higher than those of euthyroid subjects (p,0.001). However, the right and left ITA-PSV and EDV in pregnant patients with GD were lower than those of nonpregnant patients with GD (p,0.001). The right and left ITA-RI were not different among the four groups of study participants (p.0.05). The mean, minimum and maximum ITA-flow velocities of all groups of study participants are shown in Table 2.

In the current study, 30 (97%) of 31 pregnant patients with GD and 23 (96%) of 24 non-pregnant patients with GD had high TRAb levels. The TRAb levels were also significantly

Table 1 - Demographic information and laboratory values of the study participants.

Age (years) Hyperemesis gravidarum (%) Multiple pregnancy (%) TSH (0.55-4.78 mIU/ml)d,e fT3 (2.3-4.2 pg/ml)b,c fT4 (0.76-1.4 ng/dl)f TRAb (,1 IU/L)a bhCG (mIU/ml) 20 weeks TSH level 20 weeks fT3 level 20 weeks fT4 level 34 weeks TSH level 34 weeks fT3 level 34 weeks fT4 level

patients with GTT

pregnant patients with GD

non-pregnant patients with GD

healthy euthyroid subjects

(n = 41)

(n = 31)

(n = 24)

(n = 25)

29.1¡4.8 73.2 7.3 0.03¡ 0.03 3.94¡0.95 1.73¡ 0.36 0.28¡0.22 111965¡52688 1.05¡0.5 2.73¡0.29 0.92¡0.09 -

29.1¡5.12 19.4 9.7 0.03¡0.07 5.57¡2.21 2.11¡1.25 3.47¡1.43 103043¡65998 0.34¡0.43 2.95¡0.45 0.95¡0.13 0.38¡0.45 2.91¡0.44 0.99¡0.15

29.1¡3.5 0.007¡0.005 25.86¡40.97 5.86¡12.7 14.09¡11.71 -

30.8¡7.1 1.62¡0.85 3.05¡0.388 1.03¡0.13 -

a

p-value

0.29 ,0.001 0.52 0.79 ,0.001 0.71 0.058

p,0.001 when GTT was compared with pregnant patients with GD, when pregnant patients with GD were compared with non-pregnant patients with GD and when GTT was compared with non-pregnant patients with GD. b p = 0.003 when GTT was compared with pregnant patients with GD. c p.0.05 when GTT and pregnant patients with GD were compared with non-pregnant patients with GD. d p,0.001 when GTT was compared with non-pregnant patients with GD. e p.0.05 when GTT was compared with pregnant patients with GD and when pregnant patients were compared with non-pregnant patients with GD. f p.0.05 when GTT was compared with pregnant patients with GD, when pregnant patients were compared with non-pregnant patients with GD and when GTT was compared with non-pregnant patients with GD. GTT: gestational transient thyrotoxicosis, GD: Graves’ disease, TSH: thyroid-stimulating hormone, fT3: free triiodothyronine, fT4: free thyroxine, TRAb: TSH-receptor antibody, b hCG: b human chorionic gonadotropin; values in parentheses indicate normal laboratory reference ranges.

127

Doppler for thyrotoxicosis in pregnancy Zuhur SS et al.

CLINICS 2012;67(2):125-129

Table 2 - The right and left ITA blood flow velocities of the study participants. pregnant patients with GD

patients with GTT

a

Right ITA PSV cm/sec Right ITA EDV cm/seca Right ITA RIb Left ITA PSV cm/seca Left ITA EDV cm/seca Left ITA RIb

non-pregnant patients with GD

healthy euthyroid subjects

(n = 41)

(n = 31)

(n = 24)

(n = 25)

24.3¡7.3 (10.6-42) 10.39¡3.7 (3.1-20) 0.57¡0.07 (0.45-0.71) 24.75¡6.15 (11.4-36.6) 11.43¡3.93 (4.7-21.49) 0.54¡0.074 (0.4-0.8)

37.58¡10.89 (20.9-58.1) 15.9¡6.2 (7-33.6) 0.57¡0.08 (0.37-0.72) 35.19¡11.19 (21-69.7) 15.8¡5.05 (6.9-26.6) 0.54¡0.08 (0.4-0.7)

57.03¡25.7 (11.6-129.8) 24.03¡11.87 (3.9-53.8) 0.58¡0.07 (0.48-0.79) 59.4¡ 22.77(22.4-112.5) 25.97¡11.80 (7.6-58.2) 0.56¡0.07 (0.4-0.7)

17.08¡4.83 (9.4-30) 7.78¡3.07 (4.1-14.4) 0.55¡0.08 (0.43-0.73) 17.37¡ 4.81 (9-27) 8.16¡2.73 (4.4-13.7) 0.53¡0.06 (0.4-0.7)

a

p,0.001 when GTT was compared with healthy euthyroid subjects as well as pregnant and non-pregnant patients with GD, when pregnant patients with GD were compared with non-pregnant patients with GD and when pregnant and non-pregnant patients with GD were compared with healthy euthyroid subjects. b p.0.05 when GTT was compared with healthy euthyroid subjects as well as pregnant and non-pregnant patients with GD, when pregnant patients with GD were compared with non-pregnant patients with GD and when pregnant and non-pregnant patients with GD were compared with healthy euthyroid subjects.

GTT: gestational transient thyrotoxicosis, ITA: inferior thyroid artery, PSV: peak systolic velocity, EDV: end-diastolic velocity, RI: resistance index, R results are presented as mean¡SD; values in parentheses indicate the minimum and maximum ranges.

Pregnancy has a profound effect on the immune system and, as in other autoimmune diseases, the autoantibody titers decrease in autoimmune thyroid diseases during pregnancy (19,20). Although still not fully understood, it is thought that these changes in the immune system occur to protect the fetal allograft during pregnancy (19,20). Although no other studies have compared TRAb levels between pregnant and nonpregnant patients with newly diagnosed GD, the lower serum TRAb levels observed in pregnant compared with non-pregnant patients in this study are most likely related to the immunosuppressive effect of pregnancy.

In the current study, we also found a positive correlation between ITA blood flow velocities and TRAb levels. Ueda et al. previously found that the serum TRAb level was significantly and positively correlated with ITA PSV in patients whose GD had relapsed (21). Baldini et al. also found an association of increased ITA-PSV and high levels of TRAb in the relapsing form of GD and suggested that thyroid hypervascularization was most likely related to the activity of the underlying autoimmune processes (22). As demonstrated in our study, among GD patients, the lower ITA blood flow velocities in pregnant compared with nonpregnant patients may be related to the lower TRAb levels seen in pregnant patients with GD. Presumably, as a result of the lower TRAb levels seen in pregnant patients with GD as well as the stimulation of the thyroid gland stimulation by hCG or its molecular variants in patients with GTT, an overlap occurred between the ITA blood flow velocities measured with CFDUSG. Consequently, the diagnostic value of the ITA blood flow velocity measured with CFDUSG was not high during pregnancy. These results also suggest that high ITA blood flow velocities are neither pathognomonic nor specific for GD and can be seen in GTT.

DISCUSSION Determination of the etiology of thyrotoxicosis during pregnancy is crucial for providing the patient with an appropriate treatment plan. The measurement of ITA-PSV using CFDUSG has proven to be highly sensitive and specific in identifying patients with GD (11), but its capability to discriminate GTT from GD during pregnancy is not well known. According to our findings, the mean right and left ITA-PSV and EDV in patients with GTT was significantly lower than that in pregnant and non-pregnant patients with GD and significantly higher than that in healthy euthyroid subjects, which is a result that has not been found before. This increase in the mean ITA-PSV and EDV in GTT is most likely a result of the stimulation of the thyroid gland by hCG or molecular variants of hCG with higher thyrotrophic activity.

In the present study, the right and left ITA-PSV and EDV were found to be significantly lower in pregnant than in non-pregnant patients with GD. GD is an autoimmune disease, and the TRAb level plays a central role in the pathogenesis of GD (15). In clinical practice, GD can be diagnosed based upon the clinical presentation of the patients; however, TRAb level is useful for establishing a diagnosis in uncertain clinical situations, including the differential diagnosis between GTT and GD during pregnancy (16). In this study, the TRAb level was normal in all patients with GTT, and it was higher than normal in 97% and 96% of pregnant and non-pregnant patients with GD, respectively. This result was consistent with previous reports (17,18). However, similar to ITA-PSV and EDV, the TRAb level among GD patients was significantly lower in pregnant patients than in non-pregnant patients.

Table 3 - The most appropriate cutoff values for the right and left ITA-blood flow velocities for differentiating GTT from GD during pregnancy.

Right ITA PSV Right ITA EDV Left ITA PSV Left ITA EDV

Cutoff value

AUC

29.4 cm/sn 11.95 cm/sn 26.65 cm/sn 12.7 cm/sn

0.844 0.789 0.778 0.750

Sensitivity/Specificity (%) 77.4/78 74.2/70.7 74.2/68.3 74.2/68.3

PPV (%)

NPV (%)

CI (95%)

Diagnostic accuracy (%)

82.1 78.4 77.8 77.8

72.7 65.7 63.9 63.9

0.756-0.932 0.685-0.893 0.668-0.887 0.637-0.864

77 72 70 70

ITA: inferior thyroid artery, PSV: peak systolic velocity, EDV: end-diastolic velocity, AUC: area under the curve, PPV: positive predictive value, NPV: negative predictive value, CI: confidence interval.

128

CLINICS 2012;67(2):125-129

Doppler for thyrotoxicosis in pregnancy Zuhur SS et al.

The main limitation of this study is that all pregnant patients with thyrotoxicosis were diagnosed with GTT and GD based mainly on the TRAb level results. Therefore, it was difficult to compare the ITA blood flow velocity with TRAb level for differential diagnoses between GTT and GD. However, the follow-up data from our study demonstrate that the TRAb level is better than Doppler ultrasonography for the differential diagnosis of these two distinct diseases during pregnancy.

In summary, this study suggests that the measurement of ITA blood flow velocities with CFDUSG does not provide sufficient sensitivity and specificity to be recommended as an initial diagnostic test for differential diagnoses between GTT and GD during pregnancy. CFDUSG may be used as an auxiliary diagnostic tool, but the results should be interpreted with caution and in the context of additional clinical and laboratory findings.

Conflict of interest: The authors of this study declare that they have no conflicts of interest.

8.

9.

10.

11.

12.

13.

14.

AUTHOR CONTRIBUTIONS Zuhur SS contributed to the study design, implementation, data analysis ¨ zel A performed the ultrasonographic and preparation of the manuscript. O examinations and contributed to the study design. Velet S, C ¸ il E recruited and screened the patients. Bug˘daci MS, Altuntas Y contributed to the data analysis and preparation of the manuscript.

15.

16.

17.

REFERENCES 1. Burrow GN. Thyroid function and hyperfunction during gestation.

Endocr Rev. 1993;14:194-202.

2. Goldman AM, Mestman JH. Transient non-autoimmune hyperthyroidism of early pregnancy. J Thyroid Res. 2011; http://dx.doi.org/10.4061/ 2011/142413.

3. Mestman JH. Hyperthyroidism in pregnancy. Best Pract Res Clin Endocrinol Metab. 2004;18:267-88, http://dx.doi.org/10.1016/j.beem.

2004.03.005.

4. Glinoer D. The regulation of thyroid function in pregnancy: pathways of endocrine adaptation from physiology to pathology. Endocr Rev. 1997;18:404-33, http://dx.doi.org/10.1210/er.18.3.404.

5. Glinoer D, De Nayer P, Robyn C, Lejeune B, Kinthaert J, Meuris S. Serum levels of intact human chorionic gonadotropin (hCG) and its free alpha and beta subunits, in relation to maternal thyroid stimulation during normal pregnancy. J Endocrinol Invest. 1993;16:881-8.

6. Kimura M, Amino N, Tamaki H, Ito E, Mitsuda N, Miyai K, et al.

Gestational thyrotoxicosis and hyperemesis gravidarum: possible role of hCG with higher stimulating activity. Clin Endocrinol (Oxf). 1993;38:34550, http://dx.doi.org/10.1111/j.1365-2265.1993.tb00512.x.

7. Goodwin TM, Montoro M, Mestman JH, Pekary AE, Hershman JM. The role of chorionic gonadotropin in transient hyperthyroidism of hyperemesis

18.

19.

20.

21.

22.

129

gravidarum. J Clin Endocrinol Metab. 1992;75:1333-7, http://dx.doi.org/ 10.1210/jc.75.5.1333.

Matthews DC, Syed AA. The role of TSH receptor antibodies in the management of Graves’disease. Eur J Intern Med. 2011;22:213-6, http:// dx.doi.org/10.1016/j.ejim.2011.02.006.

Yoshimura NJ, Miyazaki N, Ito K, Takeda K, Hiramatsu S, Morita S, et al.

Evaluation of a new rapid and fully automated electrochemiluminescence immunoassay for thyrotropin receptor autoantibodies. Thyroid.

2008;18:1157-64, http://dx.doi.org/10.1089/thy.2008.0119.

Erdogan MF, Anil C, Cesur M, Baskal N, Erdogan G. Color flow Doppler sonography for the etiologic diagnosis of hyperthyroidism. Thyroid.

2007;17:223-8, http://dx.doi.org/10.1089/thy.2006.0104.

Hari Kumar KV, Pasupuleti V, Jayaraman M, Abhyuday V, Rayudu BR, Modi KD. Role of thyroid Doppler in differential diagnosis of thyrotoxicosis. Endocr Pract. 2009;15:6-9.

Hari Kumar KV, Vamsikrishna P, Verma A, Muthukrishnan J, Meena U, Modi KD. Evaluation of thyrotoxicosis during pregnancy with color flow Doppler sonography. Int J Gynaecol Obstet. 2008;102(2):152-5, http:// dx.doi.org/10.1016/j.ijgo.2008.03.014.

Abalovich M, Amino N, Barbour LA, Cobin RH, DeGroot LJ, Glinoer D, et al. Management of thyroid dysfunction during pregnancy and postpartum: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2007;92:1-47, http://dx.doi.org/10.1210/jc.2007-0141.

Liu C, Hermsen D, Domberg J, Graeber C, Hautzel H, Duan Y, et al.

Comparison of M22-based ELISA and human-TSH-receptor-based luminescence assay for the measurement of thyrotropin receptor antibodies in patients with thyroid diseases. Horm Metab Res. 2008;40:479-83, http://dx.doi.org/10.1055/s-2008-1077051.

McKenna TJ. Graves’ disease. Lancet. 2001;357:1793–6, http://dx.doi. org/10.1016/S0140-6736(00)04906-0.

Davies TF, Roti E, Braverman LE, DeGroot LJ. Thyroid controversystimulating antibodies. J Clin Endocrinol Metab. 1998;83:3777–85, http:// dx.doi.org/10.1210/jc.83.11.3777.

Vos XG, Smit N, Endert E, Tijssen JG, Wiersinga WM. Frequency and characteristics of TBII-seronegative patients in a population with untreated Graves’ hyperthyroidism: a prospective study. Clin Endocrinol (Oxf).

2008;69:311-7, http://dx.doi.org/10.1111/j.1365-2265.2008.03192.x.

Theodoraki A, Jones G, Parker J, Woolman E, Martin N, Perera S, et al.

Performance of a third generation TSH-receptor antibody (TRAb) in a UK clinic. Clin Endocrinol (Oxf). 2011;75:127-33, http://dx.doi.org/10.1111/ j.1365-2265.2011.04022.x.

Lazarus JH. Hyperthyroidism during pregnancy: etiology, diagnosis and management. Womens Health (Lond Engl). 2005;1:97-104, http:// dx.doi.org/10.2217/17455057.1.1.97.

Kamijo K. TSH-receptor antibodies determined by the first, second and third generation assays and thyroid-stimulating antibody in pregnant patients with Graves’ disease. Endocr J. 2007;54:619-24, http:// dx.doi.org/10.1507/endocrj.K06-196.

Ueda M, Inaba M, Kumeda Y, Nagasaki T, Hiura Y, Tahara H, et al. The significance of thyroid blood flow at the inferior thyroid artery as a predictor for early Graves’ disease relapse. Clin Endocrinol (Oxf).

2005;63:657-62, http://dx.doi.org/10.1111/j.1365-2265.2005.02397.x.

Baldini M, Castagnone D, Rivolta R, Meroni L, Pappalettera M, Cantalamessa L. Thyroid vascularization by color doppler ultrasonography in Graves’ disease. Changes related to different phases and to the long-term outcome of the disease. Thyroid. 1997;7:823-8, http:// dx.doi.org/10.1089/thy.1997.7.823.

CLINICS 2012;67(2):131-134

DOI:10.6061/clinics/2012(02)07

CLINICAL SCIENCE

Liver enzymes in patients with chronic kidney disease undergoing peritoneal dialysis and hemodialysis Isabella Ramos de Oliveira Liberato,I Edmundo Pessoa de Almeida Lopes,I Maria Alina Gomes de Mattos Cavalcante,II Tiago Costa Pinto,I Izolda Fernades Moura,III Luiz Loureiro Ju´niorIII I

Universidade Federal de Pernambuco (UFPE), Department of Internal Medicine, Recife/PE, Brazil. II Nefroclı´nica Dialysis Unit, Recife/PE, Brazil.

Universidade Federal de Pernambuco (UFPE), Hospital das Clı´nicas, Central Laboratory, Recife/PE, Brazil.

III

OBJECTIVE: The present study was designed to analyze the serum levels of aspartate and alanine aminotransferases, gamma-glutamyl transferase, and the hematocrit in patients with chronic kidney disease who were undergoing peritoneal dialysis or hemodialysis.

PATIENTS AND METHODS: Twenty patients on peritoneal dialysis and 40 on hemodialysis were assessed, and the patients were matched according to the length of time that they had been on dialysis. Blood samples were collected (both before and after the session for those on hemodialysis) to measure the enzymes and the hematocrit.

RESULTS: In the samples from the patients who were undergoing peritoneal dialysis, the aspartate and alanine aminotransferase levels were slightly higher compared with the samples collected from the patients before the hemodialysis session and slightly lower compared with the samples collected after the hemodialysis session. The levels of gamma-glutamyl transferase in the hemodialysis patients were slightly higher than the levels in the patients who were undergoing peritoneal dialysis. In addition, the levels of aminotransferases and gamma-glutamyl transferase that were collected before the hemodialysis session were significantly lower than the values collected after the session. The hematocrit levels were significantly lower in the patients who were on peritoneal dialysis compared with the patients on hemodialysis (both before and after the hemodialysis session), and the levels were also significantly lower before hemodialysis compared with after hemodialysis.

CONCLUSION: The aminotransferase levels in the patients who were undergoing peritoneal dialysis were slightly higher compared with the samples collected before the hemodialysis session, whereas the aminotransferase levels were slightly lower compared with the samples collected after the session. The hematocrits and the aminotransferase and gamma-glutamyl transferase levels of the samples collected after the hemodialysis session were significantly higher than the samples collected before the session. Taken together, the present data suggest that hemodilution could alter the serum levels of liver enzymes.

KEYWORDS: Alanine aminotransferase; Liver enzymes; Hemodialysis; Peritoneal dialysis; Chronic renal failure.

Liberato IRO, Lopes EPA, Cavalcante MAGM, Pinto TC, Moura IF, Loureiro Jr L. Liver enzymes in patients with chronic kidney disease undergoing peritoneal dialysis and hemodialysis. Clinics. 2012;67(2):131-134.

Received for publication on August 1, 2011; First review completed on September 28, 2011; Accepted for publication on October 4, 2011 E-mail: epalopes@uol.com.br Tel.: 55 81 2126-8517

Generally, ALT levels are below the upper limit of the normal (ULN) range but higher than the levels found in noninfected HD patients (3-5).

Even in noninfected HCV patients, ALT serum levels are lower in patients with CKD undergoing HD compared with the population that has normal renal function (6,7). ALT levels may be lower because of a deficiency in vitamin B6, which is a coenzyme of ALT, or hemodilution, which occurs because of water retention in patients with CKD before an HD session (8-10).

In regard to patients who do not have CKD, there has been a proposal to reduce the ULN of ALT to approximately 70% of the ‘‘conventional limits’’ when screening for liver abnormalities. Clinicians have argued that the current

INTRODUCTION Patients with chronic kidney disease (CKD) who are undergoing hemodialysis (HD) and are infected with hepatitis C virus (HCV) have been shown to present with lower serum levels of alanine aminotransferase (ALT) than HCV-infected patients with normal renal function (1,2).

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

131

Liver enzymes in patients on dialysis Liberato IRO et al.

CLINICS 2012;67(2):131-134

The patients were excluded if they had previously been diagnosed with hepatitis B and/or if they consumed $40 g/ day of alcohol.

After explaining the aims of the study to the patients, interviews were conducted by a researcher (IROL), and blood samples were collected and sent to the Central Laboratory at the Hospital das Clı´nicas – Universidade Federal de Pernambuco.

ALT, AST, and GGT serum levels were determined by an automated kinetic method for blood samples, which were taken from each patient immediately before and after a single HD session. In addition, samples were obtained from the patients who were undergoing PD after they completed the questionnaire.

The serum concentration for each patient was divided by the ULN (according to gender) of ALT (31 IU/L for women and 41 IU/L for men), AST (31 IU/L for women and 35 IU/ L for men), and GGT (38 IU/L for women and 55 IU/L for men). The Ht was measured using a CELL-DYN 4000 analyzer. Antibodies (anti-HCV) against the HCV and surface antigen (HBsAg) of hepatitis B virus (HBV) were determined by enzyme immunoassays (AbbottH).

We compared the Ht and the median serum levels of AST, ALT, and GGT in the patients on PD with the values from the patients on HD before and after the dialysis session. In addition, we compared the Ht and the median serum levels of the enzymes in the 40 patients who were undergoing HD before and after the dialysis session. Excel software was used, and the Mann-Whitney U test was applied to compare the median (P25; P75) values. The chi-squared test or Fisher’s exact test was used to compare frequencies, and statistical significance was set as p,0.05.

The study was approved by the Ethics Committee of the Health Sciences Center at the Universidade Federal de Pernambuco and followed the guidelines established by the Declaration of Helsinki.

Table 1 - Demographic characteristics and etiologies of chronic renal failure for the 60 patients who were undergoing dialysis (20 on PD and 40 on HD).

N Age¡SD (years) Range (years) Female (%) Anti-HCV+ Etiology of chronic renal failure Systemic arterial hypertension Diabetes mellitus SAH + DM Systemic lupus erythematosus Others Undetermined

Peritoneal dialysis

Hemodialysis

20 48¡17 (24 - 78) 11 (55%) 3 (15%)

40 54¡15 (21 - 86) 20 (50%) 3 (7.5%)

3 (15%)

12 (30%)

0.343

3 (15%) 0 (0%) 3 (15%)

4 (10%) 2 (5%) 2 (5%)

0.676 0.548 0.322

1 (5%) 10 (50%)

10 (25%) 10 (25%)

0.081 0.100

p-value

0.168 0.927

anti-HCV – hepatitis C virus antibody; SAH – systemic arterial hypertension; DM – diabetes mellitus.

limits, which were originally defined approximately 25 years ago, may consider some individuals to be ‘‘healthy’’ when they actually have steatosis or an HCV infection.

When the ULN was reduced, there was an increase in the sensitivity of ALT from 55% to 76% for the diagnosis of ‘‘discrete’’ liver diseases; however, the specificity was reduced from 97% to 89% (11). A study of a large Korean population revealed that the risk of mortality from liver disease would be higher in individuals with normal levels of ALT that were close to the ULN compared with individuals with lower ALT levels. A proposal was put forward to reduce the ULN level to approximately 75% of the current ULN level of ALT (12).

In an attempt to clarify whether lower levels of aminotransferases in patients with CKD are a result of renal failure or the type of dialysis treatment, the present preliminary study analyzed the ALT serum levels in patients who were undergoing peritoneal dialysis (PD) and HD. We also measured the levels of aspartate aminotransferase (AST) and gamma-glutamyl transferase (GGT). In addition, the hematocrit (Ht) was assessed to investigate the possibility of hemodilution.

RESULTS Table 1 provides a description of the demographic characteristics, the occurrence of anti-HCV and the etiology of CKD in the 60 patients who were included in the present study (the patients were divided into two groups according to the type of dialysis). Among the 20 PD patients, 12 (60%) had received HD before beginning the PD treatment. In these 20 PD patients, the AST and ALT serum levels were each higher than the ULN in three patients (15% for each enzyme). In addition, the GGT levels in eight patients (40%) were higher than the ULN. Among the 40 HD patients, the AST serum level was higher than the ULN in two (5%) patients before the dialysis session and three patients (7.5%) after the dialysis session, and similar findings were observed for the levels of ALT (i.e., the ALT serum level was higher in three (7.5%) patients before dialysis and five (12.5%) patients postdialysis). The GGT serum level was higher than the ULN in 17 (42.5%) patients before dialysis and 21 (52.5%) patients postdialysis.

Table 2 provides a description of the median Ht in the 60 patients with CKD and the median values and ranges of the AST, ALT, and GGT serum levels divided by the ULN. It should be emphasized that the median values of the serum levels of the aminotransferases were below the ULN in the patients who were undergoing PD and HD (both before and after the session). The median values of GGT were near the

PATIENTS AND METHODS The present study, which was conducted as a case series with an internal comparison group, involved 60 patients with CKD who were in renal replacement therapy and attended a dialysis unit in the city of Recife, Brazil. Twenty of the 26 patients who were undergoing PD were included; six were excluded because they either refused to take part in the study or switched their type of dialysis. Concurrently, 40 out of the 211 patients who were undergoing HD in the Nefroclinica Dialysis Unit were randomly selected for the present study, and the HD patients were paired in a 152 ratio according to the period of time that they had been on renal replacement therapy.

To be included in the present study, patients had to be at least 18 years old, have CKD, and have been undergoing dialysis therapy by HD or PD for a minimum of six months.

132

CLINICS 2012;67(2):131-134

Liver enzymes in patients on dialysis Liberato IRO et al.

The Ht rates were significantly lower in the patients on PD compared with the patients on HD (both before and after the session), and the Ht rates were also significantly lower before dialysis compared with after the HD session (Table 2).

DISCUSSION Studies have revealed that ALT serum levels are lower in patients with CKD compared with patients with normal renal function, which raises the question of whether the lower levels are related to CKD factors or to the HD treatment (1,2,10,13-15).

Researchers have assumed that there is a correlation between the ALT serum levels and the severity of renal failure caused by glomerular lesions. Indeed, Fabrizi et al. (8) reported lower ALT levels in patients who were undergoing HD compared with patients with CKD who were undergoing conservative treatment (predialysis), which demonstrated that the ALT serum levels were reduced concomitantly with the progression of renal dysfunction.

Nevertheless, given the possibility that hypoaminotransferasemia could be related to hemodilution, the present preliminary study assessed patients who were undergoing HD and patients who were undergoing PD. We observed that the ALT serum levels were slightly higher in the 20 patients with CKD who were undergoing PD compared with the 40 patients who were undergoing HD when the samples were collected before the dialysis session. A comparison of the mean ALT serum levels for the patients on PD and the postdialysis measurements for the HD patients showed that the ALT levels after therapy were slightly higher. These findings suggest that hemodilution could be involved in reducing ALT levels. Interestingly, the patients on HD presented with fluid retention before the dialysis session; however, the fluid retention regressed after the session ended. If the present study included a larger number of patients, the differences in the median values of aminotransferases may have been even greater.

Indeed, a recent study has confirmed that the Ht and the ALT level are lower before HD and higher after dialysis, which is inversely proportional to the loss of water weight (10). The patients on PD, however, did not present with fluid accumulation or rapid fluid loss, which normally occurs after HD sessions. In addition, the patients on PD evolved with stable and higher serum levels of aminotransferases.

Figure 1 - Median (P25; P75), minimum and maximum values of the serum levels of AST, ALT, and GGT divided by the ULN for the 20 patients on PD and the 40 patients on HD (before and after the HD session).

ULN in patients on PD and in samples collected before the HD session; however, they were higher than the ULN in the samples collected after the HD session (Figure 1).

In the patients who were undergoing PD, the median AST (0.53 IU/L) and ALT (0.39 IU/L) serum levels were slightly higher compared with the samples collected from the HD patients before their HD session (0.47 and 0.37 IU/L, respectively) and slightly lower than samples collected after the session (0.68 and 0.44 IU/L, respectively). The GGT serum levels were slightly lower in the patients who were undergoing PD compared with the samples collected from the patients who were undergoing HD (both before and after the session).

We also analyzed the median Ht and the median AST, ALT, and GGT serum levels divided by the ULN before and after dialysis for the 40 patients with CKD who were undergoing HD. The AST, ALT, and GGT serum levels that were collected before the dialysis session were significantly lower than the values that were observed after dialysis (Table 2).

Table 2 - The median (P25; P75) Ht and the median serum levels of AST, ALT, and GGT divided by the ULN for the 20 patients on PD compared with the 40 patients on HD (before and after the HD session).

Liver enzymes AST/ULN (range IU/L) ALT/ULN (range IU/L) GGT/ULN (range IU/L) Ht (%)

Peritoneal dialysis Median (P25; P75) 0.53 (0.41; 0.61) (4 – 121) 0.39 (0.22; 0.71) (6 – 125) 0.83 (0.55; 1.48) (13 – 155) 32.9 (29.8; 35.5)

Before hemodialysis Median (P25; P75) a

0.47 (0.41; 0.55) (8 – 93) 0.37 (0.21; 0.56) b (6 – 66) 0.88 (0.43; 1.55) c (9 – 318) 36.7 (32.1; 41.3) d

a) p,0.001 b) p,0.001 c) p,0.001 d) p,0.001.

AST – aspartate aminotransferase; ALT – alanine aminotransferase; GGT – gamma-glutamyl transferase; Ht – hematocrit; ULN – upper limit of normal.

133

p-value 0.54 0.87 0.74 0.03

After hemodialysis Median (P25; P75) a

0.68 (0.46; 0.77) (3 – 48) 0.44 (0.27; 0.77) b (6 – 87) 1.14 (0.51; 2.10) c (4 – 372) 41.3 (33.1; 47.1) d

p-value 0.13 0.42 0.68 0.001

Liver enzymes in patients on dialysis Liberato IRO et al.

CLINICS 2012;67(2):131-134

The possibility of hemodilution is corroborated when the mean values of AST and ALT in the same 40 patients are compared in the samples collected before and after the HD session. There was a significant increase in both the AST and the ALT serum levels after the dialysis session.

Moreover, a significant increase in the Ht after the HD session was also observed, which provided support for the hypothesis that patients with CKD retain water before HD. Few authors have discussed AST and GGT levels in patients with CKD who were undergoing dialysis treatment (4). The data from the present study illustrated that AST and GGT levels behave in a similar manner as ALT, which excludes the possibility of an alteration in aminotransferase production, such as a reduction of synthesis because of vitamin B6 deficiency (9).

Importantly, the GGT levels exceeded the ULN in the two groups of patients with a greater frequency than the aminotransferases. Furthermore, the median values of the GGT serum levels were near the ULN and were higher than those for AST and ALT in the three measurements.

Similarly, Souza et al. evaluated 87 patients with CKD and observed that the GGT serum levels were higher than the aminotransferase levels (16).

The high GGT levels in patients who were undergoing dialysis may have been induced by the use of medication or been related to the origins of CKD, such as diabetes mellitus, which can cause steatohepatitis (17). In addition, high GGT serum levels could be related to malnutritioninflammation-atherosclerosis (MIA syndrome), which has been observed in patients with CKD who were undergoing dialysis because of systemic arterial hypertension or diabetes mellitus (18).

There are only a few articles, however, that describe the GGT levels in patients with CKD (16,19,20), and further studies are needed to assess the possibility of other mechanisms that may be involved in the changes of this liver enzyme.

Unlike the aminotransferases, the Ht was lower in the patients on PD compared with the patients on HD (both before and after the HD session). This finding could be related to the improved control of anemia in patients on HD and to the more appropriate replacement of iron supplementation and erythropoietin because the patients on HD have more frequent contact with the dialysis unit than the patients on PD. In conclusion, the aminotransferase serum levels were slightly higher in the patients with CKD who were undergoing PD compared with the patients on HD when the samples were collected before the HD session. In addition, the median values of the samples collected after the HD session were significantly higher than the values collected before dialysis. Because this was the first study to analyze these three enzymes in patients on HD and patients on PD, further research is needed to confirm whether hemodilution is the cause of these differences or whether other factors are involved in the alteration of the AST, ALT and GGT serum levels.

responsible for the dose strategies and provided suggestions regarding the manuscript.

REFERENCES 1. Al-Wakeel J, Mailk GH, Al-Mohaya S, Mitwalli A, Baroudi F, El Gamal H, et al. Liver disease in dialysis patients with antibodies to hepatitis C virus. Nephrol Dial Transplant. 1996;11:2265-8.

2. Yuki N, Ishida H, Inoue T, Tabata T, Matsushita Y, Sasaki Y, et al.

Reappraisal of biochemical hepatitis C activity in hemodialysis patients.

J Clin Gastroenterol. 2000;30:187-94, http://dx.doi.org/10.1097/ 00004836-200003000-00012.

3. Guh JY, Lai YH, Yang CY, Chen SC, Chuang WL, Hsu TC, et al. Impact of decreased serum transaminase levels on the evaluation of viral hepatitis in hemodialysis patients. Nephron. 1995;69:459-65, http://dx.doi.org/ 10.1159/000188520.

4. Yasuda K, Okuda K, Endo N, Ishiwatari Y, Ikeda R, Hayashi H, et al.

Hypoaminotransferasemia in patients undergoing long-term hemodialysis: clinical and biochemical appraisal. Gastroenterology. 1995;109:1295300, http://dx.doi.org/10.1016/0016-5085(95)90591-X. 5. Lopes EP, Gouveia EC, Albuquerque ACC, Sette LH, Mello LA, Moreira RC, et al. Determination of the cutoff value of serum alanine aminotransferase in patients undergoing hemodialysis to identify biochemical activity in patients with hepatitis C viremia. J Clin Virol.

2006;35:298-302, http://dx.doi.org/10.1016/j.jcv.2005.09.010.

6. Fabrizi F, Lunghi G, Ganeshan SV, Martin P, Messa P. Hepatitis C virus infection and the dialysis patient. Semin Dial. 2007;20:416-22, http:// dx.doi.org/10.1111/j.1525-139X.2007.00311.x.

7. Alberti A, Noventa F, Benvegnu` L, Boccato S, Gatta A. Prevalence of liver disease in a population of asymptomatic persons with hepatitis C virus infection. Ann Inter Med. 2002;137:961-4.

8. Fabrizi F, Lunghi G, Finazzi S, Colucci P, Pagano A, Ponticelli C, et al.

Decreased serum aminotransferase activity in patients with chronic renal failure: impact on the detection of viral hepatitis. Am J Kidney Dis. 2001;38:1009-15, http://dx.doi.org/10.1053/ajkd.2001.28590.

9. Ono K, Ono T, Matsumata T. The pathogenesis of decreased aspartate amino-transferase and alanine aminotransferase activity in the plasma of hemodialysis patients: the role of vitamin B6 deficiency. Clin Nephrol.

1995;43:405-8.

10. Lopes EP, Sette LH, Sette JB, Luna CF, Andrade AM, Moraes M, et al.

Serum alanine aminotransferase levels, hematocrit rate and body weight correlations before and after hemodialysis session. Clinics (Sao Paulo).

2009;64:941-5.

11. Prati D, Taioli E, Zanella A, Torre ED, Butelli S, Vecchio ED, et al.

Updated definitions of healthy ranges for serum alanine aminotransferase levels. Ann Inter Med. 2002;137:1-9.

12. Kim HC, Nam CM, Jee SH, Han KH, Oh DK, Suh I. Normal serum aminotransferase concentration and risk of mortality from liver diseases: prospective cohort study. Br Med J. 2004;328:983-6, http://dx.doi.org/ 10.1136/bmj.38050.593634.63.

13. Trevizoli JE, Menezes R, Velasco LF, Amorim R, de Carvalho MB, Mendes LS, et al. Hepatitis C is less aggressive in hemodialysis patients than in nonuremic patients. Clin J Am Soc Nephrol. 2008;3:1385-90, http://dx.doi.org/10.2215/CJN.01330308.

14. Hung KY, Lee KC, Yen KD, Tsai TJ, Che WY. Revised cut off values of serum aminotransferase in detecting viral hepatitis among CAPD patients: experience from Taiwan, an endemic area for hepatitis B. Nephrol Dial Transplant. 1997;12:180-3, http://dx.doi.org/10.1093/ndt/12.1.180.

15. Cotler SJ, Diaz G, Gundlapalli S, Jakate S, Chawla A, Mital D, et al.

Characteristics of hepatitis C in renal transplant candidates. J Clin Gastroenterol. 2002;35:191-5, http://dx.doi.org/10.1097/00004836-2002 08000-00013.

16. Souza JF, Longui CA, Miorin LA, Sens YA. Gamma-glutamyltransferase activity in chronic dialysis patients and renal transplant recipients with hepatitis C virus infection. Transplant Proc. 2008;40:1319-23, http:// dx.doi.org/10.1016/j.transproceed.2008.03.157.

17. Ferreira VS, Pernambuco RB, Lopes EP, Morais CN, Rodrigues MC, Arruda MJ, et al. Frequency and risk factors associated with non-alcoholic fatty liver disease in patients with type 2 diabetes mellitus. Arq Bras Endocrinol Metabol. 2010;54:362-8, http://dx.doi.org/10.1590/S000427302010000400004.

18. Dummer CD, Thome´ FS, Veronese FV. Doenc¸a renal croˆnica, inflamac¸a˜o e aterosclerose: Novos conceitos de um velho problema. Rev Assoc Med Bras.

2007;53:446-50, http://dx.doi.org/10.1590/S0104-42302007000500022.

19. Fabrizi F, De Vecchi AF, Qureshi AR, Aucella F, Lunghi G, Bruchfeld A, et al. Gamma-glutamyltransferase activity and viral hepatitis in dialysis population. Int J Artif Organs. 2007;30:6-15.

20. Nishida C, Uto H, Oketani M, Tokunaga K, Nosaki T, Fukumoto M, et al.

Clinical significance of alanine aminotransferase levels and the effect of ursodeoxycholic acid in hemodialysis patients with chronic hepatitis C. J Gastroenterol. 2010;45:326-34, http://dx.doi.org/10.1007/s00535-0090149-0.

AUTHOR CONTRIBUTIONS Liberato IRO was responsible for the project design, material collection, data analysis, and manuscript writing. Lopes EPA was responsible for supervising the project and correcting the manuscript. Cavalcante MAGM was responsible for the patients and participated in meetings to discuss the strategies for data collection and study methodology. Pinto TC helped with the questionnaires and the data collection. Moura IF was responsible for the biochemical assays and analyses of all the results. Loureiro Jr L was

134

CLINICS 2012;67(2):135-143

DOI:10.6061/clinics/2012(02)08

CLINICAL SCIENCE

Gamma-tocotrienol modulation of senescenceassociated gene expression prevents cellular aging in human diploid fibroblasts Suzana Makpol,I Azalina Zainuddin,I Kien Hui Chua,II Yasmin Anum Mohd Yusof,I Wan Zurinah Wan NgahI I

Universiti Kebangsaan Malaysia, Faculty of Medicine, Department of Biochemistry, Jalan Raja Muda Abdul Aziz, Kuala Lumpur, Malaysia. II Universiti Kebangsaan Malaysia, Faculty of Medicine, Department of Physiology, Jalan Raja Muda Abdul Aziz, Kuala Lumpur, Malaysia.

OBJECTIVE: Human diploid fibroblasts undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular aging. The beneficial effects of vitamin E in aging have been established, but studies to determine the mechanisms of these effects are ongoing. This study determined the molecular mechanism of c-tocotrienol, a vitamin E homolog, in the prevention of cellular aging in human diploid fibroblasts using the expression of senescence-associated genes.

METHODS: Primary cultures of young, pre-senescent, and senescent fibroblast cells were incubated with ctocotrienol for 24 h. The expression levels of ELN, COL1A1, MMP1, CCND1, RB1, and IL6 genes were determined using the quantitative real-time polymerase chain reaction. Cell cycle profiles were determined using a FACSCalibur Flow Cytometer.

RESULTS: The cell cycle was arrested in the G0/G1 phase, and the percentage of cells in S phase decreased with senescence. CCND1, RB1, MMP1, and IL6 were upregulated in senescent fibroblasts. A similar upregulation was not observed in young cells. Incubation with c-tocotrienol decreased CCND1 and RB1 expression in senescent fibroblasts, decreased cell populations in the G0/G1 phase and increased cell populations in the G2/M phase. c-Tocotrienol treatment also upregulated ELN and COL1A1 and downregulated MMP1 and IL6 expression in young and senescent fibroblasts.

CONCLUSION: c-Tocotrienol prevented cellular aging in human diploid fibroblasts, which was indicated by the modulation of the cell cycle profile and senescence-associated gene expression.

KEYWORDS: Vitamin E; Molecular mechanism; Cellular aging; Senescence-associated genes; Human diploid fibroblasts.

Makpol S, Zainuddin A, Chua KH, Yusof YAM, Ngah WZW. Gamma-tocotrienol modulation of senescence-associated gene expression prevents cellular aging in human diploid fibroblasts. Clinics. 2012;67(2):135-143.

Received for publication on September 8, 2011; First review completed on October 13, 2011; Accepted for publication on October 20, 2011 E-mail: suzanamakpol@yahoo.com Tel.: 603-92897296

growth properties, the expression of senescence-associated b-galactosidase (SA-b-gal) (4), an enlarged and flattened morphology with a concomitant increase in the nucleus and nucleoli, an increase in the number of lysosomes and Golgi, the appearance of vacuoles in the cytoplasm and endoplasmic reticulum and an increase in the number of cytoplasmic microfilaments (5). In addition to these morphological changes, the number of multinucleated senescent cells increases (6). The gradual loss of replicative potential reduces harvest cell and cell saturation densities (7). A variety of cell cycle regulation genes, including those involved in immunity and inflammation, the cytoskeleton, stress responses and metabolism, are altered during cellular senescence (8). Serial assessments of gene expression have been used to explore senescence-associated genes to gain insight into the molecular mechanisms of senescence.

Several senescence-linked genes have been identified and cloned based on the changes in mRNA expression levels between young and senescent cells (9). Senescent cells

INTRODUCTION The free radical theory of aging suggests that the aging process involves the accumulation of oxidative damage to cells and tissues, which progressively increases morbidity and mortality (1). Human aging can be studied in vitro using normal human diploid fibroblasts (HDFs), which undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest; this process is termed replicative senescence (2).

Replicative senescence is characterized by the loss of responsiveness to mitogen-induced proliferation (3), altered

Copyright Ă&#x; 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

135

c-Tocotrienol and replicative senescence Makpol S et al.

CLINICS 2012;67(2):135-143

accumulate in human tissues with age, which suggests that these cells contribute to organismal aging (10).

Natural vitamin E occurs in eight different forms: a-, b-, c-, and d-tocopherols and a-, b-, c-, and d-tocotrienols.11 Vitamin E is the major chain-breaking antioxidant that prevents the propagation of oxidative stress, especially in biological membranes (12). a-Tocopherol modulates signal transduction and gene expression in an antioxidant and nonantioxidant manner (11). The non-antioxidant properties of a-tocopherol, including its roles in cellular signaling, gene expression, the immune response, and apoptosis, are also important (13,14).

The tocotrienol isomers have gained increasing scientific interest due to their prominent antioxidant effects and a non-antioxidant activity profile that differs from tocopherols (12) Tocotrienols are abundant in plant foods, such as rice bran and palm oil (15). Tocotrienol is more uniformly distributed in the membrane bilayer, is more efficiently recycled from its corresponding chromanoxyl radical form, and exhibits better interaction with lipid radicals compared to tocopherol. Tocotrienol exhibits better antioxidant activity than tocopherol (16). Therefore, this study determined the changes in several molecular markers of cellular aging, cell cycle profile, and senescence-associated gene expression in c-tocotrienol-treated HDFs to elucidate the molecular mechanism involved in the prevention of cellular aging.

yl)-5-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium (MTS), and the electron coupling agent, phenazine methosulphate (PMS). MTS is reduced to a soluble formazan product by the dehydrogenase enzymes in metabolically active cells. The amount of colored formazan product is proportional to the number of viable cells. Briefly, 20 mL of MTS solution was added to each well and was incubated in a humidified incubator at 37 ˚C in 5% CO2 for 24 h. The quantity of formazan product was determined by measuring the absorbance at 490 nm in a microtiter plate reader (VeraMax Molecular Devices, USA). The viability assay was performed to obtain the optimum dose of ctocotrienol (Malaysian Palm Oil Board) for subsequent experiments. Young and pre-senescent HDFs were incubated with 50 mM c-tocotrienol, and senescent HDFs were incubated with 70 mM c-tocotrienol for 24 h. Untreated cells were cultured in DMEM containing 10% FBS (PAA, Austria). The media for the untreated cells were changed in parallel to the treated cells. The untreated and treated cells were harvested on the same day.

Morphology analysis and senescence-associated (SA) b-galactosidase staining The cells were divided into six experiment groups: i) young HDFs; ii) young HDFs treated with c-tocotrienol; iii) presenescent HDFs; iv) pre-senescent HDFs treated with ctocotrienol; v) senescent HDFs; and vi) senescent HDFs treated with c-tocotrienol. SA-b-gal activity, the molecular marker of HDF cellular aging in vitro, was determined using a senescent cell staining kit (Sigma, USA) according to the manufacturer’s instructions. Blue staining was visible after 4 h of incubation with a b-galactosidase staining solution containing 5-bromo-4chloro-3-indolyl-b-D-galactosidase (X-gal) at 37˚C. The percentage of blue cells in 100 observed cells was calculated under a light microscope.

METHODS Cell culture and the induction of senescence The Universiti Kebangsaan Malaysia Ethical Committee approved this research (Approval Project Code: FF-1042007). Primary HDFs were derived from the foreskins of three 9- to 12-year-old boys after circumcision. Written consents were obtained from the parents of all subjects. The samples were aseptically collected and washed several times with 75% alcohol and phosphate-buffered saline (PBS) containing 1% antibiotic–antimycotic (PAA, Austria). The epidermis was removed, and the pure dermis was cut into small pieces and transferred to a Falcon tube containing 0.03% collagenase type I solution (Worthington Biochemical Corporation, USA). The pure dermis was digested in an incubator shaker at 37 ˚C for 6-12 h. The cells were rinsed with PBS and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) (PAA, Austria) and 1% antibiotic–antimycotic at 37 ˚C in a 5% CO2 humidified incubator. The cultured HDFs were harvested after 5–6 days using trypsinization and were culture-expanded in new T25 culture flasks (Nunc, Denmark) with an expansion degree of 154. Serial passages were performed when the subcultures reached 80–90% confluency using trypsinization, and the number of population doublings (PDs) was monitored until the HDFs reached senescence. The cells were used at passage 4 (young cells, population doubling; PD,12), passage 20 (pre-senescent cells, 30,PD,40), or passage 30 (senescent cells, PD.55) in subsequent experiments.

Cell cycle analysis The six groups of HDFs were subcultured in 10 cm2 tissue culture dishes. The cells were prepared after 24 h of incubation with c-tocotrienol using the CycleTESTTM PLUS DNA Reagent Kit (Becton Dickinson, USA) according to the manufacturer’s instructions. Cell cycle status was analyzed by flow cytometry using propidium iodide (PI) as a specific fluorescent dye probe. The PI fluorescence intensity of 10,000 cells was measured in each sample using a Becton–Dickinson FACSCalibur Flow Cytometer.

Primer design The primers for human GAPDH, COL1A1, ELN, MMP1, IL6, RB1, and CCND1 were designed using Primer 3 software and were blasted with GenBank database sequences to obtain high-specificity primers. The efficiency and specificity of each primer set were confirmed using a standard curve (Ct value versus serial dilution of total RNA) and melting profile evaluation. The primer sequences for quantitative gene expression analysis are shown in Table 1.

Total RNA extraction

Effects of various concentrations of c-tocotrienol on cell viability

Total RNA from HDFs in different groups was extracted using TRI Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions. Polyacryl Carrier (Molecular Research Center) was added to each extraction to precipitate the total RNA. The extracted total

Cellular viability was assessed using the CellTiter 96* Aqueous Non-Radioactive Cell Proliferation Assay (MTS, Promega, USA). The MTS employs 3-(4,5-dimethylthiazol-2-

136

CLINICS 2012;67(2):135-143

c-Tocotrienol and replicative senescence Makpol S et al.

Table 1 - Primer sequences for quantitative real-time RT-PCR. Gene

Sequence (59R39) Sense and antisense primers

Accession number

GAPDH

NM_002046

MMP1

NM_002421

IL6

NM_000600

CCND1

NM_053056

ELN

NM000501

RB1

NM_000321

COL1A1

NM_000088

F: tcc ctg agc tga acg gga ag R: gga gga gtg ggt gtc gct gt F: gct cct ttg gct tcc cta ga R: gca tca act ttg tgg cca at F: tgg ctg aaa aag atg gat gc R: tgc agg aac tgg atc agg ac F: aga cct tcg ttg ccc tct gt R: cag tcc ggg tca cac ttg at F: ggc ctg gag gca aac ctc tt R: cca cca act cct ggg aca cc F: cag acc cag aag cca ttg aa R: ctg ggt gct cag aca gaa gg F: agg gct cca acg aga tcg aga tcc g R: tac agg aag cag aca ggg cca acg

RNA pellet was washed with 75% ethanol and dried prior to dissolution in RNase- and DNase-free distilled water. Total RNA was stored at -80 ˚C immediately after extraction. A Nanodrop (Thermo Scientific, USA) was used to determine the yield and purity of the extracted RNA.

217 187 181 181 189 115 222

HDFs (Figure 1). c-Tocotrienol concentrations of 50 and 70 mM exhibited the greatest percentages of viable cells in young and senescent HDFs, respectively. Therefore, subsequent experiments used 50 mM c-tocotrienol in young and pre-senescent HDFs and 70 mM c-tocotrienol in senescent HDFs. Higher concentrations of c-tocotrienol reduced the viability of HDFs.

Quantitative real-time RT-PCR The expression levels of matrix metalloproteinase 1 (MMP1), inflammation mediator (IL6), extracellular matrix (COL1A1 and ELN), and cell cycle regulatory genes (RB1 and CCND1) were quantitatively analyzed using a one-step RT-PCR technique. The expression level of each targeted gene was normalized to that of GAPDH (17). The reaction was performed using 100 ng of total RNA, a concentration of 400 nM for each primer and the iScript One-Step RT-PCR kit with SYBR Green (Bio-Rad, Canada) according to the manufacturer’s instructions. Reactions were performed in a Bio-Rad iCycler with the following reaction profile: cDNA synthesis for 30 min at 50 ˚C; predenaturation for 2 min at 94 ˚C; and PCR amplification for 38 cycles of 30 sec at 94 ˚C, 30 sec at 61 ˚C. A melt curve analysis determined the reaction specificity. Agarose gel electrophoresis was performed to confirm the PCR products. The relative expression values of target genes were calculated using the 22DDCt method of relative quantification (18) and the following equation: Relative expression value ~ 2 Ct value of GAPDH

Product size, base pairs (bp)

Cellular morphology and SA-b-galactosidase expression Morphological changes were observed in aging HDFs. Young HDFs displayed the normal spindle shape of fibroblast cells. However, the original fibroblastic shape was lost with senescence, and HDFs became large and flattened (Figure 2). No morphological differences were detected in the c-tocotrienol-treated group compared to the untreated group in different passages of HDFs. The positive blue SA-b-galactosidase stain primarily appeared in HDFs at passage 30, which suggested the presence of senescent cells (Figure 3A). Quantitative

Ct value gene of interest

Statistical Analysis Each experiment was performed in triplicate using at least three independent cultures with comparable results. Data are reported as the means¡SD of at least three experiments.

ANOVA and the Student’s t-test (two-tailed) were used to compare differences between groups. p,0.05 was considered statistically significant.

Figure 1 - Dose response of c-tocotrienol in young and senescent HDFs after incubation for 24 h at 37 ˚C. Incubation with ctocotrienol produced a significant increase in the viability of HDFs. The percentages of viable cells were highest at ctocotrienol concentrations of 50 mM in young HDFs and 70 mM in senescent HDFs. However, HDF viability decreased with high concentrations of c-tocotrienol. * Denotes p,0.05 compared to control. Data are presented as means ¡ SD, n = 9.

RESULTS Effect of c-tocotrienol on cells viability Incubation with increasing concentrations of c-tocotrienol for 24 h (10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mM) significantly increased the viability of young and senescent

137

c-Tocotrienol and replicative senescence Makpol S et al.

CLINICS 2012;67(2):135-143

Figure 2 - Morphological changes in HDFs in culture. Young (A), pre-senescent (B), and senescent (C) HDFs compared to c-tocotrienoltreated HDFs (D–F). Senescent HDFs lost their original fibroblastic shape and acquiring a flattened morphology (indicated by arrow).

The size of the nucleus and cell increased in treated and untreated groups. Micrographs are shown at 100X magnification.

c-tocotrienol significantly increased (p,0.05) the percentage of senescent HDFs in the G2/M phase (Figure 5).

analysis revealed that the percentage of SA-b-gal-positive cells increased (p,0.05) in senescent cells compared to young and pre-senescent HDFs. The incubation of senescent cells with 70 mM c-tocotrienol did not alter the percentage of SA-b-gal-positive cells (Figure 3B).

Effect of c-tocotrienol on the expression of senescence-associated genes Six pairs of senescence-associated gene primers were designed. Agarose gel electrophoresis revealed that each PCR product appeared as a single band (Figure 1S). The melting curve analysis demonstrated a single and narrow peak for each PCR product (Figure 2S), which indicated that the primers and RT-PCR protocols were specific.

Senescent cells exhibited significant increases in MMP1, IL6, CCND1, and RB1 mRNA expression levels compared to young HDFs (p,0.05). However, c-tocotrienol produced significant downregulations of MMP1 mRNA expression in

Cell cycle progression The cell populations in the S phase were significantly lower (p,0.05) in pre-senescent and senescent HDFs compared to young HDFs. The cell populations in the G0/ G1 phase in pre-senescent and senescent HDFs were significantly higher (p,0.05) compared to young HDFs (Figure 4). c-Tocotrienol significantly decreased (p,0.05) the percentage of senescent HDFs in the G0/G1 phase. In contrast,

138

CLINICS 2012;67(2):135-143

c-Tocotrienol and replicative senescence Makpol S et al.

Figure 3 - Senescence-associated b-galactosidase (SA b-gal) staining in young (a), pre-senescent (b), and senescent HDFs (c). Positive blue stains of SA b-gal appeared in senescent HDFs as indicated by arrows. Micrographs are shown at 100X magnification (A). Quantitative analysis of positive b-galactosidase-stained cells in HDFs during cellular aging. The percentage of cells positive for SA-b-gal staining was significantly increased in senescent cells. The incubation of senescent cells with c-tocotrienol (70 mM for 24 h) did not produce significant changes in the percentage of SA-b-gal-positive cells. aDenotes p,0.05 compared to untreated young HDFs, bp,0.05 compared to untreated pre-senescent HDFs. Data are presented as means ยก SD, n = 6.

pre-senescent and senescent HDFs compared to untreated cells (p,0.05) (Figure 6A). c-Tocotrienol produced significant (p,0.05) upregulations of COL1A1 in young, pre-senescent, and senescent HDFs (Figure 6B). c-Tocotrienol also significantly upregulated ELN mRNA in young and senescent HDFs (p,0.05) (Figure 6C). c-Tocotrienol significantly downregulated IL6 (Figure 6D), CCND1 (Figure 6E) and RB1 (Figure 6F) mRNA expression in senescent HDFs (p,0.05).

DISCUSSION This study evaluated the effects of c-tocotrienol in the modulation of cellular aging in HDFs. Our results demonstrated clear changes in cellular morphology, decreased cellular proliferation, and increased senescence-associated b-galactosidase activity in senescent HDFs. Cellular and organism morphological changes are typical features of a

139

c-Tocotrienol and replicative senescence Makpol S et al.

CLINICS 2012;67(2):135-143

Figure 4 - Quantitative analysis of cell cycle progression in HDFs. aDenotes p,0.05 compared to young HDFs (G0/G1 phase), bp,0.05 compared to young HDFs (S phase), and cp,0.05 compared to pre-senescent HDFs (S phase). Data are presented as means ยก SD, n = 6.

Our data demonstrated an increase in cell populations in the G0/G1 phase and a decrease in S-phase cells during HDF aging. Although senescent cells display some characteristics of a late G1 arrest, these cells are actually arrested in a distinct state within a specific pathway (26). Senescent HDFs contain higher levels of oxidative DNA lesions compared to early passage cells, which suggests that oxidative damage triggers the onset of cell cycle checkpoints in senescent cells (27). The presence of damaged DNA activates checkpoint pathways, which inhibit the progression of cells through the G1 and G2 phases and induce a delay in cell cycle progression through the S phase. These checkpoints allow sufficient time to repair the damaged DNA prior to the resumption of cell cycle progression (28). Therefore, an increase in the number of cells in the G2/M phase in c-tocotrienol treated senescent HDFs suggested that c-tocotrienol decreased the amount of damaged DNA in aged cells and modulated the expression of cell cycle regulatory genes. Our previous results showed that a tocotrienol-rich fraction protected against DNA damage in senescent HDFs via the prevention of oxidative stressinduced DNA damage or the enhancement of the DNA repair mechanism (29).

The reduction in S-phase cells with senescence suggests a slowing of cellular proliferation when entering replicative senescence. The increase in the expression of the cell cycle regulatory genes RB1 and CCND1 that was observed in this study may explain this reduction. RB1 mRNA is intimately involved in cellular aging in senescent HDFs. Senescent cells do not phosphorylate the Rb protein, which leads to inactivation of Rb and cellular progression into the S phase (30). The impairment of this mechanism in senescent cells increases the expression of Rb and retains Rb in a hypophosphorylated inhibitory form. Our results demonstrated that c-tocotrienol decreased the expression of RB1 mRNA in senescent HDFs. Alterations in the expression or activity of gene products that are normally active in the G0/G1 to S progression likely play a pivotal role in cellular senescence (29). Our findings demonstrated an upregulation of CCND1 RNA in senescent HDFs. Senescent cells were arrested at G0/G1 phase where the expression of cyclin D1 was at the highest. Our finding was similar to a previous report which suggested that senescent cells might lack a feedback control mechanism

senescent phenotype (19). The enlargement and flattening of senescent HDFs is accompanied by changes in nuclear structure, gene expression, protein processing and metabolism (4) and an increase in the activity of senescenceassociated b-galactosidase at pH 6 (6,20). The flattened cell appearance may be due to the significant upregulation of matrix metalloproteinase 1 (MMP1) mRNA and an increase in extracellular matrix degradation in aged cells. Previous study reported the overexpression of matrix metalloproteinases (MMPs) gene in senescent cells which results in the loss of proteins that maintain the ultrastructural shape. The relative overproduction of collagenase in aging cells suggests a matrix-degrading phenotype of senescent cells (21).

Our results demonstrated that c-tocotrienol downregulated MMP1 mRNA expression in pre-senescent and senescent HDFs. This result suggested that c-tocotrienol reduced the aging process via the inhibition of extracellular matrix degradation. Senescent HDFs exhibit lower expression levels of several extracellular matrix components, such as collagen I-1a, collagen III-1a and elastin, and increased expression levels of collagenase and stromelysin, which degrade the extracellular matrix. These data are consistent with the upregulation of MMP1 mRNA in senescent HDFs in our study. The increased expression of COL1A1 mRNA in young, pre-senescent, and senescent HDFs and the increased ELN mRNA expression levels in young and senescent HDFs demonstrated an important role for ctocotrienol in the maintenance of extracellular matrix architecture during various stages of cellular aging.

Senescent cells disrupt normal tissue structures and functions in complex cell culture models. For example, senescent stromal fibroblasts derange the normal organization and specialized function of mammary epithelial cells (22,23). The effects of senescent cells on mammary epithelial cells were produced by the secretion of MMPs, which is similar to the effects of senescent cells on fibrosis resolution.

Osteoblasts undergo age-related cellular senescence because of the increase in oxidative stress in aged bones (24).

Senescent osteoblasts alter the bone microenvironment, which contributes to the development of age-related osteoporosis (24,25). Therefore, the c-tocotrienol-induced downregulation of MMP1 and upregulation of COL1A1 and ELN in our study suggest a medicinal role in the maintenance of normal tissue structures and functions.

140

CLINICS 2012;67(2):135-143

c-Tocotrienol and replicative senescence Makpol S et al.

Figure 5 - Cell cycle analysis with c-tocotrienol treatment. *Denotes p,0.05 compared to control. Data are presented as means ยก SD, n = 6.

that negatively regulates cyclin D1 expression. However, ctocotrienol downregulated CCND1 mRNA in senescent HDFs, reduced the cellular population in the G0/G1 phase

and increased the population in the G2/M phase. These results suggest that c-tocotrienol inhibited cell growth arrest and enhanced cellular proliferation in senescent HDFs. Our

141

c-Tocotrienol and replicative senescence Makpol S et al.

CLINICS 2012;67(2):135-143

Figure 6 - Effect of c-tocotrienol on MMP1 (A), COL1A1 (B), ELN (C), IL6 (D), CCND1 (E), and RB1 (F) mRNA expression levels in untreated and c-tocotrienol-treated young, pre-senescent and senescent HDFs. aDenotes p,0.05 compared to control young HDFs, cp,0.05 compared to control pre-senescent HDFs, and ep,0.05 compared to control senescent HDFs. Data are presented as means ยก SD, n = 6.

These results support the role of c-tocotrienol in the prevention of cellular aging in HDFs. c-Tocotrienol prevented the cellular aging of human diploid fibroblasts via modulation of the cell cycle profile and senescence-associated gene expression.

recent findings demonstrated that a tocotrienol-rich fraction (TRF) prevented cellular aging in HDFs by reversing the cell cycle arrest that is associated with cellular senescence and promoted cell cycle progression at different stages of cellular aging (29).

Stress-like conditions and inflammation are chronic during aging (31). Cytokines are important proinflammatory mediators, and aged individuals exhibit increased serum levels of proinflammatory cytokines, such as IL-1, IL6, TNF-a, and IL-8, compared to young individuals (32). Our data demonstrated an upregulation of IL6 in senescent HDFs compared to the young control group. However, ctocotrienol downregulated IL6 mRNA in senescent HDFs. In summary, c-tocotrienol decreased the percentage of cells in the G0/G1 phase and increased the percentage of cells in the G2/M phase. c-Tocotrienol also modulated senescence-associated gene expression; the expression levels of MMP1, IL6, CCND1, and RB1 were downregulated, but COL1A1 and ELN expression levels were upregulated.

ACKNOWLEDGMENTS This study was funded by the Ministry of Science, Technology and Innovation under the E-Science Fund 02.01.02. SF0027 and the Universiti Kebangsaan Malaysia grant FF-328-2009.

AUTHOR CONTRIBUTIONS Makpol S designed and approved the study, provided critical analysis and interpretation of data and was also responsible for the revision of the manuscript. Zainuddin A was responsible for the acquisition, analysis and interpretation of data and draft of the manuscript. Chua KH was responsible for the HDF primary culture optimization and significantly contributed to the acquisition of the data. Yusof YAM designed the study and was responsible for the acquisition, and interpretation of the data.

142

CLINICS 2012;67(2):135-143

c-Tocotrienol and replicative senescence Makpol S et al.

17. Zainuddin A, Chua KH, Abdul Rahim N, Makpol S. Effects of experimental treatment on GAPDH mRNA expression as a housekeeping gene in human diploid fibroblasts. BMC Molecular Biology. 2010;11:59.

18. Carthagena L, Bergamaschi A, Luna JM, David A, Uchil PD, MargottinGoguet F, Mothes W, Hazan U, Transy C, Pancino G, Nisole S. Human TRIM gene expression in response to interferons. PloS One. 2009;4:e4894.

19. Cho KA, Ryu SJ, Oh YS, Park JH, Lee JW, Kim HP, et al. Morphological Adjustment of Senescent Cells by Modulating Caveolin-1 Status. J Biol Chem. 2004;279:42270-8.

20. Krishna DR, Sperker B, Fritz P, Klotz U. Does pH 6 b-galactosidase activity indicate cell senescence? Mech Ageing Dev. 1999;109:113-23.

21. Mawal-Dewan M, Lorenzini A, Frisoni L, Zhang H, Cristofalo VJ, Sell C. Regulation of Collagenase Expression during Replicative Senescence in Human Fibroblasts by Akt-Forkhead Signaling. J Biol Chem.

2002;277:7857-64.

22. Parrinello S, Coppe JP, Krtolica A, Campisi J. Stromal–epithelial interactions in aging and cancer: senescent fibroblasts alter epithelial cell differentiation. J Cell Sci. 2005;118:485–96.

23. Tsai KK, Chuang EY, Little JB, Yuan ZM. Cellular mechanisms for lowdose ion-izing radiation-induced perturbation of the breast tissue microenvironment. Cancer Res. 2005;65:6734–44.

24. Manolagas SC. From estrogen-centric to aging and oxidative stress: a revised perspective of the pathogenesis of osteoporosis. Endocr Rev. 2010;31:266–300.

25. Kassem M, Marie PJ. Senescence-associated intrinsic mechanisms of osteoblast dysfunctions. Aging Cell. 2011;10:191–7.

26. Tresini M, Lorenzini A, Torres C, Cristofalo VJ. Modulation of Replicative Senescence of Diploid Human Cells by Nuclear ERK Signaling. J Biol Chem. 2007;282:4136-51.

27. Chen Q, Fischer A, Reagan JD, Yan LJ, Ames BN. Oxidative DNA Damage and Senescence of Human Diploid Fibroblast Cells. Proc Natl Acad Sci USA. 1995;92:4337-41.

28. Iliakis G, Wang Y, Guan J, Wang H. DNA damage checkpoint control in cells exposed to ionizing radiation. Oncogene. 2003;22:5834–47.

29. Makpol S, Durani LW, Chua KH, Mohd Yusof YA, Wan Ngah WZ. Tocotrienol-Rich Fraction Prevents Cell Cycle Arrest and Elongates Telomere Length in Senescent Human Diploid Fibroblasts. J Biomed Biotechnol. 2011;2011:506171.

30. Hinds PW, Mittnacht S, Dulic V, Arnold A, Reed SI, Weinberg RA. Regulation of retinoblastoma protein functions by ectopic expression of human cyclins. Cell. 1992;70:993-6.

31. Mocchegiani E, Costarelli L, Giacconi R, Cipriano C, Muti E, Tesei S, et al.

Nutrient-gene interaction in ageing and successful ageing: A single nutrient (zinc) and some target genes related to inflammatory/immune response. Mechanisms of Ageing and Development. 2006;127:517-25.

32. Bruunsgaard HM, Pedersen BK. Aging and proinflammatory cytokines.

Curr Opin Hematol. 2001;8:131–6.

Ngah WZW interpreted the data and provided critical analysis of the intellectual content of the manuscript. All the authors read and approved the final version of the manuscript.

REFERENCES 1. Beckman KB, Ames BN. The free radical theory of aging matures.

Physiol Rev. 1998;78:547-81.

2. Trougakos IP, Saridak A, Panayotou G, Gonos ES. Identificaton of differentially expressed proteins in senescent human embryonic fibroblast. Mech Ageing Dev. 2006;127:88-92.

3. Park Y, Park JS, Cho KA, Kim DI, Ko YG, Seo JS, et al. Up-regulation of caveolin attenuates epidermal growth factor signaling in senescent cells.

J Biol Chem. 2000;275:20847-52.

4. Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA. 1995;92:9363-67.

5. Wagner M, Hampel B, Bernhard D, Hala M, Zwerschke W, Jansen-Durr P. Replicative senescence of human endothelial cells in vitro involves G1 arrest, polyploidization and senescence-associated apoptosis. Exp Gerontol. 2001;36:1327-47.

6. Matsumura T. Multinucleation and polyploidization of aging human cells in culture. Adv Exp Med Biol. 1980;129:31–8.

7. Cristofalo VJ, Volker C, Francis MKF, Tresini M. Age-Dependent Modifications of Gene Expression in Human Fibroblasts. Crit Rev Eukaryot Gene Exp. 1998;8:43-80.

8. Zhang H, Pan KH, Cohen SN. Senescence-specific gene expression fingerprints reveal cell-type-dependent physical clustering of upregulated chromosomal loci. Proc Natl Acad Sci USA. 2003;100:3251-56.

9. Yoon IK, Kim HK, Kim YK, Song IH, Kim W, Kim S, et al. Exploration of replicative senescence-associated genes in human dermal fibroblasts by cDNA microarray technology. Exp Gerontol. 2004;39:1369-78.

10. Campisi J. Cancer, aging and cellular senescence. In Vivo. 2000;14:183-8.

11. Zingg JM. Modulation of signal transduction by vitamin E. Mol Aspects Med. 2007;28:481-6.

12. Sebastian S, Mu¨ller WE, Eckert GP. Tocotrienols: Constitutional Effects in Aging and Disease. J Nutr. 2005;135:151-4.

13. Azzi A, Ricciarelli R, Zingg JM. Non-antioxidant molecular functions of a-tocopherol (vitamin E). FEBS Lett. 2002;519(1-3):8–10.

14. Brigelius-Flohe R, Kelly FJ, Salonen JT, Neuzil J, Zingg JM, Azzi A. The European perspective on vitamin E: current knowledge and future research. Am J Clin Nutr. 2002;76(4):703-16.

15. Theriault A, Chao JT, Wang Q, Gapor A, Adeli K. Tocotrienol: a review of its therapeutic potential. Clin Biochem. 1999;32:309-19.

16. Serbinova E, Kagan V, Han D, Packer L. Free radical recycling and intramembrane mobility in the antioxidant properties of alpha-tocopherol and alpha-tocotrienol. Free Radic Biol Med. 1991;10:263-75.

143

CLINICS 2012;67(2):145-149

DOI:10.6061/clinics/2012(02)09

CLINICAL SCIENCE

Visual acuity and retinal function in patients with Bardet-Biedl syndrome Adriana Berezovsky, Daniel Martins Rocha, Paula Yuri Sacai, Sung Song Watanabe, Nı´vea Nunes Cavascan, Solange Rios Saloma˜o Universidade Federal de Sa˜o Paulo/Escola Paulista de Medicina– UNIFESP/EPM, Departamento de Oftalmologia, Sa˜o Paulo/SP, Brazil.

OBJECTIVE: Bardet-Biedl syndrome is a genetic, multisystem disorder that causes severe visual impairment. This condition is characterized by retinal dystrophy, obesity, digit anomalies, renal disease, and hypogonadism. The purpose of this study was to analyze visual acuity and full-field electroretinogram findings in patients with the Bardet-Biedl syndrome phenotype.

METHODS: The visual acuity of a group of 23 patients (15 males) with ages ranging from 6-36 years (mean = 15.8¡6.4; median = 14.7) was assessed. Retinal function was evaluated by full-field electroretinography, and dark-adapted thresholds were assessed.

RESULTS: Visual acuity in the better-seeing eye was 20/40 or better in 5 patients (21.7%), 20/50-20/150 in 13 (56.5%) patients, 20/200-20/400 in 2 (8.7%) patients and worse than 20/400 in one (4.3%) patient. The mean acuity in the better-seeing eye was 0.7¡0.6 logMAR (20/100, Snellen equivalent). Scotopic rod and maximal responses were nondetectable in 21 (91.3%) patients, and cone responses were non-detectable in 15 (65.2%) patients. Elevated darkadapted visual thresholds were observed in all 19 patients who were able to be assessed, with 10 (52.6%) patients having thresholds greater than 30 dB. CONCLUSIONS: In a relatively young cohort of patients with Bardet-Biedl syndrome, only 21% had 20/40 or better vision. ERG scotopic responses were absent in the majority of cases, with cone responses being observed in less than half of cases. These findings showed the early deleterious effects in retinal function and visual acuity caused by this condition.

KEYWORDS: Bardet-Biedl; Visual Acuity; Electroretinography; Retinal Dystrophy; Genetic Disorder.

Berezovsky A, Rocha DM, Sacai PY, Watanabe SS, Cavascan NN, Saloma˜o SR. Visual acuity and retinal function in patients with Bardet-Biedl syndrome. Clinics. 2012;67(2):145-149.

Received for publication on September 6, 2011; First review completed on September 29, 2011; Accepted for publication on October 6, 2011 E-mail: aberezovsky@oftalmo.epm.br Tel.: 55 11 5085-2046

outer retina (6). BBS patients variably display all common ciliopathy features: polydactyly, cystic kidneys, retinal degenerations, and situs inversus. To date, mutations in any one of 16 different genes can cause this phenotype (7-11).

Ocular manifestations of BBS include a marked reduction of electroretinogram (ERG) amplitude and notable degree of retinal pigmentary degenerative changes in early childhood (1). Retinal function in BBS patients has been studied by fullfield ERG and shows a variable expression of rod-cone or cone-rod dysfunction (1,5,12-16). In BBS carriers, abnormal rod-mediated responses have been detected by full-field ERG, and areas of local dysfunction in the central retina have been observed by multifocal ERG (17,18).

The natural course of BBS is characterized by poor visual prognosis, with central vision declining by 1 line per year on the logMAR chart and peripheral vision deteriorating 0.19 log units per year in dark-adapted thresholds (12). The optic disks and retinal vessels are normal in infancy; disk pallor and attenuated retinal vessels develop with age. Pigmentary changes are observed in the peripheral fundus (19,20). Night blindness usually manifests by eight years of age and progresses to complete blindness by 16-20 years of age (7).

INTRODUCTION Bardet-Biedl Syndrome (BBS), first described by Bardet in 1920 and Biedl in 1922, is a rare autosomal recessive disorder that affects numerous tissues and causes severe visual impairment (1). The estimated prevalence of BBS ranges from 1:125,000 in Europe to 1:18,500 in certain isolated populations in Canada (2,3). The clinical diagnosis of BBS is based on the presence of at least four of five cardinal features: retinal dystrophy, dystrophic extremities (polydactyly, syndactyly, brachydactyly), obesity, hypogenitalism in men only, and renal disease (4,5). BBS is currently considered a ‘ciliopathy,’ with the underlying defects affecting the basal bodies of ciliated cells, such as the connecting cilium in the

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

145

Visual acuity and retinal function in Bardet-Biedl syndrome Berezovsky A et al.

CLINICS 2012;67(2):145-149

placed on the inside surface of the contact lens for protection and to ensure good electrical contact. A gold cup ground electrode was applied to the earlobe. All stimuli were presented in a Ganzfeld dome (LKC Technologies Inc., Gaithersburg, MD, USA). Signals were amplified (gain, 910,000; 0.3–500 Hz), digitized, averaged, saved, and displayed by a digital plotter (UTAS E-3000 System, LKC Technologies Inc., Gaithersburg, MD, USA). ERGs were recorded using the standard International Society for Clinical Electrophysiology of Vision (ISCEV) protocol (22).

Stimulus and recording details have been described previously (23). The peak-to-peak amplitude (mV) and the implicit time (ms) from each step of the ISCEV standard protocol were determined. The oscillatory potential amplitude was calculated as the sum of each wavelet and automatically analyzed by the UTAS E-3000 system.

Dark-adapted thresholds. Dark-adapted thresholds were obtained using the Scotopic Sensitivity Tester (SST-1) (LKC Technologies Inc., Gaithersburg, MD, USA). The SST-1 uses a full-field green LED (lmax = 572 nm) as its stimulus in dark adaptometry mode, with a maximum intensity of -2.15 log cd/m2 at 30 dB. The SST-1 stimulus intensity decreases in 1-dB increments and increases in 5-dB increments, which range from 0 to 30 dB. The SST-1 thresholds were obtained using a two-alternative forced-choice staircase protocol (stimulus vs. non-stimulus, n = 3, decreasing in 1-dB increments). The dark-adapted final threshold was obtained by repeating this protocol three times and averaging the results (24).

Although different forms of BBS can correlate with specific mutations, demonstrating the genetic heterogeneity in this condition, several studies have suggested that molecular investigations do not show clear phenotypegenotype correlations. Therefore, the diagnosis is still made using clinical data (10,21). Moreover, BBS may be characterized by profound inter- and intrafamilial clinical variability, which may be partially explained by the presence of secondsite modifiers (9). Because BBS has a variable clinical presentation, the purpose of this retrospective study was to analyze visual acuity (VA) and full-field ERG findings in patients with the BBS phenotype recruited from a public university hospital in Brazil.

MATERIALS AND METHODS Participants. Participants were patients with the BardetBiedl syndrome phenotype who were referred to the Electrophysiology of Vision Laboratory at the Department of Ophthalmology of the Federal University of Sa˜o Paulo, Sa˜o Paulo, Brazil for assessment from October 1998 to August 2010. During this time frame, 3,282 patients were referred to our laboratory, and the Bardet-Biedl syndrome group comprised 0.8% of these patients. A history of consanguinity was obtained by a personal interview. Nonocular abnormalities were determined by history and/or available medical records; these cardinal features were not specifically assessed in these patients. This study was approved by the Federal University of Sa˜o Paulo’s Committee of Ethics in Research and followed the tenets of the Declaration of Helsinki. Written informed consent was obtained from each patient or his/her parents or guardians after researchers explained the purpose, procedures, and possible risks and benefits of the study.

Retinal dystrophy type was classified according to the standard clinical criteria, considering history, fundus examination and previous full-field ERG responses (recorded from both eyes with bipolar contact lens electrodes) as rodcone or cone-rod dystrophy. Inclusion criteria were the presence of at least four of the following cardinal features: retinal dystrophy, obesity, extremity abnormalities, hypogenitalism in males, and renal abnormalities.

Statistical Analysis Data from ERG parameters (peak-to-peak amplitude and implicit time) from each step according to the ISCEV standard protocol were measured and compared with normative data from our own laboratory. A normality test was used to confirm the data distribution. A Spearman’s rank correlation coefficient was performed to investigate possible associations between a) BCVA in the better-seeing eye and age, b) BCVA and peak-to-peak amplitude for cone responses, and c) BCVA and peak-to-peak amplitude for 30Hz flicker responses. A result was considered statistically significant if the p-value was less than 0.05.

Procedures

RESULTS

An ophthalmic examination of both eyes was performed by a retina specialist (SSW), including slit-lamp, refraction, and dilated indirect ophthalmoscopy. Best-corrected visual acuity (BCVA) was measured in each eye using a retro-illuminated Early Treatment Diabetic Retinopathy Study Chart built with Tumble ‘‘E’’ optotypes presented at a distance of 4 m.

Ocular motility was assessed using cover/uncover and alternate cover testing for distance and near vision testing, with and without glasses, with emphasis on the diagnosis of strabismus and nystagmus.

Full-Field Electroretinography. A minimum pupil diameter of 6 mm was obtained after administering a drop of tropicamide 1% with a drop of phenylephrine 10%, and all subjects were dark-adapted for 30 min. Under dim red illumination, a bipolar contact lens electrode (Burian-Allen bipolar electrode, Hansen Ophthalmic Development Lab, Coralville, IA, USA) was placed on the corneal surface. The corneal surface was anesthetized with two drops of tetracaine 1.0%, and a drop of methylcellulose 2% was Ophthalmic Examination.

We examined 23 patients (15 males) from 22 families with ages ranging from 6-36 years (mean = 15.8¡6.4; median = 14.7). A summary of the major clinical characteristics is presented in Table 1. A history of consanguinity was found in 11 participants (47.8%). The patients’ self-reported race was white in 14 (61%) patients, mixed white/black in eight patients (35%), and black in one patient (4%). The most frequent presenting visual symptoms were as follows: night vision loss in 22 patients (95.6%), visual acuity loss in 19 patients (82.6%), and visual field loss in 13 patients (56.5%).

A 12-year-old male patient had no visual symptoms (case #8).

High ametropias were found in six patients (26.0%), with four patients reporting high myopia (SE#-6.0 D) and two reporting high hyperopia (SE$6.0 D). Severe retinal dystrophy was found in all patients. A rod-cone pattern was observed in 20 patients (86.9%), with the remaining three patients showing cone-rod dystrophy. Ophthalmoscopic changes were classified according to fundus

146

CLINICS 2012;67(2):145-149

Visual acuity and retinal function in Bardet-Biedl syndrome Berezovsky A et al.

Table 1 - Clinical characteristics of 23 patients with Bardet-Biedl Syndrome.

History of Case Age Consan# (yrs.) Gender Race guinity 1 2 3 4 5 6 7 8 9* 10 11 12 13* 14 15 16 17 18 19 20 21 22 23 *

6 8 9 10 11 11 12 12 12 13 14 14 14 15 15 16 17 17 18 19 20 28 36

M M M M M M F M M F M M M F F F M F M M F M F

mixed white mixed white white white mixed white white white mixed white white white mixed white black white white mixed white mixed mixed

N N N Y N Y N N Y N Y Y Y N N Y Y Y N Y Y N N

Presenting Symptoms VA, NV loss VA, NV loss VA, NV loss VA, NV loss VA, NV loss NV, VF loss VA, NV, VF loss None VA, NV, VF loss NV, VF loss VA, NV, VF loss VA, NV, VF loss VA, NV, VF loss VA, NV, VF loss VA, NV loss VA, NV loss VA, NV, VF loss VA, NV, VF loss VA, NV, VF loss NV, VF loss VA, NV loss VA, NV loss VA, NV, VF loss

BCVA Refraction (better-seeing eye) (Mean SE) Uncooperative 20/100 20/40 20/160 20/40 20/125 20/125 20/40 20/40 20/125 20/160 20/100 20/50 20/80 20/125 20/80 20/63 20/125 20/50 20/25 20/200 Uncooperative LP

NA +7.00 +11.75 -11.50 +4.25 +2.25 +0.50 NA -2.50 -0.50 +4.50 +2.00 -2.00 -8.50 NA -9.25 +1.50 -4.75 NA +2.25 NA NA -6.50

Ophthalmoscopic Changes Uncooperative MA AV, DP, WRPEA WRPEA WRPEA AV, DP, WRPEA WRPEA AV, DP, MA AV, WRPEA MA, PP AV, MA, PP, WRPEA MA, WRPEA AV, MA AV, MA WRPEA AV, DP, WRPEA AV, DP, WRPEA AV, DP, MA, WRPEA AV, DP, MA, PP, WRPEA AV, DP, PP AV, WRPEA AV, DP, PP NA

Retinal Dystrophy rod-cone rod-cone cone-rod rod-cone rod-cone rod-cone rod-cone cone-rod rod-cone rod-cone cone-rod rod-cone rod-cone rod-cone rod-cone rod-cone rod-cone rod-cone rod-cone rod-cone rod-cone rod-cone rod-cone

Nystagmus Strabismus Y N N Y N N N N N Y Y N N N N N N N N N N Y Y

Ortho Ortho Ortho XT Ortho Ortho X(T) Ortho ET XT Ortho Ortho ET Ortho Ortho Ortho Ortho Ortho Ortho Ortho Ortho ET Ortho

brothers from a consanguineous marriage; mixed — black/white; BCVA – best-corrected visual acuity; SE – spherical equivalent; VA – visual acuity; NV – night vision; VF – visual field; NA – not available; MA – macular abnormalities; AV – attenuated vessels; DP – disk pallor; WRPEA – widespread retinal pigmented epithelium abnormalities; PP – peripheral pigmentation; ET – esotropia; XT – exotropia; and X(T) – intermittent exotropia.

(mean¡2 SD), as represented by rectangles, similar to the previously shown graphic model (13,25). Rod scotopic responses were non-detectable in 20 patients, with only three patients reporting remaining rod function below the lower normal limit. Four patients had detectable mixed scotopic responses, with two patients responding (cases #8 and #3) within the normal limits and two patients (cases #9 and #16) responding below the lower normal limits; the remaining patients had non-detectable responses. Cone function assessed by single-flash cone responses was present in nine patients; all patients except case #2 had responses below the normal limits. Cone responses for 30-Hz flicker were detected in 16 patients and non-detectable in seven patients; all detected responses were below the normal limits. No correlation was found between BCVA and peak-to-peak amplitude for cone responses (p = 0.08) or between BCVA and peak-to-peak amplitude for 30-Hz flicker responses (p = 0.76).

photographs (when available) or fundus descriptions. Data were available from 21 of the 23 examined patients. The most frequent fundus changes were attenuated vessels in 14 patients (67.0%), widespread retinal epithelium abnormalities in 14 patients (67.0%), disk pallor in nine patients (42.8%), macular abnormalities in nine patients (42.8%), and peripheral pigmentation in five patients (23.8%).

Six patients (26.0%) presented manifest bilateral nystagmus. On the cover test, three subjects presented esotropia, two presented exotropia, and one presented intermittent exotropia; the remaining 17 subjects had orthoposition of the visual axes. Both nystagmus and strabismus were found in three patients (13.0%).

Visual Acuity The BCVA distribution from 21 BBS patients is shown in Table 1. BCVA in the better-seeing eye was 20/40 or better in five patients (21.7%), 20/50-20/150 in 13 patients (56.5%), 20/200-20/400 in two patients (8.7%), and worse than 20/400 in one patient (4.3%). Two patients were uncooperative in the visual acuity measurement. The mean VA was 0.7¡0.6 logMAR (20/100, Snellen equivalent) in the better-seeing eye and 0.9¡0.6 logMAR (20/160, Snellen equivalent) in the worseseeing eye. No significant correlation was found between age and BCVA (logMAR) in the better-seeing eye (p = 0.24).

Dark-adapted thresholds Elevated dark-adapted visual thresholds were found in all 19 patients who were able to be assessed, with 10 patients (52.6%) having thresholds higher than 30 dB. The mean threshold was 22.7¡9.04 dB with a median of 30 dB.

Non-ocular findings Of the 23 patients, 21 patients (91.3%) were obese, 18 patients (78.3%) presented digital anomalies, 18 patients (78.3%) had mental retardation, and 7 patients (30.4%) had renal anomalies.

Full-field ERG ERG abnormalities were found in all 23 patients.

Representative standard full-field ERG recordings from one eye from a healthy normal subject, one patient with rod-cone dystrophy (case #2), and one patient with cone-rod dystrophy (case #3) are shown in Figure 1. Figure 2 summarizes the ERG parameters for scotopic and photopic retinal function from BBS patients compared with our own normative data

DISCUSSION The present study showed no correlation between BCVA in the better-seeing eye and age, thereby corroborating

147

Visual acuity and retinal function in Bardet-Biedl syndrome Berezovsky A et al.

CLINICS 2012;67(2):145-149

Figure 1 - Representative standard full-field ERG waveforms (rod, mixed, oscillatory potentials, cone, and 30 Hz-flicker responses) with amplitudes in mV (Y axis) and time in ms (X axis) from a healthy age-matched subject (upper panels), a patient with Bardet-Biedl syndrome (case #2) presenting rod-cone dystrophy (middle panels), and a patient with Bardet-Biedl syndrome (case #3) presenting cone-rod dystrophy (lower panels).

BBS patients presenting nystagmus, but a tendency to exotropia was not found in our cohort (20,21).

The lack of correlations between BCVA in the betterseeing eye and peak-to-peak amplitude for cone responses and between BCVA and peak-to-peak amplitude for 30-Hz flicker responses was not unusual. Because full-field ERG may not be sensitive enough to detect minimal regional responses from the central retina, these findings confirm previous reports (16,19).

The rod-cone degeneration with no rod responses and elevated dark-adapted thresholds observed in the vast majority of patients confirm previous reports (13,15).

Measurable cone-flicker responses were detected in 28/36 patients, and these responses can be used to objectively follow the natural history of the disease (13). In the current

previous reports in cohorts of three genetic BBS variants (1,16). However, other reports have shown decreased vision in the first decades of life, as found in our studied sample (5,21). Good BCVA in the better-seeing eye ($20/40) was found in only five patients (21.7%) in our study. These results are similar to those results reported in a group of 10 BBS patients, with four patients showing good visual acuity (16). Legal blindness (BCVA in the better-seeing eye #20/ 200) was present in two (8.7%) older patients (cases #21 and 23). Longitudinal data from these patients should be pursued to detect the visual acuity loss rate (in lines/year), as previously demonstrated (12). The frequency of nystagmus or strabismus was 26%. In six patients with strabismus, there was a fair distribution between esotropias and exotropias. Similar findings have been described in young

Figure 2 - ERG parameters for rod, mixed, cone, and 30-Hz flicker responses recorded from one eye of 23 patients with Bardet-Biedl syndrome compared with normative data from our own laboratory (rectangle = meanยก2 SD).

148

CLINICS 2012;67(2):145-149

Visual acuity and retinal function in Bardet-Biedl syndrome Berezovsky A et al.

study, all patients had severely reduced or extinguished ERG parameters. These findings were also seen in previous studies in young patients and a series of patients with BBS mutations, with younger patients showing less photoreceptor degeneration (1,12). These data show that BBS retinal disease, which is primarily a degeneration of photoreceptors, must affect functional processes common to both rods and cones. Both central (acuities) and more peripheral (thresholds) functions are affected (12). Beside functional abnormalities measured by ERG, disrupted inner and outer segment layers and thinned RPE have been described in vivo

by Fourier-domain OCT with inner retinal layer preservation (6,16).

Male predominance was found in our cohort, with a male-to-female ratio of 1558 being observed. These results are consistent with previously reported findings in both adults and pediatric BBS patients (20,21). However, other studies have described a slight female predominance (5).

These discrepancies could be explained by the low incidence of this recessive condition, which leads to studies with a limited number of participants.

In most developing countries, access to modern molecular diagnostic techniques is extremely limited, reinforcing the need for a detailed phenotypic description to achieve precise clinical diagnoses of several genetic eye disorders, including BBS. In this highly variable disease, not all features are present, as described in a recent case report of two brothers with proven BBS1 mutation without obesity (26). Efforts to make these molecular diagnostic techniques available to all patients with genetic ocular diseases should be pursued.

ERG scotopic responses were absent in the vast majority of cases, and cone responses remained in less than half of patients. These results confirm the early deleterious effects in retinal function and visual acuity caused by BBS.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

ACKNOWLEDGMENTS This study was supported by 1) Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) grants #04/02669-6 and 05/56459-5 to AB; 2) Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Brası´lia, Brazil grant #474251/2009-8 to AB; and 3) Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq, Brası´lia, Brazil) research scholarships to AB and SRS.

17.

AUTHOR CONTRIBUTIONS

20.

18.

19.

Berezovsky A designed, conducted and analyzed the material; prepared, reviewed and approved the manuscript. Saloma˜o SR designed the study and analyzed the material; prepared, reviewed and approved the manuscript.

Rocha DM collected and analyzed data; reviewed and approved the manuscript. Sacai PY collected data; reviewed and approved the manuscript.

Watanabe SS collected and analyzed data; reviewed and approved the manuscript. Cavascan NN collected data, reviewed and approved the manuscript.

21.

22.

23.

REFERENCES

24.

1. He´on E, Westall C, Carmi R, Elbedour K, Panton C, MacKeen L, et al.

Ocular phenotypes of three genetic variants of Bardet-Biedl syndrome.

Am J Med Gen. 2005;132A:283-7, http://dx.doi.org/10.1002/ajmg. a.30466.

2. Beales P, Warner AM, Hitman GA, Thakker R, Flinter FA. Bardet-Biedl syndrome: a molecular and phenotypic study of 18 families. J Med Genet. 1997;34:92-8, http://dx.doi.org/10.1136/jmg.34.2.92.

3. Moore SJ, Green JS, Fan Y, Bhogal AK, Dicks E, Fernandez BA, et al.

Clinical and genetic epidemiology of Bardet–Biedl syndrome in Newfoundland: a 22-year prospective, population-based, cohort study.

25.

26.

149

Am J Med Gen. 2005;132A:352-60, http://dx.doi.org/10.1002/ajmg. a.30406.

Schachat AP, Maumenee IH. Bardet-Biedl syndrome and related disorders. Am j Ophthalmol. 1982;100:285-8, http://dx.doi.org/ 10.1001/archopht.1982.01030030287011.

Green JS, Parfrey PS, Harnett JD, Farid NR, Cramer BC, Johnson G, et al.

The cardinal manifestations of Bardet-Biedl syndrome, a form of Laurence-Moon-Biedl syndrome. New Eng J Med. 1989;321:1002-9, http://dx.doi.org/10.1056/NEJM198910123211503.

Gerth C, Zawadski RJ, Werner JS, He´on E. Retinal morphology in patients with BBS1 and BBS10 related Bardet–Biedl syndrome evaluated

by Fourier-domain optical coherence tomography. Vision Res. 2008;48:392-9, http://dx.doi.org/10.1016/j.visres.2007.08.024.

Baker K, Beales PL. Making sense of cilia in disease: the human ciliopathies. Am J Med Genet Part C Semin Med Genet. 2009;151C:281– 95, http://dx.doi.org/10.1002/ajmg.c.30231.

Janssen S, Ramaswami G, Davis EE, Hurd T, Arik R, Kasanuki K, et al.

Mutation analysis in Bardet-Biedl syndrome by DNA pooling and massive parallel resequencing in 105 individuals. Hum Gen. 2011;129:70-90.

Chen J, Smaoui N, Hammer MB, Jiao X, Riazuddin SA, Harper S, et al.

Molecular analysis of Bardet-Biedl syndrome families: report of 21 novel mutations in 10 genes. Invest Ophthalmol Vis Sci. 2011;52(8):5317-24, http://dx.doi.org/10.1167/iovs.11-7554.

Mockel A, Perdomo Y, Stutzmann F, Letsch J, Marion V, Dollfus H. Retinal dystrophy in Bardet-Biedl syndrome and related syndromic ciliopathies. Prog Retin Eye Res. 2011;30:258-74, http://dx.doi.org/ 10.1016/j.preteyeres.2011.03.001.

Deveault C, Billingsley G, Duncan JL, Bin J, Theal R, Vincent A, et al. BBS Genotype–Phenotype Assessment of a Multiethnic Patient Cohort Calls for a Revision of the Disease Definition. Hum Mutat. 2011;32(6):610-9, http://dx.doi.org/10.1002/humu.21480.

Fulton AB, Hansen RM, Glynn RJ. Natural course of visual functions in the Bardet-Biedl syndrome. Arch Ophthalmol. 1993;111:1500-6, http:// dx.doi.org/10.1001/archopht.1993.01090110066026.

Riise R, Andre´asson S, Tornqvist K. Full-field electroretinograms in individuals with the Laurence-Moon-Bardet-Biedl syndrome. Acta Ophthalmol Scand. 1996;74:618-20, http://dx.doi.org/10.1111/j.16000420.1996.tb00747.x.

Ianaconne A, Vingolo EM, Rispoli E, De Propis G, Tanzilli P, Pannarale MR. Electroretinographic alterations in the Laurence-Moon-Bardet-Biedl phenotype. Acta Ophthalmol Scand. 1996;74:8-13, http://dx.doi.org/ 10.1111/j.1600-0420.1996.tb00673.x.

Fleischhauer J, Njoh WA, Niemeyer G. Syndromic retinitis pigmentosa: ERG and phenotypic changes. Klin Monbl Augenheilkd. 2005;222:186-90, http://dx.doi.org/10.1055/s-2005-857973.

Azari AA, Aleman TS, Cideciyan AV, Schwartz SB, Windsor EAM, Sumaroka A, et al. Retinal disease expression in Bardet-Biedl syndrome1 (BBS1) is a spectrum from maculopathy to retina-wide degeneration.

Invest Ophthalmol Vis Sci. 2006;47(11):5004-10, http://dx.doi.org/ 10.1167/iovs.06-0517.

Cox GF, Hansen RM, Quinn N, Fulton AB. Retinal function in carriers of Bardet-Biedl syndrome. Arch Ophthalmol. 2003;121:804-10, http:// dx.doi.org/10.1001/archopht.121.6.804.

Kim LS, Fishman GA, Seiple WH, Szlyk JP, Stone EM. Retinal dysfunction in carriers of Bardet-Biedl syndrome. Ophthalmic Genet.

2007;28:163-8, http://dx.doi.org/10.1080/13816810701537440.

Riise R, Andre´asson S, Wright AF, Tornqvist K. Ocular findings in Laurence-Moon-Bardet-Biedl syndrome. Acta Ophthalmol Scand.

1996a;74:612-7, http://dx.doi.org/10.1111/j.1600-0420.1996.tb00746.x.

Ianaconne A, De Propis G, Roncati S, Rispoli E, Del Porto G, Pannarale MR. The ocular phenotype of the Bardet-Biedl syndrome. Ophthalmic Genet. 1997;18:13-26, http://dx.doi.org/10.3109/13816819709057879.

Spaggiari E, Salati R, Nicolini P, Borgatti R, Pozzoli U, Polenghi F. Evolution of ocular clinical and electrophysiological findings in pediatric Bardet-Biedl syndrome. Int Ophthalmol. 1999;23:61-7, http:// dx.doi.org/10.1023/A:1026560721525.

Marmor MF, Fulton AB, Holder GE, Miyake Y, Brigell M, Bach H. ISCEV standard for full-field clinical electroretinography (2008 update). Doc Ophthalmol. 2009;118:69-77, http://dx.doi.org/10.1007/s10633-0089155-4.

Berezovsky A, Moraes NSB, Nusinowitz S, Saloma˜o SR. Standard full-field electroretinography in healthy preterm infants. Doc Ophthalmol.

2003;107:243-9, http://dx.doi.org/10.1023/B:DOOP.0000005333.76622.c2.

Peters AY, Locke KG, Birch DG. Comparison of the Goldmann-Weekers dark-adaptometer and LKC Technologies scotopic sensitivity tester-1. Doc Ophthalmol. 2000;101:1-9, http://dx.doi.org/10.1023/A:1002765024774.

Pereira JM, Mendieta L, Sacai PY, Saloma˜o SR, Berezovsky A. Estudo normativo do eletrorretinograma de campo total em adultos jovens. Arq Bras Oftalmol. 2003;66:137-44, http://dx.doi.org/10.1590/S000427492003000200005.

Cannon PS, Clayton-Smith J, Beales PL, Lloyd IC. Bardet-Biedl syndrome: an atypical phenotype in brothers with a proven BBS1 mutation. Ophthalmic Genet. 2008;29:128-32, http://dx.doi.org/ 10.1080/13816810802216464.

CLINICS 2012;67(2):151-156

DOI:10.6061/clinics/2012(02)10

CLINICAL SCIENCE

Classification of the severity of diabetic neuropathy: a new approach taking uncertainties into account using fuzzy logic Andreja P. Picon,I Neli R. S. Ortega,II Ricky Watari,I Cristina Sartor,I Isabel C. N. SaccoI I

Faculdade de Medicina da Universidade de Sa˜o Paulo, Physical Therapy, Speech and Occupational Therapy Department, Sa˜o Paulo/SP, Brazil. II Faculdade de Medicina da Universidade de Sa˜o Paulo, Center of Fuzzy Systems in Health, Sa˜o Paulo/SP, Brazil.

OBJECTIVE: This study proposes a new approach that considers uncertainty in predicting and quantifying the presence and severity of diabetic peripheral neuropathy.

METHODS: A rule-based fuzzy expert system was designed by four experts in diabetic neuropathy. The model variables were used to classify neuropathy in diabetic patients, defining it as mild, moderate, or severe. System performance was evaluated by means of the Kappa agreement measure, comparing the results of the model with those generated by the experts in an assessment of 50 patients. Accuracy was evaluated by an ROC curve analysis obtained based on 50 other cases; the results of those clinical assessments were considered to be the gold standard.

RESULTS: According to the Kappa analysis, the model was in moderate agreement with expert opinions. The ROC analysis (evaluation of accuracy) determined an area under the curve equal to 0.91, demonstrating very good consistency in classifying patients with diabetic neuropathy.

CONCLUSION: The model efficiently classified diabetic patients with different degrees of neuropathy severity. In addition, the model provides a way to quantify diabetic neuropathy severity and allows a more accurate patient condition assessment.

KEYWORDS: Diabetic Neuropathies; Fuzzy sets; Diabetes mellitus; Expert systems.

Picon AP, Ortega NRS, Watari R, Sartor C, Sacco ICN. Classification of the severity of diabetic neuropathy: a new approach taking uncertainties into account using fuzzy logic. Clinics. 2012;67(2):151-156.

Received for publication on November 8, 2011; First review completed on November 20, 2011; Accepted for publication on November 23, 2011 E-mail: icnsacco@usp.br Tel.: 55 11 3091-8426

static posture in diabetic neuropathic patients (3-5). Sensory, motor, and autonomic deficits may increase the risk of falling and ulcer formation in these patients (6,7).

The diagnosis of DSPN is generally made based on neurologic signs and symptoms and electrophysiologic measurements (8). However, health experts define the degree of neuropathy in a subjective way. Because neuropathy onset is insidious and manifests differently for each patient, expert experience plays an important role in the classification of neuropathy.

Many screening instruments with numerous composite scores are used to evaluate neuropathy. The tools that are most frequently used in the literature are the Michigan Neuropathy Screening Instrument questionnaire (MNSIq) and Physical Assessment (9) and the Diabetic Neuropathy Symptom (DNS) score, both of which are used solely for screening (10). Neither instrument provides a disease severity rating. Other instruments, such as the Neuropathy Disability Score (NDS) (11), Neuropathy Impairment Score (NIS) (12), Toronto Clinical Scoring System (TCSS) (13), and Clinical Neuropathy Examination (CNE) (14,15), assign patients to different disease levels.

Although these questionnaires are widely used in clinical practice, there is no consensus regarding which tool is best

INTRODUCTION Diabetic sensorimotor polyneuropathy (DSPN) is the most common chronic complication associated with diabetes mellitus. DSPN encompasses a group of clinical and subclinical syndromes with varied etiologies and clinical and laboratorial manifestations, defined by the progressive diffuse or focal degeneration of peripheral somatic and autonomic nerve fibers (1). Its prevalence is directly related to diabetes duration, patient age, and metabolic control.

Approximately 20% of diabetic patients will develop clinically significant neuropathy within 10 years of diabetes onset, and this proportion can increase to 50% after 10 or 15 years (2).

Somatosensory inputs decrease as diabetic neuropathy advances, and motor output becomes progressively more impaired. These issues may lead to the higher instability and kinematic abnormalities observed during locomotion and

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

151

Diabetic neuropathy and fuzzy logic Picon AP et al.

CLINICS 2012;67(2):151-156

suited for disease diagnosis or severity evaluation. Thus, it is common for individual clinics to develop their own protocols, in which specialized laboratory tests are used in conjunction with questionnaires (16).

The gold standard for detecting DSPN is the nerve conduction velocity test. It can diagnose sensory and motor losses due to neuropathy even when dysfunction is subclinical, and it can predict ulceration and mortality in diabetic patients (8,17,18). However, this invasive, painful test must be performed by specialists, and it is not commonly available in public health units.

The 10-g monofilament evaluation is another specialized test that is strongly recommended by the International Consensus on the Diabetic Foot. Monofilament evaluation is a good tool to assess the loss of protective sensation related to diabetic neuropathy (19). It has a high reproducibility and specificity and may be used to predict ulceration and amputation risks (20).

Glycemic control may be considered as an auxiliary measure for predicting chronic diabetes mellitus complications, including DSPN. However, this information is questionable because daily glycemic rates are highly variable. The clinical measurement of glycosylated hemoglobin (HbA1c) is more recommended for diabetes management. Maintaining HbA1c levels below 6.5% is a major goal of diabetes management because A1c levels correlate well with diabetes complications risks (21).

The tests considered here involve some uncertainties with respect to the measurement process and subsequent diagnosis. Furthermore, the boundary between sickness and health is not always clear. When this boundary is defined, the classification of severity follows binary logic, ignoring the fact that complications can represent continuous processes. Furthermore, severity classification depends on a subjective analysis by the examiner. It is important to consider these uncertainties to obtain a precise classification of DSPN severity. One of the most powerful tools to deal with this type of data is fuzzy set theory.

Fuzzy set theory was introduced by Lotfi Zadeh in 1965. It is a mathematical entity that was developed to handle problems in which there is uncertainty about whether an element is part of a certain set. Fuzzy sets structure makes it possible to deal with the concept of partial truths and the elaboration of linguistic variables (22). Thus, this theory allows us to assess the degree of diabetic neuropathy.

In this paper, we propose a fuzzy expert-based rule system for quantifying diabetic neuropathy to categorize patients into severity subsets.

Model development Fuzzy rule-based models have a simple structure and consist of four major components: 1) a fuzzification module, which translates crisp inputs (classical measurements) into fuzzy values through linguistic variables; 2) an if-then fuzzy rule base, which consists of a set of conditioned fuzzy propositions; 3) an inference method, which applies fuzzy reasoning mechanisms to obtain outputs (i.e., carries out the computation using fuzzy rules); and 4) a defuzzification module, which translates fuzzy outputs back to crisp values, if necessary (22).

The fuzzy model was elaborated based on the experience and knowledge of four diabetic neuropathy experts. These experts considered the following inputs as the most important variables: symptom assessment based on the MNSI questionnaire score, sign examination based on the MNSI Physical Assessment score, level of HbA1c, and the duration of diabetes, measured in years. The chosen tests require only simple procedures and can thus be applied in a clinical environment without the use of sophisticated equipment or specialized personnel.

The experts defined limiting values to establish each fuzzy set. They considered the impact of each assessment item on disease degree based on specific literature, their own daily experience in diagnosing these patients and the simplicity of the screening method. For the MNSI questionnaire, items that assess nocturnal worsening of symptoms and past ulcers were weighted as 2.0 points, while questions about numbness, burning, tingling, pins and the diagnosis of neuropathy from another health professional were weighted as 1.0 point. The physical evaluation of the MNSI foot inspection scored 0.5 point, and the monofilament and vibration perception tests scored 1.0 point each.

Each foot was evaluated separately.

The input variables were fuzzified using the following linguistic terms: (a) (b) (c) (d)

Symptom assessment (score): (1) absent, (2) low, (3) moderate, or (4) high; Sign assessment (score): (1) absent, (2) low, (3) moderate, or (4) high; HbA1c levels: (1) absent or (2) present; Diabetes duration (measured in years): (1) short, (2) moderate, or (3) long.

The fuzzy output sets, shown in Figure 1, indicate the degree of neuropathy. The sets were validated by expert opinion using the following linguistic terms: absent neuropathy, mild neuropathy, moderate neuropathy, and severe neuropathy (Figure 2).

Based on the fuzzy input sets, linguistic rules were elaborated using a combinatory analysis of those variables.

The consequence of each fuzzy rule was determined by the experts, and 96 rules were elaborated in the form of the following example: ‘‘IF symptom assessment is high AND sign assessment is moderate AND HbA1c is present AND diabetes duration is long THEN neuropathy is severe’’.

Using this set of rules and the system input and output values, it is possible to assess the level of diabetic neuropathy of any patient from which those input variables were measured. Neuropathy severity was assessed by means of a Mamdani inference process, and the Center of

METHODS Data acquisition The dataset was composed of 50 real cases from the Hospital of the University of Sa˜o Paulo (Ethics Committee protocol 0305/08).

The inclusion criteria were: diagnosis of type II diabetes; less than 65 years old; no lower limb amputation or severe orthopedic foot deformities (e.g., Charcot arthropathy) confirmed by radiography; no peripheral or central vestibulopathy or any other neurological disease unrelated to diabetes. These criteria were verified by a staff physician from the University Hospital.

152

CLINICS 2012;67(2):151-156

Diabetic neuropathy and fuzzy logic Picon AP et al.

Figure 1 - Linguistic terms used in the model input sets.

Area defuzzification method was applied. This produces a quantitative value for the neuropathy level, defined as a number between 0 and 10.

We used a dataset comprising 50 hypothetical cases of diabetic neuropathy elaborated by the experts to refine the fuzzy model. To illustrate the mathematical function determined by the rule base of the model, Figure 3 shows the surface representing the severity of the disease based on the inputted signs and symptoms.

Performance evaluation We evaluated the model performance in two different ways: i) we assessed the agreement between the model and expert classifications with the Kappa test; and ii) we evaluated the model sensitivity and specificity with an ROC analysis, which is considered the gold standard for neuropathy diagnosis.

For the agreement analysis, four experts evaluated a separate set of 50 real cases based on clinical data gathered

Figure 2 - Linguistic terms used in the model output sets.

153

Diabetic neuropathy and fuzzy logic Picon AP et al.

CLINICS 2012;67(2):151-156

Figure 3 - Surface representing the severity of the disease based on symptoms and signs.

Figure 4 shows the ROC performance of the model. The area under the curve was 0.91, indicating that the model is able to identify neuropathy in diabetic patients.

from patient charts. Each case received a linguistic classification indicating the degree of neuropathy (absent, mild, moderate, or severe). The model produces numerical values, which were sorted into the following classes: (i) 0 – 2.5: absent neuropathy; (ii) 2.6 – 4.5: mild neuropathy; (iii) 4.6 – 7.5: moderate neuropathy; and (iv) 7.6 – 10.0: severe neuropathy.

The model results were compared with those provided by each expert, and the expert analyses were compared with each other. Thus, we were able to determine whether model performance was consistent with human expert assessments.

RESULTS Table 1 shows the Kappa values for test agreement between the model and the experts. The level of agreement among experts was only moderate (23), highlighting the uncertainty in classifying the degree of diabetic neuropathy in these patients. The agreement between the model and the experts was also moderate, i.e., the model agrees with the experts as much as they agree with each other. In all statistical tests, p,0.001.

Table 1 - Kappa coefficient values between expert and model ratings (p,0.001).

Expert 2 Expert 3 Expert 4 Model

Expert 1

Expert 2

Expert 3

Expert 4

0.48 0.75 0.44 0.53

0.42 0.22 0.47

0.51 0.50

0.34

Figure 4 - ROC curve composed based on the gold standard database.

154

CLINICS 2012;67(2):151-156

Diabetic neuropathy and fuzzy logic Picon AP et al.

This paper proposes a fuzzy rule-based model to create an expert system to support the classification of diabetic neuropathy into different levels based on severity. The model showed an adequate level of agreement with the expert classifications, as well as high assessment accuracy in real patients. The model is able to classify patients with different degrees of neuropathy, which may improve treatment effectiveness and aid health professionals in managing this syndrome. This system supports the concept of classifying patients with different degrees of diabetic neuropathy, rather than identifying them as simply neuropathic or non-neuropathic.

This model is able to match the ability of clinical experts in classifying neuropathy severity in diabetic patients, and it does not require sophisticated testing or equipment. This model can be implemented in any public health system and may serve as an important instrument for preventing complications due to disease progression. The model also enables immediate intervention in early stages of neuropathy to maintain patient quality of life.

DISCUSSION There is no doubt that the process of identifying and classifying diabetic patients as neuropathic is unclear and subjective. Uncertainties in both symptom measurement and diagnoses complicate this process. The skill and experience of the expert evaluating the patient and the available instruments are important factors. Thus, each clinic sets its own protocols without an established consensus.

Bus et al. (24) identified neuropathy based solely on the 10-g monofilament test or vibratory sensitivity. Allet et al.

(25) categorized neuropathy using only the vibratory sensitivity test. However, the isolated use of different identification and classification methods can lead to bias because mild neuropathies often cannot be identified.

Moreover, symptoms may vary from patient to patient, especially in the early stages of the disease. Although the disease is symmetrical and distal, there can be differences in the plantar areas affected by sensory impairments, the type of foot deformity (cavus, planus, hammer toes, etc.) and the extent of motor impairment. This means that one patient may have clinical features that cannot be compared directly to those of another patient, even if both patients were diagnosed with the same degree of neuropathy.

Few studies have proposed the combination of several techniques to identify diabetic neuropathy. For example, Bacarin et al. (26) suggested the use of the MNSIq, 10-g monofilament sensitivity test, and disease duration as criteria for neuropathic severity. However, this proposal does not take the vagueness of the diagnostic process into account.

Few studies have sought to address these problems.

Duckstein et al. used fuzzy set structure to classify diabetic neuropathy based on an invasive electrophysiological test (27). However, this equipment requires specific knowledge and appropriate professional training, and it is thus not widely used in public health systems, especially in poorer countries. Furthermore, although this test is very reliable, it cannot detect neuropathy in the early stages of the disease.

Indeed, the electrophysiological test only provides clinically useful information when neural degeneration has already begun.

Conversely, the fuzzy model proposed here was based on expert knowledge and four simple variables that are easily determined in routine clinical assessments, avoiding the necessity of sophisticated and invasive methods that require a specially trained staff. The model was able to address diagnostic and inter-measurement uncertainties, identify neuropathy, and quantify its severity.

The agreement analysis presented here showed that this model is able to classify the severity of the disease in the same manner as human experts. This suggests that the system would pass the Turing test. The moderate agreement among the interviewed experts shows how difficult it is to come to consensus on the clinical severity of neuropathy based solely on physical anamnesis. Therefore, fuzzy theory can be used to address this problem.

One of the most important contributions of this model is its ability to detect diabetic neuropathy early in its progression. The literature suggests that diabetic neuropathy may be irreversible (2); therefore, timely preventive intervention at early stages may help to ensure a better quality of life for diabetic patients.

ACKNOWLEDGMENTS We would like to acknowledge funding from CNPq (Picon PhD scholarship 140848/2009-6, Watari Master scholarship 556374/2010-0) and CAPES (Sartor scholarship). We would also like to thank Aline Arcanjo Gomes and Tatiana de Almeida Bacarin for their participation as experts in the development of the fuzzy model.

AUTHOR CONTRIBUTIONS All the authors participated in the study design, results discussion, and final revision. Picon AP and Ortega NRS wrote the first draft and were responsible for fuzzy model development and statistical analysis. Sartor C and Watari R acquired patient data at the hospital. Sacco ICN and Sartor C coordinated the research and participated as experts in fuzzy model development.

REFERENCES 1. Greene DA, Sima AAF, Stevens MJ, Feldman EL, Lattimers A. Complications: neuropathy, pathogenetic considerations. Diabetes Care. 1992;15:1902-25, http://dx.doi.org/10.2337/diacare.15.12.1902.

2. Rathur HM, Boulton AJM. Recent advances in the diagnosis and management of diabetic neuropathy. J Bone Joint Surg. 2005;87B(12): 1605-10.

3. Shaw JE, Van Schie CHM, Carrington AL, Abbot CA, Boulton AJM. An analysis of dynamic forces transmitted through the foot in diabetic neuropathy. Diabetes Care. 1998;21(11):1955-9, http://dx.doi.org/ 10.2337/diacare.21.11.1955.

4. Akashi PM, Sacco IC, Watari R, Hennig E. The effect of diabetic neuropathy and previous foot ulceration in EMG and ground reaction forces during gait. Clin Biomech (Bristol, Avon). 2008;23:584-92, http:// dx.doi.org/10.1016/j.clinbiomech.2007.11.015.

5. Sacco ICN, Hamamoto AN, Gomes AA, Onodera AN, Hirata RP, Hennig EM. Role of ankle mobility in foot rollover during gait in individuals with diabetic neuropathy. Clin Biomech. 2009;24(8):687-92, http:// dx.doi.org/10.1016/j.clinbiomech.2009.05.003.

6. Perry JE, Hall JO, Davis BL. Simultaneous measurement of plantar pressure and shear forces in diabetic individuals. Gait Posture.

2002;15:101-7, http://dx.doi.org/10.1016/S0966-6362(01)00176-X. 7. Davies M, Brophy S, Williams R, Taylor A. The Prevalence, severity and impact of painful diabetic peripheral neuropathy in type II diabetes.

Diabetes Care. 2006;29(7):1518-22, http://dx.doi.org/10.2337/dc05-2228.

8. Dyck PJ, Albers JW, Andersen H, Arezzo JC, Biessels G, Bril V, et al.

Diabetic polyneuropathies: update on research definition, diagnostic criteria and estimation of severity. Diabetes Metab Res Rev. In press 2011, http://dx.doi.org/10.1002/dmrr.1226.

9. MNSI – Michigan Neuropathy Screening Instrument. Michigan Diabetes Research and Training Center. 2010 [cited 2010 Aug]. Available from: URL: http://www.umich.edu/mdrtc/survey/index/html#mnsi.

10. Meijer JW, Smith AJ, Sonderen EV, Groothoff JW, Eisma WH, Links TP. Symptom scoring systems to diagnose distal polyneuropathy in diabetes:

155

Diabetic neuropathy and fuzzy logic Picon AP et al.

11.

12.

13.

14.

15.

16.

17.

18.

CLINICS 2012;67(2):151-156

the diabetic neuropathy symptom score. Diabet Med. 2002;19(11):962-5, http://dx.doi.org/10.1046/j.1464-5491.2002.00819.x.

Service FJ, Rizza RA, Daube JR, O’Brien PC, Dyck PJ. Near normoglycaemia improved nerve conduction and vibration sensation in diabetic neuropathy. Diabetologia. 1985;28(10):722-7.

Dyck PJ, Davies JL, Daube J, Litchy WJ, O’Brien P. Longitudinal assessment of diabetic polyneuropathy using a composite score in the Rochester Diabetic Neuropathy Study cohort. Neurology. 1997;49(1):22939.

Bril V, Perkins BA. Validation of the Toronto Clinical Scoring System for diabetic polyneuropathy. Diabetes Care. 2002;25(11):2048-52, http:// dx.doi.org/10.2337/diacare.25.11.2048.

Valk GD, de Sonnaville JJ, van Houtum WH, Heine RJ, van Eijk JT, Bertelsmann FW. The assessment of diabetic polyneuropathy in daily clinical practice: reproducibility and validity of Semmes Weinstein monofilaments examination and clinical neurological examination.

Muscle Nerve. 1997;20(1):116-8, http://dx.doi.org/10.1002/(SICI)10974598(199701)20:1,116::AID-MUS19.3.0.CO;2-2. van de Poll-Franse LV, Valk GD, Renders CM, Heine RJ, van Eijk JT. Longitudinal assessment of the development of diabetic polyneuropathy and associated risk factors. Diabet Med. 2002;19(9):771-6, http:// dx.doi.org/10.1046/j.1464-5491.2002.00778.x.

Wu EQ, Borton J, Said G, Le TK, Monz B, Rosilio M, et al. Estimated prevalence of peripheral neuropathy and associated pain in adults with diabetes in France. Curr Med Res Opin. 2007;23(9):2035-42, http:// dx.doi.org/10.1185/030079907X210516.

Tesfaye S, Boulton AJM, Dyck PJ, Freeman R, Horowitz M, Kempler P, et al. Diabetic neuropathies: update on definitions, diagnostic criteria, estimation of severity, and treatments. Diabetes Care. 2010;33:2285-93, http://dx.doi.org/10.2337/dc10-1303.

Carrington AL, Shaw JE, Van Schie CH, Abbott CA, Vileikyte L, Boulton AJ. Can motor nerve conduction velocity predict foot problems in diabetic subjects over 6-year outcome period? Diabetes Care.

2002;25(11):2010-5, http://dx.doi.org/10.2337/diacare.25.11.2010.

19. Apelqvist J, Bakker K, van Houtum WH, Schaper NC. Practical Guidelines on the management and prevention of the diabetic foot.

Based upon the International Consensus on the diabetic Foot (2007); Prepared by the International Working Group on the diabetic foot.

Diabetes Metab Res Rev. 2008;24(Suppl1):S181-7, http://dx.doi.org/ 10.1002/dmrr.848.

20. Sosenko JM, Kato M, R Soto R, Bild DE. Comparison of quantitative sensory-threshold measures for their association with foot ulceration in diabetic patients. Diabetes Care. 1990;13(10):1057-61, http:// dx.doi.org/10.2337/diacare.13.10.1057.

21. International Expert Committee. International Expert committee report on the role of the A1c assay in the diagnosis of diabetes. Diabetes Care.

2009;32:1327-34, http://dx.doi.org/10.2337/dc09-9033.

22. Massad E, Ortega NRS, Barros LC, Struchiner CJ. Fuzzy logic in action: Applications in epidemiology and beyond. New York: Springer; 2008.

23. Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics. 1977;33:159-74, http://dx.doi.org/10.2307/ 2529310.

24. Bus SA, Deursen RWM, Kanade RV, Wissink M, Manning EA, Baal JG, et al. Plantar pressure relief the diabetic foot using forefoot offloading shoes. Gait & Posture. 2009;29(4):618-22, http://dx.doi.org/10.1016/ j.gaitpost.2009.01.003.

25. Allet L, Armand S, Aminian K, Pataky Z, Golay A, Bie RA, et al. An exercise intervention to improve diabetic patients’ gait in a real-life environmental. Gait & Posture. 2010;32(2):185-90, http://dx.doi.org/ 10.1016/j.gaitpost.2010.04.013.

26. Bacarin TA, Sacco ICN, Hennig EM. Plantar pressure distribution patterns during gait in diabetic neuropathy patients with a history of foot ulcers. Clinics. 2009;64(2):113-20.

27. Duckstein L, Blinowska A, Verroust J. Fuzzy classification of patient state with application to electrodiagnosis of peripheral polyneuropathy. IEEE Trans Biomed Eng. 1995;42(8):786-92, http://dx.doi.org/10.1109/ 10.398639.

156

CLINICS 2012;67(2):157-162

DOI:10.6061/clinics/2012(02)11

CLINICAL SCIENCE

Clinical and serological manifestations associated with interferon-a levels in childhood-onset systemic lupus erythematosus Mariana Postal,I Nailu Ange´lica Sinicato,II Karina Oliveira Pelic¸ari,I Roberto Marini,III Lilian Tereza Lavras Costallat,I Simone AppenzellerI I State University of Campinas, Faculty of Medical Science, Department of Medicine, Rheumatology Unit, Campinas/SP, Brazil. II State University of Campinas, Faculty of Medical Science, Postgraduate Program of Child and Adolescent Health, Campinas/ SP, Brazil. III State University of Campinas, Faculty of Medical Science, Department of Pediatrics, Pediatric Rheumatology Unit, Campinas/SP, Brazil.

OBJECTIVE: To determine the serum levels of interferon alpha in childhood-onset systemic lupus erythematosus patients, their first-degree relatives and healthy controls and to evaluate the associations between serum interferon alpha and disease activity, laboratory findings and treatment features.

METHODS: We screened consecutive childhood-onset systemic lupus erythematosus patients in a longitudinal cohort at the pediatric rheumatology unit of the State University of Campinas between 2009 and 2010. All patients demonstrated disease onset before the age of 16. Disease status was assessed according to the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) and Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index (SDI). Interferon alpha levels were measured using an enzyme-linked immunoabsorbent assay.

RESULTS: We included 57 childhood-onset systemic lupus erythematosus patients (mean age 17.33¡4.50), 64 firstdegree relatives (mean age 39.95¡5.66), and 57 healthy (mean age 19.30¡4.97) controls. Serum interferon alpha levels were significantly increased in childhood-onset systemic lupus erythematosus patients compared to their firstdegree relatives and healthy controls. Interferon alpha levels were significantly increased in patients with positive dsDNA antibodies, patients with cutaneous vasculitis, patients with new malar rash and patients who were not receiving medication. Interferon alpha levels correlated with C3 levels and systemic lupus erythematosus Disease Activity Index scores. In addition, we observed an inverse correlation between patient age and interferon alpha levels.

CONCLUSION: Interferon alpha may play a role in the pathogenesis of childhood-onset systemic lupus erythematosus, especially in cutaneous manifestations and dsDNA antibody formation. The observation that interferon alpha levels are increased in patients who are not taking medication should be investigated in longitudinal studies to determine whether elevated interferon alpha levels may predict systemic lupus erythematosus flares.

KEYWORDS: Interferon alpha (IFN-a); SLEDAI; Childhood-onset; Systemic lupus erythematosus.

Postal M, Sinicato NA, Pelic¸ari KO, Marini R, Costallat LTL, Appenzeller S. Clinical and serological manifestations associated with interferon-a levels in childhood-onset systemic lupus erythematosus. Clinics. 2012;67(2):157-162.

Received for publication on November 14, 2011; First review completed on December 20, 2011; Accepted for publication on January 10, 2012 E-mail: appenzellersimone@yahoo.com Tel.: 55 19 3289-1818

SLE (1). The pathogenesis of SLE is multifactorial and is likely driven by a complex combination of genetic and environmental factors, which leads to an irreversible failure of immunologic self-tolerance (3).

Approximately 20% of all SLE patients experience disease onset prior to the age of 16 (4). Childhood-onset SLE demonstrates a different phenotype than adult-onset SLE. Renal (50%-67%), neurological (22-95%) and hematological (77%) involvement in addition to fever and lymphadenopathy are observed more frequently in pediatric patients than in patients with adult-onset SLE (4,5-11). In addition, childhood-onset SLE is associated with significantly more active diseases, both at disease onset and over time, compared to adult-onset SLE (9). The outcome of childhood-onset disease

INTRODUCTION Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by diverse clinical manifestations ranging from malar rash to renal involvement (1,2). Immune system disorders and abnormalities in cytokine production have been described in patients with

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

157

IFN-a and childhood-onset systemic lupus erythematosus Postal M et al.

CLINICS 2012;67(2):157-162

suppression (leukopenia ,4000 cells/mm3, thrombocytopenia ,100,000 cells/mm3, and hemolytic anemia). We also assessed the presence of malar rash, discoid lesions, subacute cutaneous lesions, cutaneous vasculitis, photosensitivity, oral ulcers, arthritis, and serositis. Neurological and psychiatric involvement was defined according to the ACR guidelines (20).

The treatment prescribed at the time of blood withdrawal and any adverse events related to medication use were recorded. Doses of oral and parenteral corticosteroids were analyzed and converted to the equivalent doses of prednisone.

is also generally worse than that of adult-onset disease (4,12).

The awareness that SLE in childhood is a potentially fatal disease, that atypical presentations are very common, and that aggressive treatment should be introduced early in the course of the disease have significantly improved survival in patients with childhood-onset SLE (13,14).

There is strong evidence indicating that cytokines play a role in the pathogenesis of SLE (15,16). The first cytokine abnormality documented in SLE was an increased serum level of interferon (IFN), subsequently characterized as IFNa, which is produced mainly by leukocytes (17). Elevated serum IFN-a has also been observed in adult-onset SLE, and IFN-a levels correlate with both disease activity and disease severity (15,17,18). Associations have also been observed between IFN-a levels and several markers of immune activation, such as complement activation and doublestranded DNA (dsDNA) antibody titers (15). However, the role of IFN-a in childhood-onset SLE has not been investigated.

The aim of our study was to determine the serum levels of IFN-a in childhood-onset SLE patients, their first-degree relatives and healthy controls. In addition, we evaluated the association of IFN-a with disease activity, laboratory findings and treatment features.

Laboratory studies Antinuclear antibody (ANA) levels were determined by indirect immunofluorescence using mouse liver as a substrate and were regarded as positive if the titers were higher than 1540. The levels of dsDNA antibodies were determined by indirect immunofluorescence using Crithidia as a substrate and were considered positive if they were higher than 1510.

The levels of precipitating antibodies to extractable nuclear antigens (ENA), including Ro (SSA), La (SSB), and Sm, were detected using a standardized enzyme-linked immunosorbent assay (ELISA) method and were considered positive if higher than 1580. The levels of IgG and IgM anticardiolipin antibodies (aCL) were measured by ELISA (21). Lupus anticoagulant (LA) activity was detected by coagulation assays in platelet-free plasma obtained by double centrifugation following the recommendations of the Scientific and Standardization Committee of the International Society of Thrombosis and Homeostasis subcommittee on LA (22).

These measurements were performed twice at an interval of 12 weeks.

METHODS Subjects Fifty-seven consecutive childhood-onset SLE patients followed at the Pediatric Rheumatology Outpatient Clinic of the State University of Campinas were invited to participate in this cross-sectional study. Patients were included in the present study if they (i) fulfilled at least four of the American College of Rheumatology (ACR) criteria (19), (ii) were younger than 16 years of age at disease onset, and (iii) had a follow-up duration of at least 6 months.

Sixty-four first-degree relatives and 57 healthy controls without a history of any chronic disease (including autoimmune diseases) were included as control groups. The healthy controls were matched for age, sex and demographic background. This study was approved by the ethics committee at our institution, and informed written consent was obtained from each participant and/or legal guardian.

Disease Activity/Cumulative Damage Evaluation Disease activity was measured by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) (23).

The SLEDAI consists of 24 weighted items grouped into 9 domains, or organ systems, as follows: central nervous system (assigned a weight of 8), vascular system (weight of 8), renal system (weight of 4), musculoskeletal system (weight of 4), serosal system (weight of 2), dermal system (weight of 2), immune system (weight of 2), constitutional (weight of 1), and hematologic system (weight of 1). The SLEDAI scores range between 0 and 105, and scores of $3 were considered to represent active disease (24).

Cumulative SLE-related damage in all patients was determined using the Systemic Lupus International Collaborating Clinics (SLICC)/ACR Damage Index (SDI) measured at the time of blood withdrawal. The SDI scores ranged from 0 to 47 (25).

Clinical features The medical histories and clinical and serological characteristics of each patient were entered into a computer database at the time of SLE diagnosis. The following variables were included in this database: onset of disease, defined as the age at which the first symptoms clearly attributable to SLE occurred; age at diagnosis, defined as the age at which the patients fulfilled four or more of the 1982 revised criteria for the classification of SLE (19); and followup time, defined as the time from disease onset until May 2010.

All clinical manifestations and laboratory findings were recorded at disease onset on a quarterly basis throughout the follow-up period and on the day of blood withdrawal.

Nephritis was defined as proteinuria exceeding 0.5 g/L with abnormal urinary sediment and/or histological findings. Nephrotic syndrome was defined as proteinuria in excess of 3 g/day. Hematological abnormalities were ascribed to lupus only in the absence of bone-marrow

IFN-a assay Peripheral venous blood was collected from each subject and allowed to clot at room temperature for 30 min.

Samples were then centrifuged for 15 min at 3000 rpm.

Separated sera were stored in aliquots at 280 ËšC for subsequent use in assays. None of the samples were taken during an episode of acute or chronic infection (26).

Commercially available kits from R&D Systems (London, UK) were used to measure serum IFN-a levels by ELISA in accordance with the manufacturer’s instructions.

158

CLINICS 2012;67(2):157-162

IFN-a and childhood-onset systemic lupus erythematosus Postal M et al.

We included 57 consecutive childhood-onset SLE patients. Fifty-four (94.7%) were female with a mean age of 17.33 years [standard deviation (SD) ยก4.50 years; range 9-37]. Disease duration was 4.71 years (SDยก4.57; range 026). We also investigated 64 first-degree relatives with a mean age of 39.95 years (SDยก5.66; range 28-52). The control group consisted of 57 healthy volunteers (52 women) with a mean age of 19.30 (SDยก4.97 years; range 6-30) (Table 1).

Patients and healthy controls were statistically comparable in terms of age and sex.

between first-degree relatives and healthy controls was observed (p = 0.484) (Figure 1a).

IFN-a levels were significantly increased in patients who were positive for dsDNA antibodies (p = 0.011) (Figure 1b), patients with cutaneous vasculitis (p = 0.001), and patients with a new malar rash (p = 0.032) or disease activity (p = 0.031). IFN-a levels were directly correlated with C3 levels (r = 0.34; p = 0.032) and SLEDAI scores (r = 0.43; p = 0.012) and indirectly correlated with age (r = -0.17; p = 0.025). IFN-a levels were significantly higher in patients who were not taking medication (mean = 13.01; SDยก6.09) compared to patients who were receiving medication (mean = 21.59; SDยก16.02; p = 0.035) (Figure 1c). In an analysis of individual medications, higher levels of IFN-a were observed in patients not taking prednisone (mean = 20.07; SDยก14.65) compared to patients taking prednisone (mean = 12.95; SDยก6.19; p = 0.042). No association between IFN-a levels and other clinical or laboratory variables (hematological or immunological) or SDI scores was observed. No difference in IFN-a levels was observed between patients with and without hydroxychloroquine or other immunosuppressants.

Clinical, laboratory, and treatment features

DISCUSSION

Statistical analyses An analysis of variance with Tukeyโ s pairwise post hoc comparisons was used to compare IFN-a levels between groups. Spearmanโ s correlation was used to correlate continuous variables (e.g., IFN-a levels and SLEDAI, SDI).

IFN-a levels and categorical variables were compared using a 2-sample t-test. For all of the analyses, p-values ,0.05 were considered statistically significant.

RESULTS Demographics

At the time of study entry, 30 (52.6%) childhood-onset SLE patients had active disease (SLEDAI$3) with mean SLEDAI scores of 8.37 (SDยก3.80, range 3-18). Patients with inactive disease [N = 27 (47.4%)] had a mean SLEDAI score of 0.39 [(SDยก0.80 range 0-2)]. Active nephritis (28.3%), new malar rash (6.6%), new alopecia (5.0%), and cutaneous vasculitis (5.0%) were the most frequently observed clinical manifestations.

At the time of blood withdrawal, 8 (13.3%) patients were not taking any medication. Thirty-nine (68.4%) patients were receiving prednisone, 32 (53.3%) were receiving hydroxychloroquine, and 22 (36.6%) patients were receiving other immunosuppressive drugs (Table 1).

Cytokines are low-molecular weight proteins that play a key role in the immunological dysregulation observed in autoimmune diseases. The increased levels of proinflammatory cytokines are believed to be critical in the pathogenesis of SLE (27). Higher cytokine levels in SLE patients may promote the inflammatory response, apoptosis and autoantibody production, which initiate and may also maintain SLE disease activity over time (16,27).

The first cytokine abnormality documented in SLE was an increased serum level of IFN-a, which is a cytokine with both antiviral and immunoregulatory functions (17,28). The contributions of IFN-a to SLE can be explained through several distinct, but related, mechanisms. In genetically susceptible individuals, B cell precursors expressing selfreactive antibodies are not removed (15). Most likely due to a mismanagement of naturally occurring apoptotic cells, nuclear material stimulates autoreactive B-cells, leading to antibody secretion and the formation of immune complexes.

Cytokine assay The mean serum IFN-a level was 13.84ยก8.46 pg/mL in childhood-onset SLE patients, 10.36ยก6.04 pg/mL (p = 0.012) in first-degree relatives and 11.68ยก6.66 pg/mL in healthy controls (p = 0.043). No difference in serum IFN-a levels

Table 1 - Demographic and clinical characteristics of the patient and control groups included in the study.

Parameter Sex Female Age (years) Disease duration (years) SLEDAI Active disease N = 30 Inactive disease N = 27 SDI Treatment No medication Prednisone Hydroxychloroquine Immunosuppressive IFN-a (pg/mL) *

Childhood-onset SLE patients N = 57

First-degree relatives N = 64

Controls N = 57

54 (94.7%) 17.33ยก4.50 (range 9-37) 4.71 ยก4.57 (range 0-26) 4.43ยก4.94 8.37ยก3.80 0.39ยก0.80 0.50ยก0.82

59 (92.18%) 39.95ยก5.66* (range 28-52) -----

52 (91.22%) 19.30ยก4.97 (range 6-30) -----

-----

-----

-----

-----

10.36ยก6.04

11.68ยก6.66

8 (14%) 39 (68.4%) 32 (56.1%) 22 (38.6%) 13.84ยก8.46*

p#0.05.

159

IFN-a and childhood-onset systemic lupus erythematosus Postal M et al.

CLINICS 2012;67(2):157-162

Figure 1 - IFN-a association in SLE. 1a- Analysis of variance with Tukey’s pairwise post-hoc comparisons among SLE patients, their firstdegree relatives and controls; 1b- 2-sample t-test comparing SLE patients positive and negative for dsDNA antibodies; 1c- 2-sample ttest comparing SLE patients taking and not taking medication. Data are presented as box plots; the box represents the 25th to 75th percentiles, the line within the box represents the 50th percentile, and the lines outside the box represent the minimum and maximum values.

antibodies (32,39). Anti-dsDNA antibodies have been associated with lupus nephritis (40), and studies have linked anti-Ro antibody to lupus-related skin findings (41).

It remains unclear whether the association between IFN-a and cutaneous and renal disease manifestations found in previous studies is primary or secondary due to an association between autoantibodies and IFN-a (39). We did not observe any associations between IFN-a levels and the levels of other antibodies, such as anti-Ro, anti-Sm, or anti-RNP. Relatives of individuals with SLE are at a higher risk of developing not only SLE but also other autoimmune diseases (3,31). A heritable predisposition to increased IFNa pathway activation in SLE patient families could explain some of the burden of both SLE and non-SLE autoimmunity in the population. Single nucleotide polymorphisms (SNPs) in the IFN-a pathway genes IFN regulatory factor 5 (IRF5) and non-receptor tyrosine-protein kinase (TYK2) (42-45) are associated with SLE, suggesting that genetic variability in endogenous IFN-a signaling may be involved in the etiology of this disease, although the impact of these polymorphisms on IFN-a activity in vivo is not known (3,43). We did not observe any differences in serum IFN-a levels between first-degree relatives of SLE patients and controls. However, the limited sample size may have affected these results.

We found an inverse correlation between patient age and IFN-a levels. Similar findings have been reported in adult SLE patients as well as in healthy controls, independent of menopause status (31). It is not clear whether the higher serum IFN-a activity observed in young SLE patients is a cause or a result of disease activity, but this correlation may explain some of the differences in the clinical and serologic manifestations of childhood-onset and adult-onset SLE patients.

In addition, we observed higher IFN-a levels in patients who were not receiving medication. None of these patients had any evidence of disease activity at the time of evaluation. Previous studies have shown a dramatic decrease in the expression of IFN-inducible genes (IFIGs) in patients who received pulse glucocorticoid (GC) therapy

These immune complexes and apoptotic bodies stimulate plasmacytoid dendritic cells to produce IFN-a, which in turn enhances antigen presentation by dendritic cells to Tcells while promoting memory T-cell expansion and survival (15,29).

Increased IFN-a serum levels are often observed in SLE patients (3,17,18,30-34). Here, we observed increased serum IFN-a in childhood-onset SLE patients compared to their first-degree relatives or healthy controls. Our data support the results of previous studies that demonstrated elevated IFN-a levels in the sera of adult-onset SLE patients (3,17,18,30-35).

The clinical significance of IFN-a pathway activation in SLE is multifaceted. IFN-a has been implicated in the pathogenesis of SLE, and therefore, therapies targeted against IFN-a are currently in clinical trials (36,37). Furthermore, IFN-a activation may determine specific subset of SLE patients with potential diagnostic, prognostic, and therapeutic implications.

Importantly, a change in IFN-a activity levels may reflect changes in disease activity and thus inform the clinical management of the disease (38). In our study, IFN-a was significantly higher in patients with active disease (SLEDAI $3) than in patients with inactive disease. Furthermore, we observed a direct correlation between SLEDAI scores and IFN-a levels, suggesting that IFN-a could be a biomarker for disease activity in childhood SLE. Similar results have been observed in adult-onset SLE (3,17,18,30-35).

Previous studies suggest that IFN-a plays an important role in the immunopathogenesis of SLE (3,17,18,30-35).

Serum IFN-a is correlated with multiple clinical and serological features of SLE (29,35). We observed that IFN-a levels were increased in patients with cutaneous manifestations of the disease. Our data also showed increased IFN-a levels in childhood-onset SLE patients positive for dsDNA antibodies as well as a direct correlation between IFN-a and C3 levels. However, no association with renal disease was observed. The increased expression of IFN-inducible genes (IFIGs) in peripheral blood mononuclear cells (PBMCs) has been associated with the presence of lupus nephritis and proteinuria, cutaneous manifestations, and the presence of anti-Ro, anti-Smith (anti-Sm), anti-RNP, and anti-dsDNA

160

CLINICS 2012;67(2):157-162

IFN-a and childhood-onset systemic lupus erythematosus Postal M et al. the manuscript writing. Appenzeller S designed the study and was also responsible for the collection and analysis of data and manuscript writing.

(46,47). Data from other studies suggest that intravenous pulse GC treatment may decrease the number of IFNproducing cells, transiently reducing the stimulus for IFIG expression (47).

Although previous studies have analyzed IFN-a levels in childhood-onset SLE, none of these studies have analyzed the clinical and laboratory features associated with increased IFN-a levels (31,45). Serum IFN-a activity was found to be higher in younger individuals in SLE family cohorts, and this tendency was accentuated in affected individuals (31). In addition, one other study revealed that childhood-onset SLE patients are characterized by a potent IFN-a response (45). Genomic approaches have shown that .95% of childhood-onset SLE patients display an ‘‘IFN signature’’, as measured by PBMC gene expression profiling (45,48). The PBMC transcriptional signature in childhood-onset SLE corresponds to neutrophil-specific genes, and the differential expression of these genes correlates with disease activity (45). Notably, SLE neutrophils undergo accelerated spontaneous apoptosis in vitro, and SLE sera induce apoptosis in healthy neutrophils, both of which are correlated with disease activity (49). In addition, the IFIG expression signature can be used to establish subgroups of patients with severe SLE characterized by renal disease, complement activation, and autoantibody production to RNA-associated autoantigens (33,50).

Genetic association studies of SLE patients have identified several genes, especially components of the pathways both upstream and downstream of IFN (mainly type I), including signal transducer and activator of transcription 4 (STAT4) and IRF5 (38,51-53). STAT4 interacts with type I IFN receptors and is directly involved in IFN signaling. IRF5 is a transcription factor that induces IFN transcription in response to Toll-like receptor (TLR) signaling. In fact, the IRF5 risk haplotype in SLE patients is associated with high serum IFN-a activity (34). These findings are consistent with the fundamental observations from a previous study that identified gene expression profiling of SLE PBMCs (38).

These experiments demonstrate a significant upregulation of IFN-regulated gene transcripts in adult and childhoodonset SLE PBMCs (32,34). This characteristic is referred to as the ‘‘IFN signature’’ and is assessed as a biomarker for disease activity (38).

In summary, our findings suggest that IFN-a may play a role in the pathogenesis of childhood-onset SLE. The higher levels observed in younger children may explain the different clinical and serologic manifestations when compared to older patients. The pattern of increased IFN-a levels in patients not taking medication should be investigated further in longitudinal studies to determine whether elevated IFN-a levels may predict SLE flares.

REFERENCES 1. Lee HM, Mima T, Sugino H, Aoki C, Adachi Y, Yoshio-Hoshino N, et al.

Interactions among type I and type II interferon, tumor necrosis factor, and beta-estradiol in the regulation of immune response-related gene expressions in systemic lupus erythematosus. Arthritis Res Ther.

2009;11:R1, http://dx.doi.org/10.1186/ar2584.

2. Kotzin B. Systemic lupus erythematosus. Cell. 1996;85:303-306.

3. Niewold TB, Hua J, Lehman TJ, Harley JB, Crow MK. High serum IFNalpha activity is a heritable risk factor for systemic lupus erythematosus.

Genes Immun. 2007;8:492-502, http://dx.doi.org/10.1038/sj.gene.636 4408.

4. Brunner HI, Gladman DD, Iban˜ez D, Urowitz MD, Silverman ED. Difference in disease features between childhood-onset and adult-onset systemic lupus erythematosus. Arthritis Rheum. 2008;58:556-62, http:// dx.doi.org/10.1002/art.23204.

5. Mina R, Brunner HI. Pediatric lupus–are there differences in presentation, genetics, response to therapy, and damage accrual compared with adult lupus? Rheum Dis Clin North Am. 2010;36:53-80, http:// dx.doi.org/10.1016/j.rdc.2009.12.012.

6. Font J, Cervera R, Espinosa G, Pallare´s L, Ramos-Casals M, Jime´nez S, et al. Systemic lupus erythematosus (SLE) in childhood: analysis of clinical and immunological findings in 34 patients and comparison with SLE characteristics in adults. Ann Rheum Dis. 1998;57:456–9, http:// dx.doi.org/10.1136/ard.57.8.456.

7. Hoffman IE, Lauwerys BR, De Keyser F, Huizinga TW, Isenberg D, Cebecauer L, et al. Juvenile-onset systemic lupus erythematosus: different clinical and serological pattern than adult-onset systemic lupus erythematosus. Ann Rheum Dis 2009;68:412–5.

8. Carreno L, Lopez-Longo FJ, Monteagudo I, Rodrı´guez-Mahou M, Bascones M, Gonza´lez CM, et al. Immunological and clinical differences between juvenile and adult onset of systemic lupus erythematosus.

Lupus. 1999;8:287–92, http://dx.doi.org/10.1191/096120399678847786.

9. Hersh AO, von Scheven E, Yazdany J, Panopalis P, Trupin L, Julian L, et al. Differences in long-term disease activity and treatment of adult patients with childhood- and adult-onset systemic lupus erythematosus.

Arthritis Rheum. 2009;61:13–20.

10. Sibbitt WL Jr, Brandt JR, Johnson CR, Maldonado ME, Patel SR, Ford CC, et al. The incidence and prevalence of neuropsychiatric syndromes in pediatric onset systemic lupus erythematosus. J Rheumatol.

2002;29:1536–42.

11. Rood MJ, ten Cate R, van Suijlekom-Smit LW, den Ouden EJ, Ouwerkerk FE, Breedveld FC, et al. Childhood-onset Systemic Lupus Erythematosus: clinical presentation and prognosis in 31 patients.

Scand J Rheumatol. 1999;28:222–6.

12. von Scheven E, Bakkaloglu A. What’s new in paediatric SLE? Best Pract Res Clin Rheumatol. 2009;23:699-708, http://dx.doi.org/10.1016/ j.berh.2009.07.008.

13. Tucker LB, Menon S, Schaller JG, Isenberg DA. Adult- and childhoodonset systemic lupus erythematosus: a comparison of onset, clinical features, serology, and outcome. Br J Rheumatol. 1995;34:866-72.

14. Appenzeller S, Marini R, Costallat LT. Damage did not independently influence mortality in childhood systemic lupus erythematosus.

Rheumatol Int. 2005;25:619-24, http://dx.doi.org/10.1007/s00296-0040552-z.

15. Ro¨nnblom L, Alm GV. Systemic lupus erythematosus and the type I interferon system. Arthritis Res Ther. 2003;5:68-75, http://dx.doi.org/ 10.1186/ar625.

16. Niewold TB, Clark DN, Salloum R, Poole BD. Interferon alpha in systemic lupus erythematosus. J Biomed Biotechnol. 2010;2010:948364.

17. Hooks JJ, Moutsopoulos HM, Geis AS, Stahl NI, Decker JL, Notkins AL. Immune interferon in the circulation of patients with autoimmune disease. N Engl J Med. 1979;301:5-8.

18. Ytterberg SR, Schnitzer TJ. Serum interferon levels in patients with systemic lupus erythematosus. Arthritis Rheum. 1982;25:401-6.

19. Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF, et al.

The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1982;25:1271–7, http://dx.doi.org/ 10.1002/art.1780251101.

20. ACR Ad Hoc Committee on Neuropsychiatric Lupus Nomencalture. The American College of Rheumatology nomenclature and case definitions for neuropsychiatric lupus syndromes. Arthritis Rheum. 1999;42:599– 608, http://dx.doi.org/10.1002/1529-0131(199904)42:4,599::AIDANR2.3.0.CO;2-F. 21. Harris EN, Gharavi AE, Patel SP, Hughes GR. Evaluation of the anticardiolipin antibody test: report of an international workshop held 4 April 1986. Clin Exp Immunol. 1987;68:215–22.

22. Brandt JT, Triplett DA, Alving B, Scharrer I, , on behalf of the Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the ISTH. Criteria for the

ACKNOWLEDGMENTS ` Pesquisa Estado Sa˜o Paulo-Brasil (FAPESP 2008/ Fundac¸a˜o de Amparo A 02917-0, 2009/06049-6, 2009/11076-2, 2010/13636-2, 2010/13637-9), Conselho Nacional Pesquisa Desenvolvimento-Brasil CNPq (300447/ 2009-4)

AUTHOR CONTRIBUTIONS Postal M designed the study and was also responsible for data collection, data analysis and manuscript writing. Sinicato NA was responsible for the collection and analysis of data. Pelic¸ari KO was responsible for the data collection. Marini R designed the study and was also responsible for the data collection and manuscript writing. Costallat LTL was responsible for

161

IFN-a and childhood-onset systemic lupus erythematosus Postal M et al.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

CLINICS 2012;67(2):157-162

39. Weckerle CE, Franek BS, Kelly JA, Kumabe M, Mikolaitis RA, Green SL, et al. Network analysis of associations between serum interferon alpha activity, autoantibodies, and clinical features in systemic lupus erythematosus. Arthritis Rheum. 2011;63:1044-53, http://dx.doi.org/10.1002/ art.30187.

40. Bastian HM, Alarcon GS, Roseman JM, McGwin G Jr, Vila´ LM, Fessler BJ, et al. Systemic lupus erythematosus in a multiethnic US cohort (LUMINA) XL II: factors predictive of new or worsening proteinuria.

Rheumatology (Oxford) 2007;46:683-9.

41. Sontheimer RD, Maddison PJ, Reichlin M, Jordon RE, Stastny P, Gilliam JN. Serologic and HLA associations in subacute cutaneous lupus erythematosus, a clinical subset of lupus erythematosus. Ann Intern Med 1982;97:664-71.

42. Graham RR, Kozyrev SV, Baechler EC, Reddy MV, Plenge RM, Bauer JW, et al. A common haplotype of interferon regulatory factor 5 (IRF5) regulates splicing and expression and is associated with increased risk of systemic lupus erythematosus. Nat Genet. 2006;38:550–5, http:// dx.doi.org/10.1038/ng1782.

43. Rullo OJ, Woo JM, Wu H, Hoftman AD, Maranian P, Brahn BA, et al.

Association of IRF5 polymorphisms with activation of the interferon alpha pathway. Ann Rheum Dis. 2010;69:611-7, http://dx.doi.org/ 10.1136/ard.2009.118315.

44. Bauer JW, Petri M, Batliwalla FM, Koeuth T, Wilson J, Slattery C, et al.

Interferon-regulated chemokines as biomarkers of systemic lupus erythematosus disease activity: a validation study. Arthritis Rheum.

2009;60:3098-107, http://dx.doi.org/10.1002/art.24803.

45. Garcia-Romo GS, Caielli S, Vega B, Connolly J, Allantaz F, Xu Z, et al.

Netting neutrophils are major inducers of type I IFN production in pediatric systemic lupus erythematosus. Sci Transl Med. 2011;3:73ra20, http://dx.doi.org/10.1126/scitranslmed.3001201.

46. Kirou KA, Lee C, George S, Louca K, Peterson MG, Crow MK. Activation of the interferon-alpha pathway identifies a subgroup of systemic lupus erythematosus patients with distinct serologic features and active disease. Arthritis Rheum. 2005;52:1491-503, http://dx.doi.org/10.1002/ art.21031.

47. Shodell M, Shah K, Siegal FP. Circulating human plasmacytoid dendritic cells are highly sensitive to corticosteroid administration. Lupus 2003; 12:222–30.

48. Bennett L, Palucka AK, Arce E, Cantrell V, Borvak J, Banchereau J, et al.

Interferon and granulopoiesis signatures in systemic lupus erythematosus blood. J. Exp. Med. 2003;197:711–23, http://dx.doi.org/10.1084/ jem.20021553.

49. Midgley A, McLaren Z, Moots RJ, Edwards SW, Beresford MW. The role of neutrophil apoptosis in juvenile-onset systemic lupus erythematosus.

Arthritis Rheum. 2009;60:2390–401, http://dx.doi.org/10.1002/art.

24634.

50. Flesher DL, Sun X, Behrens TW, Graham RR, Criswell LA. Recent advances in the genetics of systemic lupus erythematosus. Expert Rev Clin Immunol. 2010;6:461-79, http://dx.doi.org/10.1586/eci.10.8.

51. Gateva V, Sandling JK, Hom G, Taylor KE, Chung SA, Sun X, et al. A large-scale replication study identifies TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL10 as risk loci forsystemic lupus erythematosus. Nat Genet. 2009;41:1228-33, http://dx.doi.org/10.1038/ng.468.

52. Sigurdsson S, Nordmark G, Go¨ring HH, Lindroos K, Wiman AC, Sturfelt G, et al. Polymorphisms in the tyrosine kinase 2 and interferon regulatory factor 5 genes are associatedwith systemic lupus erythematosus. Am J Hum Genet. 2005;76:528-37.

53. International Consortium for Systemic Lupus Erythematosus Genetics (SLEGEN), Harley JB, Alarco´n-Riquelme ME, Criswell LA, Jacob CO, Kimberly RP, et al. Genome-wide association scan in women with systemic lupus erythematosus identifiessusceptibility variants in ITGAM, PXK, KIAA1542 and other loci. Nat Genet. 2008;40:204-10, http://dx.doi.org/10.1038/ng.81.

diagnosis of lupus anticoagulants: an update. Thromb Haemost.

1995;74:1185–90.

Bombardier C, Gladman DD, Urowitz MB, Caron D, Chang CH. Derivation of the SLEDAI. A disease activity index for lupus patients.

The Committee on Prognosis Studies in SLE. Arthritis Rheum.

1992;35:630–40.

Yee CS, Farewell VT, Isenberg DA, Griffiths B, Teh LS, Bruce IN, et al.

The use of Systemic Lupus Erythematosus Disease Activity Index-2000 to define active disease and minimal clinically meaningful change based on data from a large cohort of systemic lupus erythematosus patients.

Rheumatology (Oxford). 2011;50:982-8, http://dx.doi.org/10.1093/ rheumatology/keq376.

Gladman DD, Urowitz MB, Goldsmith CH, Fortin P, Ginzler E, Gordon C, et al. The reliability of the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index in patients with systemic lupus erythematosus. Arthritis Rheum. 1997;40:809–13, http://dx.doi.org/10.1002/art.1780400506.

Galley HF, Webster NR. The immuno-inflammatory cascade.Br J Anaesth. 1996;77:11-6.

Wozniacka A, Lesiak A, Narbutt J, McCauliffe DP, Sysa-Jedrzejowska A. Chloroquine treatment influences proinflammatory cytokine levels in systemic lupus erythematosus patients. Lupus. 2006;15:268-75, http:// dx.doi.org/10.1191/0961203306lu2299oa.

Golding A, Rosen A, Petri M, Akhter E, Andrade F. Interferon-alpha regulates the dynamic balance between human activated regulatory and effector T cells: implications for antiviral and autoimmune responses.

Immunology. 2010;131:107-17.

Zhang R, Xing M, Ji X, Gu L, Yang X, Wang H, et al. Interferon-alpha and interleukin-6 in SLE serum induce the differentiation and maturation of dendritic cells derived from CD34+ hematopoietic precursor cells.

Cytokine. 2010;50:195-203, http://dx.doi.org/10.1016/j.cyto.2010.02.017.

Kim T, Kanayama Y, Negoro N, Okamura M, Takeda T, Inoue T. Serum levels of interferons in patients with systemic lupus erythematosus. Clin Exp Immunol. 1987;70:562-9.

Niewold TB, Adler JE, Glenn SB, Lehman TJ, Harley JB, Crow MK. Ageand sex-related patterns of serum interferon-alpha activity in lupus families. Arthritis Rheum. 2008;58:2113-9, http://dx.doi.org/10.1002/ art.23619.

Baechler EC, Batliwalla FM, Karypis G, Gaffney PM, Ortmann WA, Espe KJ, et al. Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus. Proc Natl Acad Sci U S A. 2003;100:2610–5, http://dx.doi.org/10.1073/pnas.0337679100.

Kirou KA, Lee C, George S, Louca K, Peterson MG, Crow MK. Activation of the interferon-alpha pathway identifies a subgroup of systemic lupus erythematosus patients with distinct serologic features and active disease. Arthritis Rheum. 2005;52:1491–503, http://dx.doi.org/10.1002/ art.21031.

Niewold TB, Kelly JA, Flesch MH, Espinoza LR, Harley JB, Crow MK. Association of the IRF5 risk haplotype with high serum interferon-alpha activity in systemic lupus erythematosus patients. Arthritis Rheum.

2008;58:2481–7, http://dx.doi.org/10.1002/art.23613.

Dall’era MC, Cardarelli PM, Preston BT, Witte A, Davis JC Jr. Type I interferon correlates with serological and clinical manifestations of SLE. Ann Rheum Dis. 2005;64:1692-7, http://dx.doi.org/10.1136/ard.2004.

033753.

Ro¨nnblom L, Elkon KB. Cytokines as therapeutic targets in SLE. Nat Rev Rheumatol. 2010;6339-47.

Yoo DH. Anticytokine therapy in systemic lupus erythematosus. Lupus.

2010;19:1460-7, http://dx.doi.org/10.1177/0961203310376955.

Obermoser G, Pascual V. The interferon-alpha signature of systemic lupus erythematosus. Lupus. 2010;19:1012-9, http://dx.doi.org/10.1177/ 0961203310371161.

162

CLINICS 2012;67(2):163-170

DOI:10.6061/clinics/2012(02)12

BASIC RESEARCH

Comparison of two peptide radiotracers for prostate carcinoma targeting Bluma Linkowski Faintuch,I Erica A. Oliveira,I Eutimio G. F. Nunez,I Ana M. Moro,II P. K. Nanda,III Charles J. SmithIII I

Institute of Energy and Nuclear Research, (IPEN/CNEN), Radiopharmacy Center, Sa˜o Paulo, Brazil. II Butantan Institute, Laboratory of Monoclonal Antibodies, Sa˜o Paulo, SP, Brazil. III University of Missouri, School of Medicine, Department of Radiology, Columbia, MO, USA.

OBJECTIVES: Scintigraphy is generally not the first choice treatment for prostate cancer, although successful studies using bombesin analog radiopeptides have been performed. Recently, a novel peptide obtained using a phage display library demonstrated an affinity for prostate tumor cells. The aim of this study was to compare the use of a bombesin analog to that of a phage display library peptide (DUP-1) radiolabeled with technetium-99m for the treatment of prostate carcinoma. The peptides were first conjugated to S-acetyl-MAG3 with a 6-carbon spacer, namely aminohexanoic acid.

METHODS: The technetium-99m labeling required a sodium tartrate buffer. Radiochemical evaluation was performed using ITLC and was confirmed by high-performance liquid chromatography. The coefficient partition was determined, and in vitro studies were performed using human prostate tumor cells. Biodistribution was evaluated in healthy animals at various time points and also in mice bearing tumors.

RESULTS: The radiochemical purity of both radiotracers was greater than 95%. The DUP-1 tracer was more hydrophilic (log P = -2.41) than the bombesin tracer (log P = -0.39). The biodistribution evaluation confirmed this hydrophilicity by revealing the greater kidney uptake of DUP-1. The bombesin concentration in the pancreas was greater than that of DUP-1 due to specific gastrin-releasing peptide receptors. Bombesin internalization occurred for 78.32% of the total binding in tumor cells. The DUP-1 tracer showed very low binding to tumor cells during the in vitro evaluation, although tumor uptake for both tracers was similar. The tumors were primarily blocked by DUP1 and the bombesin radiotracer primarily targeted the pancreas.

CONCLUSION: Further studies with the radiolabeled DUP-1 peptide are recommended. With further structural changes, this molecule could become an efficient alternative tracer for prostate tumor diagnosis.

KEYWORDS: Bombesin; DUP-1 Peptide; Technetium-99m; Prostate Tumor; Radiolabeling.

Faintuch BL, Oliveira EA, Nunez EGF, Moro AM, Nanda PK, Smith CJ. Comparison of two peptide radiotracers for prostate carcinoma targeting. Clinics.

2012;67(2):163-170.

Received for publication on July 24, 2011; First review completed on August 23, 2011; Accepted for publication on September 5, 2011 E-mail: blfaintuch@hotmail.com Tel.: 55 11 31339531

Bombesin (BBN) is a 14-amino acid neuropeptide that was isolated from the skin of the European Bombina frog, and it shows a high affinity for the gastrin-releasing peptide receptor. BBN and its mammalian homolog, GRP, share a seven-amino-acid sequence in their C-terminal region, which is essential for receptor binding and biological activity (6,7).

Most studies that have used radiolabeled BBN have focused on prostate cancer, although some have also focused on breast cancer (8,9). Recent efforts in the field of phage display technology have led to significant drug discoveries. Phage display peptide libraries encompass high-affinity molecules that show potential for use in cancer diagnosis (10).

The synthetic peptide FRPNRAQDYNTN (DUP-1) was identified by phage display techniques. The DUP-1 peptide binds to DU-145 prostate cells and also to PC-3 cells with high affinity (11).

DUP-1 has been radiolabeled with iodine-131 (11) and also recently with indium-111 (111In) (12). However, no

INTRODUCTION Many investigations have been conducted using diagnostic radiotracers specific for prostate cancer, as prostate cancer is a condition characterized by substantial morbidity and mortality. Radiolabeled bombesin analog are preferable because they target gastrin-releasing peptide receptor (GRPR)-expressing tumors (1-2). GRPR is highly expressed on the surface of malignant human prostate cells, and its specificity for prostate cancer, compared to benign hyperplastic prostate tissue, has been demonstrated (3-5).

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

163

Radiotracers for prostate carcinoma Faintuch BL et al.

CLINICS 2012;67(2):163-170

Figure 1 - Structure of conjugated bombesin (SAMA-G-G-G-Ahx-QWAVGHLM-NH2); chemical formula: C59H89N17O15S2; molecular weight: 1340.57.

reports have focused on the use of one of the most widely used SPECT radioisotopes, technetium-99m (99mTc).

Askoxilakis et al. (12) demonstrated that the phage display linear molecule DUP-1 is generally serum-instable, which suggests the existence of targeted modifications, such as cyclization, an exchange of amino acids, such D-amino acids, and peptide acetylation.

Technetium-99m (99mTc) has many advantages, including its widespread availability, low cost and convenient physicochemical properties, such as its half-life (t1/2 = 6 h) and gamma energy (Ec = 140 keV). Moreover, it can also be provided as part of a ready-to-use lyophilized kit. Different approaches have been used to label biomolecules with 99mTc by means of bifunctional chelators, including hydrazinenicotinic acid (HYNIC) or mercaptoacetylglycyltriglycine (MAG3). The use of preformed metallic precursors, such as organometallic 99mTc(CO)3 or 99mTc-nitrido, as well as the 3+1 or 4+1 mixed ligand approach and the ‘‘click chemistry’’ procedure are additional approaches used for labeling (13).

The S-acetyl NHS-MAG3 chelator was originally used for the post-conjugation labeling of antibodies, peptides and oligomers with 99mTc; this is typically performed at a neutral pH and at room temperature.

The aim of this study was to compare the use of a bombesin analog to that of a phage display peptide (DUP-1) labeled with technetium-99m for experimental prostate carcinoma. PC3 human prostate cancer cells were used in this study, and these represent an attractive target for bombesin markers because they overexpress GRPR. Schally and co-workers have further shown that there are 44,000 bombesin receptor sites/cell on PC-3 human prostate cancer cells (14).

The peptides were conjugated with S-acetyl mercaptoacetyl-triglycine (SAMA-G3) using a six-carbon spacer, namely aminohexanoic acid (Figs. 1 and 2). It was hypothesized that both the classic bombesin tracer as well as the DUP-1 tracer

would be confirmed as potential tools for nuclear medicine diagnoses.

MATERIALS AND METHODS Materials All of the chemicals used were reagent-grade (SigmaAldrich and Merck, Sa˜o Paulo, Brazil). The peptide-conjugated bombesin analog (SAMA-G-G-G-Ahx-QWAVGHLM-NH2, abbreviated as SAMA-G3-Ahx-BBN) and the phage display peptide DUP-1 (FRPNRAQDYNTNK-SAMA-G-G-GAhx-NH2, abbreviated as SAMA-G3-Ahx-DUP-1) (Figures 1, 2) were purchased from piChem Laboratory (Graz, Austria).

99m Tc, in the form of Na99mTcO4, was eluted in saline from an alumina-based 99Mo/99mTc generator, which was supplied by the Institute of Energetic and Nuclear Research/ Brazilian Commission of Nuclear Energy (IPEN/CNEN, Sa˜o Paulo, Brazil). Silica gel strips (ITLC-SG, Gelman Science, Ann Arbor, MI, USA) were used for instant thin-layer chromatography. Reverse-phase high-performance liquid chromatography (RP-HPLC, Shimadzu, Kyoto, Japan) was also performed using a Symmetry C-18 column (5.0 mm, ˚ , 4.66250 mm, Waters, Milford, MA, USA).

100 A Radioactivity measurements were conducted using an automated, well-typed c-counter NaI(Tl) crystal (Canberra, Meriden, CT, USA). The PC-3 human prostate adenocarcinoma cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Male Swiss albino mice (20 - 25 g) and male nude mice (16 - 20 g) were used for the biodistribution studies, and the animals were provided by the Animal Facility of IPEN/CNEN in Sa˜o Paulo, Brazil.

Methods Radiolabeling of conjugated peptides and radiochemical evaluation. The conjugated peptides (10 mL of a mg/mL

Figure 2 - Structure of conjugated DUP-1 (FRPNRAQDYNTNK) (SAMA-G-G-G-Ahx)-NH2; chemical formula: C85H131N29O27S; molecular weight: 2023.19.

164

CLINICS 2012;67(2):163-170

Radiotracers for prostate carcinoma Faintuch BL et al.

of the injected dose per gram of tissue was calculated using the activity of the injected dose as a standard.

Biodistribution was analogously evaluated in tumor-bearing mice at 30 and 90 min post-injection and was followed by blocking studies, which involved the coinjection of excess amounts of cold conjugate.

Tumor imaging. Planar imaging studies were performed with anesthetized mice that were horizontally placed under the collimator of a Mediso Imaging System (Budapest, Hungary). A low-energy, high-resolution collimator was used, and images were acquired at 0.5 h and 1.5 h postinjection using a 2566256616 matrix size and a 20% energy window set at 140 keV for a period of 180 s.

Statistical analysis. Quantitative data were expressed as the mean ¡ SD. Means were compared by an independent Student’s t test. p-values less than 0.05 were considered significant.

solution) were labeled with 300 mL of technetium-99m using sodium tartrate dissolved in a labeling buffer (0.5 M ammonium bicarbonate, 0.25 M ammonium acetate, 0.18 M ammonium hydroxide, 1:1:1) along with a stannous chloride solution (chloridric acid/ascorbic acid).

The reaction was induced by heating the mixture to 100 ˚C for 20 min. Radiochemical evaluation was performed using instant thin-layer chromatography on silica gel strips, and the mobile phase consisted of methylethylketone (MEK) and a solution of 50% acetonitrile (ACN).

Each of the radiolabeled conjugates was also characterized by reverse-phase high-performance liquid chromatography analysis. All solvents used in the chromatographic analyses were HPLC-grade and had been previously filtered through 0.22-mm membrane filters (Millipore, Milford, MA, USA).

The HPLC solvents consisted of H2O, which contained 0.1% trifluoroacetic acid (Solvent A), and ACN, which contained 0.1% trifluoracetic acid (Solvent B). The HPLC gradient system began with a solvent composition of 95% A and 5% B, which was followed by a linear gradient of 30% A and 70% B from 0 to 25 min and 5% A and 95% B from 25 to 30 min.

Determination of log P values. The radiotracers (100 mL) were dissolved in a mixture of equal volumes (3 mL to 3 mL) of n-octanol and water. After the mixture had been vigorously stirred and centrifuged, samples (in triplicate) from both phases (n-octanol and water) were collected and their radioactivity was measured using a gamma counter.

In vitro cell binding assays. PC3 androgen-independent human prostate carcinoma cells were cultured in RPMI 1640 medium (Sigma-Aldrich, Sa˜o Paulo, Brazil) that was supplemented with 10% (v/v) fetal calf serum. The cells were kept at 37 ˚C in a humidified air environment containing 5% CO2. They were grown to near confluency and were then harvested by trypsinization. Cells (106 cells/ well) were seeded into six- and twelve-well culture plates for the binding studies 24 h before the experiment.

The culture plates were then filled with increasing concentrations (1 nM to 18 nM) of cold, nonlabeled conjugates along with 90 pM of 99mTc-labeled peptide and were subsequently incubated for 120 min.

Internalization studies were also performed in triplicate at 15, 30, 60, 90, and 120 min time points with and without an excess of 1 mM cold conjugated peptide for nonspecific internalization. The supernatant was collected, and cell surface-bound radioligand was removed with a two-step acid wash. Internalized radioligand was evaluated by lysing the cells with 1 M NaOH for 5 min, and radioactivity was measured with the automated c counter.

In vivo studies. All experiments were carried out in compliance with the protocol that had been approved by the local Animal Welfare Committee. Suspensions of PC3 cells (5.06106) were subcutaneously injected into the upper back regions of athymic male nude mice. The tumors were allowed to grow in vivo for 2–3 weeks, at which point the tumor diameter was approximately 1 cm.

99m Tc-conjugated peptides were injected via the tail vein into healthy Swiss mice (0.1 mL/18.5 MBq), and the biodistribution was evaluated at 5, 30, 90, and 240 min post-injection using six mice for each time point. All animals were sacrificed by cervical dislocation. Subsequently, all major organs and tissue samples were excised, weighed and counted with a NaI(Tl) gamma counter, and the percentage

RESULTS Both conjugates were radiolabeled using the same protocol. Radiolabeling with 99mTc was performed via stannous-tartrate exchange. The radiochemical purities of 99m Tc-MAG3-Ahx-BBN and 99mTc-MAG3-Ahx-DUP-1 were 96.3¡0.32% and 96.0¡0.15%, respectively, and the corresponding retention times by HPLC were 12.1 min and 13.4 min, respectively (Figure 3).

The specific activity of 99mTc-MAG3-Ahx-BBN was 148.8 MBq/nM, and that of 99mTc-MAG3-Ahx-DUP-1 was 224.7 MBq/nM. The lipophilicity of the radiotracers was determined according to their proportional distribution between octanol and water. A partition ratio of 0.4065, where log p = -0.39¡0.02, indicated moderate hydrophilicity for the 99m Tc-MAG3-Ahx-BBN. It was more remarkable for 99mTcMAG3-Ahx-DUP1, with a partition ratio of 0.00389 and log p = -2.41¡0.12.

Radioactivity associated with PC3 tumor cells for 99mTcBBN tracer was 28.87%. A sharp decrease (93%) of the binding was observed with increase of the amount of cold BBN (0 to 18 nM). For 99mTc- SAMA-G3-Ahx-DUP1 the binding value was much lower, , 1.97%, diminishing by a factor of 63% at the highest concentration of 18 nM (Figure 4).

Figure 3 - Reverse-phase HPLC profile: (A) BBN; (B) 99mTc- SAMA-G3-Ahx-DUP-1.

165

99m

Tc-SAMA-G3-Ahx-

Radiotracers for prostate carcinoma Faintuch BL et al.

CLINICS 2012;67(2):163-170

conditions, displayed a much lower rate of internalization, which was in the range between 5.78% and 9.34% and remained stable at all time points.

The biodistribution evaluation in healthy animals confirmed that kidney uptake was more robust for the DUP tracer than for the BBN tracer (p , 0.05). Blood depuration was rapid and similar for both tracers (p . 0.05). Intestinal uptake was high and also equivalent for both radiotracers (p . 0.05). Hepatobiliary excretion was predominant for the BBN tracer, whereas the hepatobiliary system and the urinary system were found to be the major excretory pathways for the DUP tracer. 99mTc-SAMA-G3-Ahx-BBN exhibited greater uptake in the pancreas (p , 0.05) than 99m Tc-SAMA-G3-Ahx-DUP-1.

Clearance from the blood and from other GRP receptornegative tissues (heart, lung, spleen, stomach, muscle, and bone) was rapid, as less than 1% ID/g was detected 30 min after the injection of either radiotracers BBN and DUP-1 (Table 1).

The biodistribution for nude mice bearing PC3 tumors is shown in Table 2 and Figures 6 and 7. Lung uptake was in the range of 1% ID/g, and that of the heart, muscle and bone was less than 1% ID/g. Tumor uptake was not significantly different for the two radiotracers (p . 0.05).

The excretion profile was also investigated for the tumorbearing mice (Figure 6). The highest level of kidney uptake for 99mTc-SAMA-G3-Ahx-DUP-1 was documented at 30 min post-injection. No differences occurred (p . 0.05) between blocked and unblocked animals (Figures 6 and 7).

The liver uptake of 99mTc-SAMA-G3-Ahx-BBN confirmed that tracer elimination was rapid, as the uptake values at 90 min were significantly decreased.

Figure 4 - Cellular uptake of the radiotracers 99mTc-SAMA-G3Ahx-BBN and 99mTc-SAMA-G3-Ahx-DUP-1 in PC-3 human prostate carcinoma cells at 37 ˚C. Percentage of total binding radioactivity vs. an increasing concentration of the respective cold peptide.

The internalization of the tracers by PC3 cells, as a function of time for the two radiolabeled peptide derivatives, is illustrated in Figure 5. Internalization was assessed both in the absence and presence (blocking experiments) of the native peptides.

The rate of internalization was time-dependent for 99mTcSAMA-G3-Ahx-BBN. Incubation at 37 ˚C resulted in an increased internalization rate, as 20.36¡0.83% of the radioactivity was internalized at 120 min. The internalization reached its maximum (78.32%) of total binding by 2 h.

99m Tc- SAMA-G3-Ahx-DUP-1, as evaluated under the same

Figure 5 - Internalization of the radiotracers: total binding represents uptake on the surface membrane plus that inside of the cell; internalization represents the activity inside of the cell only. (A) Total binding – 99mTc-SAMA-G3-Ahx-BBN; (B) Internalization – 99mTcSAMA-G3-Ahx-BBN; (C) Total binding – 99mTc- SAMA-G3-Ahx-DUP-1; (D) Internalization 99mTc- SAMA-G3-Ahx-DUP-1.

166

CLINICS 2012;67(2):163-170

Radiotracers for prostate carcinoma Faintuch BL et al.

Table 1 - Biodistribution of the

99m

Tc conjugates in healthy Swiss mice.

Tissue %ID/g

Time 5 min BBN

Blood Heart Lung Kidney Spleen Stomach Pancreas Liver L. Int. S. Int. Muscle Bone

*

1.64¡0.22 0.61¡0.20 1.56¡0.33 1.81¡0.06 1.45¡0.24 1.49¡0.20 6.58¡1.67 10.05¡2.18 4.17¡0.77 12.03¡3.37 0.33¡0.05 0.51¡0.09

30 min *

DUP**

BBN

1.21¡0.02 0.82¡0.14 1.48¡0.23 22.62¡2.03 1.37¡0.18 0.73¡0.17 1.44¡0.33 12.31¡2.19 2.92¡0.24 16.57¡3.02 0.77¡0.06 0.84¡0.17

0.36¡0.06 0.14¡0.05 0.51¡0.06 3.01¡0.78 0.64¡0.05 0.49¡0.05 2.78¡0.23 2.79¡0.47 1.00¡0.22 14.34¡2.58 0.11¡0.03 0.13¡0.01

90 min DUP

**

BBN

0.43¡0.06 0.24¡0.07 0.73¡0.16 21.06¡0.41 0.28¡0.07 0.91¡0.11 0.18¡0.11 8.58¡1.75 1.11¡0.07 13.31¡4.46 0.14¡0.04 0.20¡0.01

*

0.12¡0.03 0.06¡0.01 0.21¡0.03 0.62¡0.01 0.29¡0.08 0.83¡0.32 1.67¡0.33 1.58¡0.45 2.00¡1.21 18.17¡2.27 0.10¡0.04 0.11¡0.05

240 min DUP

**

BBN

0.08¡0.01 0.04¡0.01 0.15¡0.037 7.28¡2.07 0.09¡0.03 0.27¡0.01 0.05¡0.01 1.57¡0.57 0.33¡0.28 14.62¡2.15 0.51¡0.01 0.05¡0.02

*

0.05¡0.01 0.04¡0.02 0.13¡0.03 0.29¡0.03 0.73¡0.07 0.67¡0.05 0.35¡0.05 1.54¡0.06 19.40¡0.9 0.38¡0.11 0.06¡0.02 0.40¡0.11

DUP** 0.05¡0.01 0.03¡0.02 0.13¡0.07 3.22¡0.43 0.07¡0.02 0.29¡0.01 0.04¡0.01 1.53¡0.64 15.45¡2.8 1.43¡0.49 0.17¡0.07 0.05¡0.01

Abbreviations: L. intestine: large intestine; S. Int: small intestine; (*)99mTc-SAMA-G3-Ahx-BBN; (**)99mTc-SAMA-G3-Ahx-DUP-1.

Data are expressed as the mean ¡ SD (n = 6). The radioactivity in the stomach and intestine was evaluated after thoroughly removing the luminal contents.

improved conjugation or more favorable kinetics. The preferred chelators for peptide labeling using technetium include HYNIC, the tricarbonyl core, and N3S types, such as benzoyl or S-acetyl-MAG3 (mercaptoacetyl-Gly-Gly-Gly).

The S-acetyl group is reported to remain intact after conjugation; thus, the risk of subsequent degradation of the molecule is low (15). Degradation was not measured in the present circumstance, but it may be a useful parameter for future studies. The radiolabeling yield in this study was high, thus precluding the need for radiolabeling optimization.

Drug transportation characteristics are influenced by the partition coefficient because this coefficient affects how drugs reach the target organs and other structures at the site of action. 99mTc-SAMA-G3-Ahx-DUP-1 was more hydrophilic than 99mTc-SAMA-G3-Ahx-BBN, and this was confirmed by its stronger kidney uptake. This hydophilicity is a valuable feature of the compound that improved its solubilization and access to body systems. In the case of the prostate, too much radioactivity in the urinary system may interfere with tumor imaging; however, this limitation can be offset by simultaneous amino acid administration (6).

As shown in Figure 7, as well as in Table 2, the cold conjugate-blocked tumor uptake values for the BBN tracer were 32% and 49%, and those for the DUP tracer were 82% and 83% at 30 and 90 min post-injection, respectively. The pancreas was 93.19% blocked at 30 min and 97.06% blocked at 90 min using cold BBN, and these blockage values for the cold DUP-1 were decreased at 32.53% and 53.85%, respectively DUP-1 (Figure 7).

The scintigraphic images are shown in Figure 8. The tumor images that were acquired 90 min post-injection revealed approximately 60% less uptake than at the earlier time point. High uptake was observed in the gastrointestinal tract, and the prostate tumor was clearly visualized at 30 min post-injection. Although the image quality was somewhat better for the BBN tracer, the region of interest (ROI) measurements for both radiotracers were not significantly different (p . 0.05).

DISCUSSION The radiolabeling of many peptides requires functional groups, such as spacers, chelators, or radiometals, for Table 2 - Biodistribution of the

99m

Tc tracers in Nude mice bearing PC-3 tumors.

Tissue %ID/g

Time post-injection 30 min

BBN* Blood 0.72¡0.15 Heart 0.34¡0.08 Lung 1.20¡0.32 Spleen 1.51¡0.55 Stomach 0.94¡0.27 Muscle 0.27¡0.09 Bone 0.50¡0.12 Tumor 0.75¡0.15 Uptake ratio (tumor/normal tissue) Tumor/blood 1.04 Tumor/muscle 2.78

90 min

BBN* Block

DUP**

DUP** Block

BBN*

BBN* Block

DUP**

DUP** Block

0.98¡0.29 0.54¡0.04 1.40¡0.23 1.40¡0.12 0.90¡0.20 0.30¡0.03 0.78¡0.13 0.51¡0.17

0.69¡0.14 0.41¡0.02 1.24¡0.43 0.56¡0.13 1.05¡0.07 0.27¡0.27 0.31¡0.08 0.85¡0.19

0.58¡0.16 0.40¡0.08 0.98¡0.23 0.49¡0.11 1.23¡0.34 0.23¡0.03 0.27¡0.02 0.15¡0.03

0.20¡0.06 0.10¡0.01 1.05¡0.19 0.72¡0.31 0.65¡0.22 0.25¡0.04 0.24¡0.08 0.29¡0.05

0.13¡0.03 0.09¡0.02 1.02¡0.21 0.65¡0.13 0.31¡0.06 0.16¡0.03 0.29¡0.02 0.16¡0.02

0.23¡0.06 0.10¡0.10 0.34¡0.27 0.54¡0.19 0.72¡0.27 0.31¡0.11 0.30¡0.05 0.41¡0.11

0.12¡0.02 0.09¡0.01 0.29¡0.04 0.62¡0.11 0.66¡0.16 0.28¡0.03 0.24¡0.02 0.07¡0.01

1.23 3.15

1.45 1.16

(*)99mTc-SAMA-G3-Ahx-BBN; (**)99mTc-SAMA-G3-Ahx-DUP-1. The data are expressed as the mean ¡ SD (n = 6).

167

1.78 1.32

Radiotracers for prostate carcinoma Faintuch BL et al.

Figure 6 - Excretion organ uptake of

CLINICS 2012;67(2):163-170

99m

Tc- peptides in urethane-anesthetized mice (0.1 mL/18.5 MBq).

Cell binding assays in the current study were consistent with the higher affinity of the BBN tracer. For DUP-1, maximum binding and internalization were reached after 5 min and subsequently decreased over time. A comparable profile of cell binding and internalization has been described for 131I-DUP-1 regarding DU-145 and PC3 tumor cells (11).

Such differences between BBN and DUP-1 may be partially explained by the fact that the DUP-1 conjugate has twice the molecular weight and a greater hydrophilicity than BBN. Degradation was not considered to be a problem in this study, as higher rather than lower blood concentrations tend to occur in such circumstances. The BBN concentration in the pancreas was also significantly greater, which is in accordance with the literature. The pancreas has

PC3 human prostate cancer cells overexpress gastrinreleasing peptide receptor (GRPR), which renders them an attractive target for bombesin markers. Schally and coworkers have shown that there are 44,000 bombesin receptor sites/cell on PC-3 human prostate cancer cells (16).

Zitzmann et al. (11) used a 12-mer M13 phage display peptide library to screen for ligands of the DU-145 prostate carcinoma cell line, as well as those of PC3 cells. After six rounds of biopanning, all sequenced phage clones were found to display the same peptide, FRPNRAQDYNTN (DUP-1). The number of cell receptors for this molecule has not been estimated. Nevertheless, the Zitzman group (11) validated the binding of DUP-1 to PC-3 cells using a radioactive derivative of DUP-1 in the presence of increasing concentrations of unlabeled DUP-1 (17).

Figure 7 - Pancreatic and tumor uptake in regular, as well as blocked, urethane-anesthetized animals (0.1 mL/18.5 MBq): (A) 99mTcSAMA-G3-Ahx-BBN 30 min; (B) 99mTc-SAMA-G3-Ahx-BBN 90 min; (C) 99mTc-SAMA-G3-Ahx-DUP 30 min; (D) 99mTc-SAMA-G3-Ahx-DUP 90 min.

168

CLINICS 2012;67(2):163-170

Radiotracers for prostate carcinoma Faintuch BL et al.

elevated 131I-DUP tumor uptake at the relatively early time of 15 min. Thus, future studies should begin assessments at earlier time-points to disclose stronger cell affinities and higher concentrations than were reported here. In addition, binding affinity was reduced in the study by Askoxylakis et al., which evidenced the difficulties associated with reaching an ideal molecular design.

These authors concluded that the sequence in the middle of the NRAQDY molecule may be significant for target binding.

The occurrence of instability and degradation was suggested by Askoxylakis et al., but this was not in agreement with the findings of others. Phage display peptides have been used as vehicles for the delivery of drugs and other biomolecules, and they have achieved high success due to their affinity and selectivity for a variety of cellular targets. Attention to peptide conformation and the chemical synthesis of specific molecules have been cited as the greatest potential challenges for this technique (19,20).

As a result, stability studies were not considered a priority in the current investigation.

Functional groups are crucial for the optimization of peptide tracers, and this also applies to DUP-1. Accordingly, the glycosylation state of the peptide and the effects of various chelators need to be assessed by future studies.

Both the BBN and DUP-1 tracers exhibited low but consistent tumor uptake. The DUP-1 tumor uptake values likely experienced interference due to the experimental design, as peak values were missed at the comparatively long analysis interval of 30 min. Improvements in the design of the molecule, which may result in more favorable pharmacokinetics, along with the use of a shorter assessment timeframe may substantially enhance the potential of DUP-1 to be used as a prostate tumor radiopeptide.

ACKNOWLEDGMENTS We are grateful to Rodrigo Teodoro and Natanael Gomes da Silva for their technical support during the animal experiments and imaging procedures. We also thank Roselaine Campos Targino for her help concerning cell culture and Maria Neide Ferreira Mascarenhas for her cooperation regarding the animal facilities.

Figure 8 - Lateral scintigraphic images of urethane-anesthetized mice bearing PC3 tumors in the upper right part of the back; (A) 99m Tc-SAMA-G3-Ahx-BBN – 30 min (ROI: 0.49%); (B) 99mTc-SAMAG3-Ahx-BBN – 90 min (ROI: 0.26%); (C) 99mTc-SAMA-G3-AhxDUP-1 – 30 min (ROI: 0.71%; (D) 99mTc-SAMA-G3-Ahx-DUP-1 – 90 min (ROI: 0.27%) using a 25662 or 56616 matrix size (0.1 mL/ 18.5 MBq).

AUTHOR CONTRIBUTIONS Faintuch BL, Nanda PK, and Smith CJ designed the protocol and analyzed the results, and all the authors examined and approved the final manuscript. Oliveira EA, Nunez EGF, and Moro AM prepared the materials and conducted the technical procedures.

long been known to express a large number of GRP receptors (18). Indeed, this number is approximately eightfold greater than that of PC3 tumors (18). These properties of BBN suggest that this peptide will likely remain the first choice for prostate cancer, although this does not rule out interest in alternative agents.

The chosen time (30 min) for the evaluation of the tumorbearing mice was based on blood clearance. Blood elimination was conveniently rapid and demonstrated an uptake of less than 0.5% ID/mL. However, the optimal time for the BBN diagnosis was 90 min post-injection. Our preliminary conjugation of the molecule probably improved its stability, compared to the study of Zitzmann et al. (11) that demonstrated decreased blood uptake.

DUP-1 evaluation times for the tumors were less ideal.

The highest cell binding values occurred as early as 5 min after incubation. By 30 min, the DUP-1 tracer, which has been shown to bind very quickly, may have already been washed out. Also, Askoxylakis et al. (12) observed

REFERENCES 1. Yang M, Gao H, Zhou Y, Ma Y, Quan Q, Lang L, et al. 18F-labeled GRPR agonist and antagonists: A comparative study in prostate cancer imaging. Theranostics. 2011;1:220-9.

2. Gonza´lez N, Mantey SA, Pradhan TK, Sancho V, Moody TW, Coy DH, et al. Characterization of putative GRP-and NMB-receptor antagonists interaction with human receptors. Peptides. 2009;30:1473-86, http:// dx.doi.org/10.1016/j.peptides.2009.05.007.

3. Markwalder R, Reubi JC. Gastrin-releasing peptide receptors in the human prostate relation to neoplastic transformation. Cancer Res. 1999;59:1152-9.

4. Beer AJ, Eiber M, Souvatzoglou M, Schwaiger M, Krause BJ. Radionuclide and hybrid imaging of recurrent prostate cancer. Lancet Oncol. 2011;12:181-91, http://dx.doi.org/10.1016/S1470-2045(10)701030.

5. Nanda PK, Lane SR, Retzloff LB, Pandey US, Smith CJ. Radiolabeled regulatory peptides for imaging and therapy. Curr Opin Endocrinol Diabetes Obes. 2010;17:69-76.

169

Radiotracers for prostate carcinoma Faintuch BL et al.

CLINICS 2012;67(2):163-170

6. Faintuch BL, Teodoro R, Duatti A, Muramoto E, Faintuch S, Smith CJ. Radiolabeled bombesin analogs for prostate cancer diagnosis: preclinical studies. Nucl Med Biol. 2008;35:401-11, http://dx.doi.org/10.1016/ j.nucmedbio.2008.02.005.

7. Flores DG, Lenz G, Roesler R, Schwartzmann G. Gastrin-releasing peptide receptor signaling in cancer. Cancer Therapy. 2009;7:332-46.

8. Ferro-Flores G, Rivero IA, Santos-Cuevas CL, Sarmiento JI, Arteaga de Murphy C, Ocampo-Garcı´a BE, et al. Click chemistry for [99mTc(CO)3] labeling of Lys3-bombesin. Appl Radiot Isot. 2010;68:2274-8, http:// dx.doi.org/10.1016/j.apradiso.2010.06.014.

9. Parry JJ, Andrews R, Rogers BE. MicroPET imaging of breast cancer using radiolabeled bombesin analogs targeting the gastrin-releasing peptide receptor. Breast Cancer Res Treat. 2007;101:175-83, http:// dx.doi.org/10.1007/s10549-006-9287-8.

10. Krumpe LRH, Mori T. Potential of phage-displayed peptide library technology to identify functional targeting peptides. Expert Opin Drug Discov. 2007;2:525-43, http://dx.doi.org/10.1517/17460441.2.4.525.

11. Zitzmann S, Mier W, Schad A, Kinscherf R, Askoxylakis V, Kraemer S, et al. A new prostate carcinoma binding peptide (DUP-1) for tumor imaging and therapy. Clin Cancer Res. 2005;11:139–46.

12. Askoxylakis V, Zitzmann-Kolbe S, Zoller F, Altmann A, Markert A, Rana S, et al. Challenges in optimizing a prostate carcinoma binding peptide, identified through the phage display technology. Molecules. 2011;16:155978, http://dx.doi.org/10.3390/molecules16021559.

13. Bartoloma M, Valliant J, Maresca KP, Babich J, Zubieta J. Single amino acid chelates (SAAC): a strategy for the design of technetium and rhenium radiopharmaceuticals. Chem Commun. 2009;7:493-512, http:// dx.doi.org/10.1039/b814903h.

14. Reile H, Armatis PE, Schally AV. Characterization of high-affinity receptors for bombesin/gastrin releasing peptide on the human prostate cancer cell lines PC-3 and DU-145: Internalization of receptor bound 125I-(Tyr4) bombesin by tumor cells. Prostate. 1994;25:29–38, http:// dx.doi.org/10.1002/pros.2990250105.

15. Liu G, Hnatowich DJ. Labeling biomolecules with radiorhenium: a review of the bifunctional chelators. Anticancer Agents Med Chem.

2007;7:367-77, http://dx.doi.org/10.2174/187152007780618144.

16. Byk G, Partouche S, Weiss A, Margel S, Khandadash R. Fully Synthetic Phage-Like System for Screening Mixtures of Small Molecules in Live Cells. J Comb Chem. 2010;12:332–45, http://dx.doi.org/10.1021/ cc900156z.

17. Deschodt-Lanckman M, Robberecht P, De Neef P, Lammens M, Christophe J. In vitro action of bombesin and bombesin-like peptides on amylase secretion, calcium efflux, and adenylate cyclase activity in the rat pancreas: a comparison with other secretagogues. J Clin Invest.

1976;58:891–8, http://dx.doi.org/10.1172/JCI108542.

18. Chanda N, Kattumuri V, Shukla R, Zambre A, Katti K, Upendran A, et al.

Bombesin functionalized gold nanoparticles show in vitro and in vivo cancer receptor specificity. Proc Natl Acad Sci USA. 2010;107:8760-5, http://dx.doi.org/10.1073/pnas.1002143107.

19. Min K, Jo H, Song K, Cho M, Chun YS, Jon S, et al. Dual-aptamer-based delivery vehicle of doxorubicin to both PSMA (+) and PSMA (-) prostate cancers. Biomaterials. 2011;32:2124-32, http://dx.doi.org/10.1016/ j.biomaterials.2010.11.035.

20. Uchiyama F, Tanaka Y, Minari Y, Tokui N. Designing scaffolds of peptides for phage display libraries. J Biosci Bioeng 2005;99:448-56.

170

CLINICS 2012;67(2):171-178

DOI:10.6061/clinics/2012(02)13

BASIC RESEARCH

The protective effect of cilostazol on isolated rabbit femoral arteries under conditions of ischemia and reperfusion: the role of the nitric oxide pathway Mariana R.G.A. Santos, Andre´a C. Celotto, Verena K. Capellini, Paulo R. B. Evora, Carlos E. Piccinato, Edwaldo E. Joviliano Laboratory of Endothelial Function, Department of Surgery and Anatomy, Ribeira˜o Preto Faculty of Medicine, University of Sa˜o Paulo, Ribeira˜o Preto/SP, Brazil.

OBJECTIVES: The clinical significance of ischemia/reperfusion of the lower extremities demands further investigation to enable the development of more effective therapeutic alternatives. This study investigated the changes in the vascular reactivity of the rabbit femoral artery and nitric oxide metabolites under partial ischemia/ reperfusion conditions following cilostazol administration.

METHODS: Ischemia was induced using infrarenal aortic clamping. The animals were randomly divided into seven groups: Control 90 minutes, Ischemia/Reperfusion 90/60 minutes, Control 120 minutes, Ischemia/Reperfusion 120/90 minutes, Cilostazol, Cilostazol before Ischemia/Reperfusion 120/90 minutes, and Ischemia 120 minutes/Cilostazol/ Reperfusion 90 minutes. Dose-response curves for sodium nitroprusside, acetylcholine, and the calcium ionophore A23187 were obtained in isolated femoral arteries. The levels of nitrites and nitrates in the plasma and skeletal muscle were determined using chemiluminescence.

RESULTS: Acetylcholine- and A23187-induced relaxation was reduced in the Ischemia/Reperfusion 120/90 group, and treatment with cilostazol partially prevented this ischemia/reperfusion-induced endothelium impairment. Only cilostazol treatment increased plasma levels of nitrites and nitrates. An elevation in the levels of nitrites and nitrates was observed in muscle tissues in the Ischemia/Reperfusion 120/90, Cilostazol/Ischemia/Reperfusion, and Ischemia/ Cilostazol/Reperfusion groups.

CONCLUSION: Hind limb ischemia/reperfusion yielded an impaired endothelium-dependent relaxation of the femoral artery. Furthermore, cilostazol administration prior to ischemia exerted a protective effect on endotheliumdependent vascular reactivity under ischemia/reperfusion conditions.

KEYWORDS: Cilostazol; Nitric oxide; Ischemia/reperfusion; Femoral artery; Skeletal muscle.

Santos MRGA, Celotto AC, Capellini VK, Evora PRB, Piccinato CE, Joviliano EE. The protective effect of cilostazol on isolated rabbit femoral arteries under conditions of ischemia and reperfusion: the role of the nitric oxide pathway. Clinics. 2012;67(2):171-178.

Received for publication on November 20, 2011; First review completed on December 2, 2011; Accepted for publication on December 14, 2011 E-mail: eejov@fmrp.usp.br Tel.: 55 16 3602-2593 / 3602-2406

vascular disease patients. This compound is a selective inhibitor of phosphodiesterase 3 (PDE3), which raises intracellular cyclic adenosine monophosphate (cAMP) content and activates protein kinase A (PKA). These events culminate in antiplatelet aggregation and peripheral vasodilation (4).

The role of the NO pathway on the pharmacological action of cilostazol remains largely unknown. Therefore, this study investigated the effects on NO/endotheliumdependent femoral reactivity in vitro, as induced by an in vivo cilostazol infusion. Rabbit hind limb global ischemia was induced using infrarenal abdominal aortic crossclamping followed by reperfusion. The femoral artery wall response was examined because of its importance in the propagation of arterial blood throughout the hind limb.

INTRODUCTION The mechanisms of arterial occlusion are clearly associated with endothelial function, and these mechanisms are strongly affected by ischemia and blood reperfusion.

Endothelial dysfunction is caused by impaired nitric oxide (NO) release or a decrease in NO bioavailability, which increases the risk of blood vessel spasm and thrombosis.

Cilostazol exerts a protective effect on various vascular tissues following acute ischemia (1-3), and it alleviates the symptoms of intermittent claudication in peripheral

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

METHODS Drugs. Acetylcholine chloride (ACh), the calcium ionophore (A23187), sodium nitroprusside (SNP), phenylephrine

No potential conflict of interest was reported.

171

Cilostazol and hind limb reperfusion Santos MRGA et al.

CLINICS 2012;67(2):171-178

(Phe), indomethacin, and cilostazol (Cil) were purchased from Sigma Chemical Company (St. Louis, MO, USA).

SNP, ACh, and Phe were dissolved in distilled water.

Indomethacin was dissolved in ethanol (10-5 M), and the calcium ionophore was first dissolved in dimethylsulfoxide (DMSO) and was subsequently diluted in water. Cilostazol was first dissolved in DMSO (10 mg/ml), and the 1 mg/kg dose was prepared by the dilution of this solution with saline to a volume of 5 ml. This dose was intravenously administered for 20 minutes (5).

Experimental design. Male rabbits (2 2.5 kg) were preanesthetized with ketamine hydrochloride (KetalarH, 5 mg/ kg IM) and xylazine hydrochloride (RompunH, 10 mg/kg IM), and they were anesthetized using pentobarbital sodium (30 mg/kg IV). The animals underwent tracheostomy, and they were ventilated using an endotracheal tube (3.0 mm, Rusch, Teleflex Medical, Durham, NC, USA) with 100% O2 in a pressure-controlled mode (Takaoka 600, K. Takaoka Indu´stria e Come´rcio Ltda, Sa˜o Bernardo do Campo, SP, Brazil). The ear marginal artery was cannulated for volemic reposition with saline solution (NaCl 0.9%, 10 ml/kg/h).

The abdominal infrarenal aorta was exposed using a median transperitoneal laparotomy approach, and a vascular clamp was applied to produce ischemia. The Institutional Animal Care and Use Committee of the School of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, Brazil reviewed and approved the procedures for animal handling, which were in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996).

The animals were randomly divided into two main experimental groups, specifically, those treated without cilostazol (Group I - 4 subgroups) and with cilostazol (Group II - 3 subgroups): 1) Control 90 (anesthesia for 150

minutes); 2) Control 120 (anesthesia for 210 minutes); 3) I/R 90/60 (90 minutes of ischemia followed by 60 minutes of reperfusion); 4) I/R 120/90 (120 minutes of ischemia followed by 90 minutes of reperfusion); 5) Cil (endovenous cilostazol administered after anesthesia); 6) Cil/I/R (endovenous cilostazol administered before ischemia and submission of rabbits to 120 minutes of ischemia followed by 90 minutes of reperfusion); and 7) I/Cil/R (120 minutes of ischemia followed by cilostazol administration and reperfusion). Subgroups 1 and 2 from Group I were sham operation groups with different anesthesia times (120 and 210 minutes, respectively). Subgroups 3 and 4 from Group I (without cilostazol) underwent different durations of ischemia (90 and 120 minutes, respectively). Subgroups 5, 6, and 7 from Group II (with cilostazol) were used to investigate the isolated action of cilostazol and its protective effect when infused prior to or after 120 minutes of ischemia followed by 90 minutes of reperfusion (Figure 1).

Vascular reactivity. The femoral artery was carefully exposed through an anterior-medial hind limb incision and was immersed in Krebs solution composed of the following (in mM): NaCl 118.3, KCl 4.7, MgSO4 1.66, KH2PO4 1.22, CaCl2 2.5, NaHCO3 25.0, and glucose 11.1. Femoral artery segments (4-5 mm in length) were prepared with caution to avoid damage to the intimal surface of the vascular segments. The endothelium was removed by the gentle rubbing of the intimal surface of the blood vessel with a pair of watchmaker’s forceps to investigate vascular smooth muscle function without the influence of the endothelium.

This procedure removes the endothelium, but it does not affect vascular smooth muscle contraction. Vascular segments with and without endothelium were suspended in organ chambers (10 ml) filled with Krebs solution, maintained at 37 ˚C and bubbled with 95% O2/5% CO2 (pH = 7.4). Each ring was suspended using two stainless

Figure 1 - Diagram of study design illustrating the following subgroups within Group I (without cilostazol) and Group II (with cilostazol): 1) Control 90 (anesthesia for 150 minutes); 2) Control 120 (anesthesia for 210 minutes); 3) I/R 90/60 (90 minutes of ischemia followed by 60 minutes of reperfusion); 4) I/R 120/90 (120 minutes of ischemia followed by 90 minutes of reperfusion); 5) Cil (endovenous cilostazol administered after anesthesia); 6) Cil/I/R (endovenous cilostazol administered before ischemia and submission of rabbits to 120 minutes of ischemia followed by 90 minutes of reperfusion); and 7) I/Cil/R (120 minutes of ischemia followed by cilostazol administration and reperfusion).

172

CLINICS 2012;67(2):171-178

Cilostazol and hind limb reperfusion Santos MRGA et al.

preincubated with Phe (10-6 M), and dose-response curves for SNP, ACh, and A23187 were obtained after the rings reached a stable plateau.

Nitrite and nitrate (NOx) quantification. Blood samples from the cava vein were collected in tubes containing heparin (1520 v/v). The blood was centrifuged (30006g, 10 minutes, 4 ˚C), and plasma aliquots were stored at -70 ˚C for subsequent NOx measurement. Muscle (gracilis) tissue samples were wrapped and immediately stored at -70 ˚C. The muscle samples were homogenized in Tris-HCl (20 mM, pH 7.4, 10% w/v) for analysis. The homogenate was centrifuged (30006g, 10 minutes, 4 ˚C), and the supernatant was used to measure NOx concentrations and total protein using the modified biuret reaction (6). Plasma and muscle samples were analyzed using an ozone-based chemiluminescence assay. Briefly, the samples were treated

steel clips that passed through the lumen. One clip was anchored to the bottom of the organ chamber, and the other clip was connected to a strain gauge for the measurement of isometric force (Grass FT03; Grass Instrument Company, Quincy, MA). Each ring was stretched to the optimal lengthtension of 2 g (determined in a pilot study) and equilibrated for 60 minutes. Tissues were washed every 20 minutes during the equilibration period. Endothelial integrity was assessed by the degree of ACh-induced (10-6 M) relaxation in rings that were precontracted with Phe (10-6 M). The endothelium was present when the ACh-induced relaxation was 80% or greater. Each ring was washed and reequilibrated for 30 minutes in the presence of indomethacin (10-5 M) to exclude the cyclooxygenase effects from the analysis because the NO pathway was the target of the investigation. The femoral artery rings were

Figure 2 - Femoral artery concentration-response curves. (A, D) ACh (10-10 to 3.10-5 M); (B, E) A23187 (10-10 to 3.10-5 M); and (C, F) SNP (10-10 to 3.10-5 M) from the Control and I/R groups. * Indicates the difference between the Control 120 and I/R 120/90 groups (p,0.05).

173

Cilostazol and hind limb reperfusion Santos MRGA et al.

CLINICS 2012;67(2):171-178

with cold ethanol (152 v/v for 30 minutes at -20 ˚C) and centrifuged (40006g, 10 min). NOx levels were measured by injecting 25 mL of the supernatant into a glass purge vessel containing 0.8% vanadium (III) in HCl (1 N) at 90 ˚C, which reduces NOx to an NO gas. A nitrogen stream was bubbled through the purge vessel, which contained vanadium (III), and through NaOH (1 N) into an NO analyzer (SieversH Nitric Oxide Analyzer 280, GE Analytical Instruments, Boulder, CO, USA). The NOx concentration was calculated from a standard curve (sodium nitrate 0.5, 1.5, 10, and 50 mM), and it is expressed in mM for plasma and mM/mg protein for muscle samples.

Statistical analysis. The results are expressed as means ¡ the standard error of the mean (SEM). The dose-response curves for ACh, A23187, and SNP were derived using molar concentrations, and the logarithm of the molar concentration (log [M]) is presented in the three figures of figure 3. The EC50 was calculated from the dose-response curves of SNP), ACh, and A23187. The concentrationresponse curves were analyzed using two-way repeatedmeasures analysis of variance (ANOVA) and Bonferroni post-hoc tests. The concentrations of NOx were assessed using a one-way ANOVA (Prism 4.0, GraphPad Software,

Inc., San Diego, CA, USA). Values were considered to be statistically significant at p,0.05.

RESULTS Vascular reactivity. ACh and A23187 promoted concentration-dependent relaxation in femoral artery endothelium-intact rings, and this relaxation was reduced in the I/R 120/90 group (Figures 2A and 2B). SNP produced a concentration-dependent relaxation in femoral artery endothelium-denuded rings, and no intergroup statistical differences were observed (Figure 2C). Cilostazol treatment impaired ACh- and A23187-induced relaxation (Figures 3A and 3B), but cilostazol did not alter SNP-induced relaxation (Figure 3C). Cilostazol administration prior to the induction of ischemia partially prevented the ischemia/reperfusioninduced impairment of ACh- and A23187-induced relaxation (Figures 4A and 4B). No change in SNPinduced relaxation was observed (Figure 4C). The EC50 values of sodium nitroprusside (SNP), acetylcholine (ACh), and the calcium ionophore (A23) are listed in Table 1.

Plasma and tissue levels of NOx. No differences in the plasma levels of NOx (the Control 90 group versus the I/R

Figure 3 - Femoral artery concentration-response curves. (A) ACh (10-10 to 3.10-5 M); (B) A23187 (10-10 to 3.10-5 M); and (C) SNP (10-10 to 3.10-5 M) from the Control and Cilostazol groups. * Indicates the difference between the Control 120 and Cil groups (p,0.05).

174

CLINICS 2012;67(2):171-178

Cilostazol and hind limb reperfusion Santos MRGA et al.

Figure 4 - Femoral artery concentration-response curves. (A, D) ACh (10-10 to 3.10-5 M); (B, E) A23187 (10-10 to 3.10-5 M); and (C, F) SNP (10-10 to 3.10-5 M) from the Control, ischemia/reperfusion, and ischemia/reperfusion with cilostazol groups. * Indicates a difference from the Control 120 group, and # indicates a difference from the I/R 120/90 group (p,0.05).

compared to the levels found in the Control 120 and I/R 120/90 subgroups (Figure 6B).

90/60 group and the Control 120 group versus the I/R 120/90 group) (Figure 5A) between the Control and I/R groups were observed. Cilostazol administration prior to and after ischemia increased plasma levels of NOx compared to levels in the Control 120 group (Figure 5B).

In muscle tissues, 120 minutes of ischemia followed by 90 minutes of reperfusion increased the levels of NOx (Figure 6A). Cilostazol treatment prior to and after ischemia produced a stronger increased in NOx levels

DISCUSSION The re-establishment of blood flow to ischemic tissues or organs (i.e., reperfusion) is an essential step in many surgical procedures. However, reperfusion often leads to I/ R injury, especially after a long period of ischemia (7). I/R

Table 1 - The EC50 values of sodium nitroprusside (SNP), acetylcholine (ACh), and the calcium ionophore (A23). The data are expressed as means¡SEM.

ACh SNP A23

Cont 90

I/R 90

Cont 120

I/R 120

Cil

Cil/I/R

I/Cil/R

6.28¡0.03 5.96¡0.01 6.43¡0.01

6.23¡0.04 5.95¡0.01 6.40¡0.01

6.31¡0.03 5.93¡0.02 6.43¡0.01

5.98¡0.03 5.96¡0.03 6.52¡0.04

6.14¡0.03 5.69¡0.08 5.98¡0.14

6.47¡0.03 5.88¡0.02 6.20¡0.06

5.99¡0.03 5.92¡0.02 6.57¡0.03

175

Cilostazol and hind limb reperfusion Santos MRGA et al.

CLINICS 2012;67(2):171-178

Figure 6 - Concentration of muscle NO metabolites in the Control and ischemia/reperfusion (A) and Control, ischemia/reperfusion and ischemia/reperfusion with cilostazol (B) groups. * Indicates a difference from the Control 120 group (p,0.05).

Figure 5 - Concentration of plasma NO metabolites in the Control and ischemia/reperfusion (A) and Control, ischemia/reperfusion and ischemia/reperfusion with cilostazol (B) groups. * Indicates a difference from the Control 120 group (p,0.05).

with the results from two previous investigations that described relaxation dysfunction in canine femoral arteries after a 3 h/2 h hind limb I/R (4,10). Cilostazol administration prior to ischemia partially prevented the impaired ischemia/ reperfusion-induced relaxation found in the current model.

This endothelial dysfunction indicated that the loss of NOmediated vasorelaxation is an initial manifestation of free radical release during reperfusion. Cilostazol treatment reduces the size of myocardial infarcts (3,11). Therefore, our results showing the impairment of an endothelium-dependent vasodilation suggest that the vascular dysfunction found in our model is related to a reduced production of NO. The reperfusion of ischemic tissue induces an acute inflammatory response that results in necrosis and irreversible vascular endothelial cell injury. However, the precise role of the NO that is generated by iNOS and/or eNOS during ischemia and reperfusion is controversial. The pharmacological inhibition of eNOS NO synthesis has deleterious effects on myocardial ischemia and reperfusion injury, but NO donor treatment or the superinduction of iNOS attenuates reperfusion-induced myocardial infarction and improves contractile function (12-14). Also, iNOS inhibition abolishes the cardioprotection resulting from ischemic preconditioning (15-18).

However, the enhanced release of NO may contribute to ischemia and reperfusion injury, and NO inhibition may exert cardioprotective effects (19-21).

Our data revealed that 120 minutes of ischemia followed by 90 minutes of reperfusion increased NOx levels in muscle tissues. However, this phenomenon was not observed in plasma samples. Forty-five minutes of rat

injury is characterized by an increase in free reactive oxygen metabolites and cytokines, impaired NO production, the activation of adhesion molecules and neutrophils, and the promotion of lipid peroxidation with endothelial function impairment (8). The present study confirmed the importance of I/R duration on endothelium dysfunction; namely, an impairment of endothelium relaxation was observed in the I/R 120/90 group but not in the I/R 90/60 group. This result agrees with our previous experimental investigations that demonstrated femoral artery endothelium dysfunction in an I/R 120/90 group using an infrarenal clamp but indicated no alteration in the endothelium-dependent relaxation of the celiac trunk, renal, and superior mesenteric arteries after I/R 60/30 of the supraceliac aorta (4,9).

The present study also demonstrated no significant differences in the plasma levels of NOx between the groups.

An increase in the muscle levels of NOx was observed in the I/R 120/90 group, but no significant differences between the I/R and Cil groups were observed. The Cil/I/R group exhibited greater relaxation compared to the I/R group. The plasma levels of NOx were augmented in the Cil/I/R group.

The vascular reactivity results revealed that 120 minutes of ischemia induced by infrarenal aortic cross-clamping followed by 90 minutes of reperfusion reduced ACh- and A23187-induced endothelium-dependent relaxation, but these conditions did not affect the endothelium-independent relaxation of rabbit femoral arteries. These data are consistent

176

CLINICS 2012;67(2):171-178

Cilostazol and hind limb reperfusion Santos MRGA et al.

kidney ischemia followed by 6 hours of reperfusion boosts plasma levels of nitrite and nitrate, which are reduced by an iNOS inhibitor (22). Wild-type mice exhibit increased plasma NOx levels after myocardial I/R, but NOx species are not detected in iNOS-deficient (iNOS2/2) mice (23). In contrast, a decrease in NO below basal levels after hind limb I/R (2.5 hours/2 hours) has been observed (24). These results suggest that iNOS activation mediates the rise in NOx after reperfusion.

Cilostazol administration prior to or after ischemia attenuated vascular dysfunction and increased NOx levels in plasma and muscle tissues. Several agonists have been found to activate eNOS after an enhancement in intracellular free Ca2+ concentrations, but an alternative pathway for eNOS activation by the serine/threonine protein kinase, Akt, has also been demonstrated, which should lead to an augmented release of NO (11,25,26). Stimulation of the phosphatidylinositol-3-OH kinase/Akt pathway improves serine phosphorylation and induces eNOS activation and NO release.

Cilostazol activates Akt and eNOS to promote the protective effect of statins and reduce infarct size. In addition, cilostazol induces NO release in human aortic endothelial cells through PKA-dependent eNOS phosphorylation (26).

Therefore, the cilostazol-induced production of NOx species in the present work may be mediated by eNOS phosphorylation by Akt, the activation of protein kinase A (PKA), due to increased intracellular cAMP or an unknown pathway.

Study limitation. The present study did not use a NOS blocker to evaluate the effect of cilostazol on NO bioavailability. However, a recent study investigated the effect of systemic cilostazol administration on the ischemiainduced neovascularization process in vivo and examined the potential involvement of endothelial nitric oxide synthase (eNOS) signaling in the cilostazol-regulated neovascularization of ischemic muscles. The NOS inhibitor l-NAME abrogated the involvement of eNOS signaling in the neovascularization process (27).

In conclusion, 120 minutes of infrarenal aortic clamping followed by 90 minutes of reperfusion culminates in endothelium dysfunction, as suggested by the impaired endothelium-dependent relaxation of the femoral artery.

Moreover, cilostazol administration prior to ischemia exerted a protective effect on the endothelium-dependent vascular reactivity under ischemia/reperfusion conditions.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

ACKNOWLEDGMENTS

17.

We thank Jose´ Carlos Vanni and Agnes Afrodite Albuquerque Sumarelli for technical support. We are grateful to the Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and the Fundac¸a˜o de Apoio ao Ensino, Pesquisa e Assisteˆncia do Hospital das Clı´nicas da Faculdade de Medicina de Ribeira˜o Preto da Universidade de Sa˜o Paulo (FAEPA/ HCFMRP/USP) for financial support.

18.

19.

AUTHOR CONTRIBUTIONS 20.

Santos MRGA and Capellini VK contributed to the execution of the experiments and statistics. Celotto AC contributed to the execution of the experiments and to the article writing. Evora PRB and Piccinato CE contributed to the article writing. Joviliano EE was responsible for the project design and article writing.

21.

REFERENCES

22.

1. Iba T, Kidokoro A, Fukunaga M, Takuhiro K, Ouchi M, Ito Y. Comparison of the protective effects of type III phosphodiesterase (PDE3) inhibitor (cilostazol) and acetylsalicylic acid on intestinal

177

microcirculation after ischemia reperfusion injury in mice. Shock.

Augusta, Ga 2006; 26:522-6, http://dx.doi.org/10.1097/01.shk.000022 8800.56223.db.

Sun B, Le SN, Lin S, Fong M, Guertin M, Liu Y, et al. New mechanism of action for cilostazol: interplay between adenosine and cilostazol in inhibiting platelet activation. J Cardiovasc Pharmacol. 2002;40:577-85, http://dx.doi.org/10.1097/00005344-200210000-00011.

Bai Y, Muqier, Murakami H, Masamitsu I, Shohei S, Yoshihisa S, Hiroaki U, et al. Cilostazol protects the heart against ischaemia reperfusion injury in a rabbit model of myocardial infarction: focus on adenosine, nitric oxide and mitochondrial ATP-sensitive potassium channels. Clin Exp Pharmacol Physiol. 2011;38:658-65, http://dx.doi.org/10.1111/j.14401681.2011.05550.x.

Joviliano EE, Piccinato CE, Cherri J, Viaro F, Evora PR. Inferior canine hind limb ischemia and reperfusion impairs femoral artery endotheliumdependent wall relaxation. Vasc Endovascular Surg. 2005;39:39-46.

Fong M, Yoshitake M, Kambayashi J, Liu Y. Cilostazol increases tissue blood flow in contracting rabbit gastrocnemius muscle. Circ J. 2010; 74:181-7, http://dx.doi.org/10.1253/circj.CJ-09-0372.

Kaplan RS, Pedersen PL. Characterization of phosphate efflux pathways in rat liver mitochondria. Biochem J. 1983;212:279-88.

Krause GS, Kumar K, White BC, Aust SD, Wiegenstein JG. Ischemia, resuscitation, and reperfusion: mechanisms of tissue injury and prospects for protection. Am Heart J. 1986;111:768-80, http://dx.doi.org/ 10.1016/0002-8703(86)90114-6.

Lefer AM, Lefer DJ. The role of nitric oxide and cell adhesion molecules on the microcirculation in ischaemia-reperfusion. Cardiovasc Res. 1996;32:743-51.

Ciscato JG Jr., Capellini VK, Celotto AC, Baldo CF, Joviliano EE, Evora PR, et al. Vascular relaxation of canine visceral arteries after ischemia by means of supraceliac aortic cross-clamping followed by reperfusion.

Scand J Trauma Resusc Emerg Med. 2010;18:41, http://dx.doi.org/ 10.1186/1757-7241-18-41.

Johnson D, Freischlag JA, Lesniak R, Kelly H, Mudaliar JH, Cambria RA, et al. Endothelial damage due to ischemia and reperfusion is prevented with SIN-1. Cardiovasc Surg. 1998;6:367-72, http://dx.doi.org/10.1016/ S0967-2109(98)00026-X. Manickavasagam S, Ye Y, Lin Y, Perez-Polo RJ, Huang MH, Lui CY, et al.

The cardioprotective effect of a statin and cilostazol combination: relationship to Akt and endothelial nitric oxide synthase activation.

Cardiovasc Drugs Ther. 2007;21(5):321-30, http://dx.doi.org/10.1007/ s10557-007-6036-0.

Jones SP, Girod WG, Palazzo AJ, Granger DN, Grisham MB, Heuil DJ, et al. Myocardial ischemia-reperfusion injury is exacerbated in absence of endothelial cell nitric oxide synthase. Am J Physiol. 1999;276:H156773.

Kanno S, Lee PC, Zhang Y, Ho C, Griffti BP, Shears LL, et al. Attenuation of myocardial ischemia/reperfusion injury by superinduction of inducible nitric oxide synthase. Circulation. 2000;101:2742-8.

Pabla R, Buda AJ, Flynn DM, Blesse SA, Shin AM, Curtis MJ, et al. Nitric oxide attenuates neutrophil-mediated myocardial contractile dysfunction after ischemia and reperfusion. Circ Res. 1996;78:65-72.

Guo Y, Jones WK, Xuan YT, Tang XL, Bao W, Wu WJ, et al. The late phase of ischemic preconditioning is abrogated by targeted disruption of the inducible NO synthase gene. Proc Natl Acad Sci U S A. 1999; 96:11507-12, http://dx.doi.org/10.1073/pnas.96.20.11507.

Imagawa J, Yellon DM, Baxter GF. Pharmacological evidence that inducible nitric oxide synthase is a mediator of delayed preconditioning.

Br J Pharmacol. 1999;126:701-8, http://dx.doi.org/10.1038/sj.bjp.070 2368.

Takano H, Manchikalapudi S, Tang XL, Qiu Y, Rizvi A, Jadoon AK, et al.

Nitric oxide synthase is the mediator of late preconditioning against myocardial infarction in conscious rabbits. Circulation. 1998;98:441-9.

Xi L, Jarrett NC, Hess ML, Kukreja RC. Essential role of inducible nitric oxide synthase in monophosphoryl lipid A-induced late cardioprotection: evidence from pharmacological inhibition and gene knockout mice.

Circulation. 1999;99:2157-63.

Patel VC, Yellon DM, Singh KJ, Neild GH, Woolfson RG. Inhibition of nitric oxide limits infarct size in the in situ rabbit heart. Biochem Biophys Res Commun. 1993;194:234-8, http://dx.doi.org/10.1006/bbrc.1993.

1809.

Wildhirt SM, Weismueller S, Schulze C, Conrad N, Kornberg A, Reichart B. Inducible nitric oxide synthase activation after ischemia/reperfusion contributes to myocardial dysfunction and extent of infarct size in rabbits: evidence for a late phase of nitric oxide-mediated reperfusion injury. Cardiovascular Res. 1999;43:698-711, http://dx.doi.org/10.1016/ S0008-6363(99)00080-2.

Woolfson RG, Patel VC, Neild GH, Yellon DM. Inhibition of nitric oxide synthesis reduces infarct size by an adenosine-dependent mechanism.

Circulation. 1995;91:1545-51.

Chatterjee PK, Patel NS, Kvale EO, Cuzzocrea S, Brown PA, Stewart KN, et al. Inhibition of inducible nitric oxide synthase reduces renal ischemia/reperfusion injury. Kidney Int. 2002;61:862-71, http:// dx.doi.org/10.1046/j.1523-1755.2002.00234.x.

Cilostazol and hind limb reperfusion Santos MRGA et al.

CLINICS 2012;67(2):171-178

phosphorylation. Nature. 1999;399:601-5, http://dx.doi.org/10.1038/ 21224.

26. Hashimoto A, Miyakoda G, Hirose Y, Mori T. Activation of endothelial nitric oxide synthase by cilostazol via a cAMP/protein kinase A- and phosphatidylinositol 3-kinase/Akt-dependent mechanism. Atherosclerosis. 2006;189:350-7, http://dx.doi.org/10.1016/j.atherosclerosis.2006.01.

022.

27. Hori A, Shibata R, Morisaki K, Murohara T, Komori K. Cilostazol stimulates revascularisation in response to ischaemia via an enos-dependent mechanism. Eur J Vasc Endovasc Surg. 2011 Aug 16. [Epub ahead of print].

23. Zingarelli B, Hake PW, Yang Z, O’Connor M, Denenberg A, Wong HR. Absence of inducible nitric oxide synthase modulates early reperfusioninduced NF-kappaB and AP-1 activation and enhances myocardial damage. Faseb J. 2002;16:327-42, http://dx.doi.org/10.1096/fj.01-0533com.

24. Huk I, Nanobashvili J, Neumayer C, Punz A, Mueller M, Afkhampour K, et al. L-arginine treatment alters the kinetics of nitric oxide and superoxide release and reduces ischemia/reperfusion injury in skeletal muscle. Circulation. 1997;96:667-75.

25. Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt-dependent

178

CLINICS 2012;67(2):179

DOI:10.6061/clinics/2012(02)14

READERS OPINION

The detection of cysto-biliary communications during surgery for liver hydatid cysts: let’s speak the unspoken Oktay ˙Irko¨ru¨cu¨, Enver Reyhan, Hasan Erdem Department of General Surgery, Adana Numune Training and Research Hospital, Turkey.

Email: oktaytip@yahoo.com Tel.: 90 32235500-5498

We read with great interest the article by Kayaalp et al.

(1), whose work identified biliary orifices using a bile test and described the use of suturing cysto-biliary communications to reduce postoperative biliary complications significantly following hydatid liver surgery. We would like to express our opinion that testing using the injection of normal saline into the biliary system has several disadvantages based on three important concerns. First, normal saline is colorless, and in some cases, small or hidden cystobiliary communications may not be detected. Why did the authors not use a colored agent, such as methylene blue?

Second, small daughter cysts and minor fragments of cysts in the biliary tree may not be detected by injecting normal saline. How did the authors evaluate the whole biliary tree in patients with cysto-biliary communications? Third, in some cases, especially for deep cysts with multiple connections, the whole cavity may not be observable by the naked eye using a colorless agent. How did the authors evaluate the cyst cavities in these patients?

During routine surgical practice, we prefer injecting water-soluble contrast media and using cholangiography in patients with suspected cysto-biliary communications (Figure 1). If we detect leaks using cholangiography, we can inject methylene blue into the biliary system to find hidden orifices (2). There are certain advantages to using this technique. First, the surgeon can see the location and the diameter of the cysto-biliary communications and understand the severity of the problem. Second, using cholangiography, small daughter cysts or minor fragments of endocysts in the biliary tree can be detected. Third, hidden biliary-thoracic communications can be visualized. Fourth, the use of cholangiography may prevent the complete suture capture and occlusion of biliary branches when suturing cysto-biliary communications.

Figure 1 - Intraoperative transcystic cholangiography.

REFERENCES 1. Kayaalp C, Aydin C, Olmez A, Isik S, Yilmaz S. Leakage tests reduce the frequency of biliary fistulas following hydatid liver cyst surgery.

Clinics. 2011;66(3):421-4, http://dx.doi.org/10.1590/S1807-5932201100 0300010.

2. Atahan K, Ku¨peli H, Deniz M, Gu¨r S, C ¸ o¨kmez A, Tarcan E. Can occult cystobiliary fistulas in hepatic hydatid disease be predicted before surgery. Int J. Med. Sci. 2011;8(4):315-20.

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

179

CLINICS 2012;67(2):181-183

DOI:10.6061/clinics/2012(02)15

RAPID COMMUNICATION

Prevalence of erectile dysfunction in chronic renal disease patients on conservative treatment Jose´ Fernando Pereira Mesquita,I Thiago Faria Ramos,II Felipe Pereira Mesquita,II Jose´ Murillo Bastos Netto,III Marcus Gomes Bastos,IV Andre´ Avarese de FigueiredoV I

Faculdade de Medicina da Universidade Federal de Juiz de Fora, Curso de Po´s Graduac¸a˜o em Sau´de Brasileira, Juiz de Fora/MG, Brasil. II Faculdade de Medicina da Universidade Federal de Juiz de Fora, Juiz de Fora/MG, Brasil. III Faculdade de Medicina da Universidade Federal de Juiz de Fora, Departamento Materno Infantil e da Resideˆncia Me´dica em Urologia, Juiz de Fora/MG, Brasil. IV Faculdade de Medicina da Universidade Federal de Juiz de Fora Departamento de Clı´nica Me´dica, Juiz de Fora/MG, Brasil. V Universidade Federal de Juiz de Fora, Departamento de Anatomia do Instituto de Cieˆncias Biolo´gicas, Juiz de Fora/MG, Brasil.

Email: jfpmesquita@uol.com.br Tel.: 55 32 3231-2129

We assessed 81 patients with a mean age of 61.7¡11.6 years (range: 21-84). The results included an estimated ED rate of 70%, significance level of 0.05, presumed error margin of 5%, and a population of 163 elements. The study databank was statistically analyzed with SPSS 13.0 software.

For descriptive and bivariate analyses, only the valid cases were used. Logistic regression was used to characterize the relative weight of several variables studied on outcomes among the group of interviewed subjects. This study was approved by the ethics committee of the University Hospital of the Federal University of Juiz de Fora. During the historytaking visit, each subject received an explanation of the study and the shared patient/researcher responsibility in the study’s success. Informed consent authorizing data collection was then obtained.

INTRODUCTION Erectile dysfunction (ED), the inability to maintain sufficient penile erection for satisfactory sexual performance, is highly prevalent in the general population, affecting almost 50% of men in the 40-70 years age range, which corresponds to approximately 150 million men worldwide (1,2). More recently, ED has been considered a manifestation of a functional and/or structural abnormality affecting penile circulation as part of a more generalized vascular disorder (2).

Erectile dysfunction, therefore, has been associated with signs of generalized arterial disease, as it frequently coexists with diseases with a high component of endothelial dysfunction, such as coronary artery disease, idiopathic systemic arterial hypertension, atherosclerosis, and endstage chronic kidney disease (ESCKD). ED is also associated with cardiovascular disease risk factors, such as diabetes mellitus, dyslipidemia, and smoking (1-3).

Erectile dysfunction prevalence rates in men with chronic kidney disease (CKD) may reach 70-80%, with similar rates having been reported for those with ESCKD. A number of factors have been implicated in the development of ED in these patients, including the actual presence of a chronic disease, drugs, increased parathyroid hormone (PTH) serum levels, zinc deficiency, decreased testosterone serum levels, increased serum prolactin levels, and psychological factors (3,4).

RESULTS The prevalence of erectile dysfunction among those individuals aged 61 years or older was 85.1% compared with 66.7% of those ranging from 21 to 60 years in age (p = 0.052). The sample size may not have been sufficiently large to demonstrate a significant difference between the groups, as indicated in the literature. The prevalence of ED was 76.5% (62 patients), with 72.3% classified as stage 3 CKD, 81.5% as stage 4 CKD, and 85.7% as stage 5 CKD, although no statistically significant difference was found.

Nineteen patients (23.5%) had no ED; 24 (29.6%) had mild ED; 15 (18.5%) had mild/moderate ED; 13 (16%) had moderate ED; and 10 (12.3%) had severe ED. Our study did not demonstrate any association between ED and most comorbidities considered to impart important cardiovascular risk, except diabetes mellitus, which reached significance as both a cause (p = 0.004) of kidney disease and comorbidity (p = 0.006; Tables 1 and 2).

Of the 81 patients assessed, 59 were aware of their CKD for 60 months at most. Among these patients, 86.4% had ED, while 50% of those aware of their CKD for longer than 61 months had ED (p = 0.001). Sixty-three patients were followed-up as outpatients for 60 months at most; 82.5% had ED compared with 55.6% of those followed up for longer than 61 months (p = 0.017; Table 3).

The logistic regression model revealed that among subjects with ED, those with diabetes mellitus were 4.05 times more likely to have ED than non-diabetic subjects

MATERIALS AND METHODS This study assessed the prevalence rate of ED in stage 3, 4, and 5 CKD outpatients and sought to identify the associations among ED, CKD stage, and comorbidities. ED was graded according to the International Index of Erectile Function (IIEF), which was validated for use in Portuguese (5).

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

181

Prevalence of Erectile Dysfunction Mesquita JFP et al.

CLINICS 2012;67(2):181-183

Table 1 - Bivariate analysis of the association between erectile dysfunction and diabetes mellitus.

With dysfunction N (%)

p-value

YES

29 (93.6)

0.004*

NO

33 (66.0)

Variable Diabetes mellitus

Table 3 - Bivariate analysis of the association among erectile dysfunction, the length of disease and the length of follow-up.

With dysfunction

p-value

Less than 60 months

51(86.4)

0.001*

Equal to or more than 61 months Less than 60 months

11(50)

Variable Length of disease

(p = 0.048). However, subjects aware of their CKD for a period of less than 60 months were 3.5 times more likely to have ED than those who were aware of their CKD for longer periods (p = 0.012; Table 4).

Length of follow-up

52(82.5)

Equal to or more than 61 months *

0.017*

10(55.6)

p-value,0.05.

DISCUSSION The high prevalence of ED (76.5%) among CKD patients is compatible with published reports (4). The prevalence of ED among those who were aware of their CKD for less than 60 months was higher than that among subjects who were aware of their CKD for 61 months or more. The former group was 3.5 times more likely to have ED (p = 0.012; CI 1.4– 14.489). These data may indicate greater clinical, metabolic, and inflammatory imbalances among those who were more recently aware of their CKD and consequently had shorter follow-up times, a condition that theoretically favors a higher prevalence of ED (4,7-9). Therefore, this trend may also reflect a major public health issue, because these patients are likely to require specialized treatment. This study found a 93.6% ED prevalence rate among 31 patients with diabetes mellitus as a cause of their CKD, compared with a 66% ED prevalence rate among non-diabetic subjects (p = 0.004). For those with diabetes mellitus as a comorbidity, the prevalence of ED was 93,5%, which is in agreement with the literature (3,4).

Diabetic patients were 4.05 times more likely to have ED compared with non-diabetic subjects.

In conclusion, our study found a high prevalence rate of ED among CKD patients on conservative treatment. There

Table 4 - Logistic regression model for the association between erectile dysfunction, diabetes mellitus and the duration of chronic kidney disease.

Diabetes Mellitus Length of Disease

2

DM Caucasian Smoker (current or former) SAH1 Dyslipidemia CAD3 PVD4 Stroke5 ACEI5 ARB6 CCB7 Diuretics ALPHA-ADREN8 BETA-BLOCK9 Vasodilator Nitrate

% with Erectile Dysfunction

p-value

93.5 77.2 78.9 77.6 71.4 85.7 100 66.7 79.3 76.3 69.4 77.6 83.3 82.8 100 100

0.006 * 0.832 0.631 0.367 0.619 0.549 0.125 0.553 0.351 0.964 0.177 0.619 0.683 0.324 0.256 0.329

p-value

CI (95%)

Yes

4.05

0.048*

1.017 – 25.085

No Less than 60 months

1 3.50

0.012*

1.400 – 14.489

Equal to or more than 61 months

1

Analysis in relation to the ‘‘with erectile dysfunction’’ group of individuals. * p-value,0.05.

was a strong association with diabetes mellitus, either as a cause of the renal disease or as a comorbidity, and disease duration. We understand that inflammation, which is common in these patients (6-8), may account for the high prevalence rate of ED in this study. The presence of inflammation may point to the possibility of an early diagnosis of a generalized endothelial disease that is amenable to screening at the first nephrological consultation through a simple but efficient clinical approach.

The utilization of 5-phosphodiesterase inhibitors represents a novel therapeutic approach used to improve endothelial function (10). When administered daily to CKD patients, this treatment can prevent or delay the development of kidney disease as a consequence of reduced inflammation in the endothelium.

Table 2 - Bivariate analysis of the association between erectile dysfunction and other variables.

Variables

Odds ratio

Variable

ACKNOWLEDGMENTS This study was supported by the IMEPEN Foundation (Instituto Mineiro de Ensino e Pesquisa em Nefrologia) through the efforts of physicians, nurses, social assistants, psychologists, nutritionists, and supporting staff.

AUTHOR CONTRIBUTIONS Mesquita JFP was the researcher in charge of gathering and analyzing data as well as orienting students involved with this work; he is an expert on Brazilian healthcare. Ramos T and Mesquita FP are medical students from Faculdade de Medicina da Universidade Federal de Juiz de Fora who are in charge of interviewing patients and obtaining data. Netto JMB and Bastos MG are co-advisors on this study. Figueiredo AA is an advisor on this study.

*

p-value,0.05.

1-Systemic Arterial Hypertension; 2-Diabetes Mellitus; 3-Coronary Artery Disease; 4-Peripheral Vascular Disease; 5-Angiotensin-converting Enzyme Inhibitor; 6-Angiotensin Receptor Blocker; 7-Calcium-Channel Blocker; 8Alpha-adrenergics; 9-Beta-blocker.

182

CLINICS 2012;67(2):181-183

Prevalence of Erectile Dysfunction Mesquita JFP et al.

5. Ferraz MB, Ciconelli RM. Traduc¸a˜o e adaptac¸a˜o cultural do ´Indice Internacional de Func¸a˜o Ere´til para a lı´ngua portuguesa. Rev Bras Me´d.

1998;55:35-40.

6. Azadzoi KM, Goldstein I. Erectile Dysfunction due to Atherosclerotic Vascular Disease: the development of a animal model. J Urol. 1992; 147:1675-81.

7. Vlachopoulos C, Rokkas K, Stefanadis C. Inflammation, Metabolic Syndrome, Erectile Dysfunction and Coronary Artery Disease: Common Links. Eur Urol. 2007;52:1590-600, http://dx.doi.org/10.1016/j.eururo.

2007.08.004.

8. Stenvinkel P. Inflammatory and Atherosclerotic Interactions in the Depleted Uremic Patient. Blood Purif. 2001;19:53-61, http://dx.doi. org/10.1159/000014479.

9. Finkelstein FO, Shirani S, Wuerth D, Finkelstein SH. Therapy Insight: sexual Dysfunction in patients with chronic kidney disese. Ature Clinical Practice Nephrology. 2007;3(4):200-7, http://dx.doi.org/10.1038/ ncpneph0438.

10. Rosano GMC, Aversa A, Vitale C, Fabbri A, Fini M, Spera G. Chronic Treatment with Tadalafil Improves Endothelial Function in Men with Increased Cardiovascular Risk. Eur Urol. 2005;47:214-21, http:// dx.doi.org/10.1016/j.eururo.2004.10.002.

REFERENCES 1. Valchopoulos C, Aznaouridis K, Loakeimidis N, Rokkas K, Tsekoura D, Vasiliadou C, et al. Arterial function and intima-media thickness in hypertensive patients with erectile dysfunction. Journal of Hypertension.

2008;26:1829-36, http://dx.doi.org/10.1097/HJH.0b013e3283050886.

2. Yaman O, Gulpinar O, Hasan T, Ozdol C, Ertas FS, Ozgenci E. Erectile Dysfunction may predict coronary artery disease: relationship between coronary artery calcium scoring and erectile dysfunction severity. Int Urol Nephrol. 2008;40:117-23, http://dx.doi.org/10.1007/s11255-0079293-8.

3. Krishnan R, Izatt S, Bargman JM, Oreopoulos D. Prevalence and determinants of erectile dysfunction in patients on peritoneal dialysis.

International Urology and Nephrology. 2003;35:553-6, http:// dx.doi.org/10.1023/B:UROL.0000025649.54702.a6.

4. Navaneethan S D, Vecchio M, Chem P, Johnson DW, Saglimbene V, Graziano G, et al. Prevalence and Correlates of Self-Reported Sexual Dysfunction in CKD: A Meta-analysis of Observation Studies. American Journal of Kidney Diseases. 2010;56(4):670-85, http://dx.doi.org/ 10.1053/j.ajkd.2010.06.016.

183

CLINICS 2012;67(2):185-189

DOI:10.6061/clinics/2012(02)16

RAPID COMMUNICATION

Interobserver concordance in the BI-RADS classification of breast ultrasound exams Maria Julia G. Calas,I,II Renan M.V.R. Almeida,I Bianca Gutfilen,II Wagner C.A. PereiraI I Programa de Engenharia Biome´dica – COPPE, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. II Departamento de Radiologia, Faculdade de Medicina, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

Email: mjgcalas@terra.com.br Tel.: +55 21 2562 8629

of Radiology (ACR) standards (12). The image evaluation criteria were based on six BI-RADS–US categories (12): incomplete (0), negative (1), benign (2), probably benign (3), suspicious (4), and highly suggestive of malignancy (5). The surgical histopathological data were also obtained.

Fourteen breast-imaging radiologists participated in the study. They worked in different institutions but had similar numbers of ultrasounds, mammography exams, and biopsy procedures and had 4 to 23 years of breast radiology experience (,5 years, n = 2; 5–10 years, n = 8; .10 years, n = 4). The retrospective review was performed on hard copies of the digitized sonographic images. Each observer received a compact disc with images from 40 lesions and a form with the ultrasound morphological criteria and BIRADS classification. They were instructed to classify the lesions according to this system and were given 30 days to return the material. While no specific training was provided before the study, all of the readers had been using the BIRADS lexicon since 2005. The observers had no access to clinical or histopathological information from the patients, and all of them complied with the instructions provided. To ensure patient anonymity, the names were removed from the images and materials, and each patient was identified by a code.

The observers’ BI-RADS analyses were classified according to their level of concordance: a) total, the same BI-RADS category was assigned; b) partial, different categories were assigned but grouped into negative (2 or 3) and positive (4 or 5) categories so that the biopsy recommendations were the same; and c) disagreement, different categories were assigned (at least for one observer) and produced recommendations for different management plans (biopsy or follow-up). We considered category 0 (incomplete) as partial agreement with the negative (2 or 3) and positive (4 or 5) categories in the sense that the patient would have to be submitted to further studies to define the final classification.

The proportions of discordant classifications according to the experience time categories (,5 years, 5–10 years, and .10 years) were analyzed using Chi-square tests.

The modified Fleiss’ kappa index was used to analyze concordance because the data were grouped into six categories (13). The index values give the following interpretations: poor (k,0), slight (k = 0.0–0.20), fair (k = 0.21– 0.40), moderate (k = 0.41–0.60), substantial (k = 0.61–0.80), and almost perfect (k = 0.81–1.00). The analyses stratified by the BI-RADS categorization and by the data grouping described above. The statistical analysis was performed using the R Project for Statistical Computing software (14).

INTRODUCTION Breast ultrasound is an important complement to the clinical/mammographic investigation of breast lesions. This operator-dependent method entails real-time image detection and analysis and requires extensive training and experience in identifying and differentiating between benign and malignant lesions (1-4).

Lesion contour and shape are considered to be the main features that allow differentiating benign and malignant lesions, the former with high sensitivity and the latter with high specificity. Many authors believe that combined ultrasound methods may yield greater accuracy (5-10).

However, using morphological characteristics for lesion differentiation demands a high rate of interobserver agreement, an issue that has been extensively examined for mammography but that has been given less attention for ultrasound. Interobserver agreement is thus a matter of strong concern in clinical radiological practice (3-11).

To better characterize the interobserver agreement in breast ultrasound, this study examined a group of 14 breast imagers who used the Breast Imaging Reporting and Data System (BI-RADS) ultrasound classification on 40 breast lesions. The study was exclusively concerned with lesion categorization agreement among the observers according to the BI-RADS lexicon. The accuracy of the observers was not directly assessed through comparisons with the final lesion histology.

MATERIALS AND METHODS This study used 40 B-mode echographic images of lesions obtained from 40 patients who were examined at a private institution and who subsequently underwent surgery as indicated by their referring physicians.

The study was approved by an Institutional Ethics Committee, and all of the patients provided written informed consent. All of the examinations were performed by one radiologist using Logic 5 ultrasound equipment (GE Medical Systems, Inc., Milwaukee, WI, USA) with a 12 MHz transducer. Short- and long-axes orthogonal images were recorded for each patient according to the American College

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

185

Interobserver concordance in breast ultrasound Calas MJG et al.

CLINICS 2012;67(2):185-189

recommendation for tissue sampling. In one of these cases, an observer classified a carcinoma as BI-RADS 0; this classification was considered to be partially concordant because this category demanded further studies. One additional carcinoma in the partial agreement group was incorrectly classified as benign, and two fibroadenomas were classified as BI-RADS 2 or 3 by 13 observers (with one observer choosing BI-RADS 0, which was considered to be partial agreement) (Table 1).

In the 24 cases of disagreement (Table 2), the histopathological analyses confirmed that 15 cases were benign and nine were malignant lesions. In 5 of the 15 benign cases, only one observer disagreed; 13 agreed. A single observer disagreed in three of the nine malignant cases. However, nine observers disagreed on a benign hematoma case (longest lesion axis = 18.2 mm), and eight observers disagreed on a carcinoma case (longest lesion axis = 26.9 mm).

The proportions of discordant classifications were not significantly different by the experience time categories (11%, 12%, 15%; p = 0.62).

The kappa value for the original BI-RADS categories was 0.389 (fair agreement). This value was 0.612 when the categories were grouped as previously described, indicating substantial agreement.

RESULTS The average age of the 40 subjects was 50.7 years, ranging from 16 to 88 years. The lesion sizes ranged from 6 to 27.0 mm (mean diameter, 15.4 mm); 19 were benign, and 21 were malignant. The concordance analysis identified three cases of total agreement, 13 of partial agreement, and 24 cases of total disagreement (Table 1).

In the three cases of total agreement among all the reviewers, two fibroadenomas were classified as BI-RADS 3, and one carcinoma was classified as BI-RADS 4 (Table 1). In the 13 cases of partial agreement, 10 carcinomas were assessed by all of the reviewers as BI-RADS 4 or 5, with a Table 1 - The histopathological results and BI-RADS classifications for the individual observers. The results are grouped according to the level of observer concordance.

Observers and assigned categories

Concordance Complete Fibroadenoma 1 2 Carcinoma Partial Fibroadenoma 1 2 Carcinoma 1 2 3 4 5 6 7 8 9 10 11 Discordance Fibroadenoma 1 2 3 4 5 6 7 8 9 Hematoma Cyst 1 2 3 4 5 Carcinoma 1 2 3 4 5 6 7 8 9

1

2

3

4

5

6

7

8

9

10 11 12 13 14

3 3 4

3 3 4

3 3 4

3 3 4

3 3 4

3 3 4

3 3 4

3 3 4

3 3 4

3 3 4

3 3 4

3 3 4

3 3 4

3 3 4

3 3

3 3

3 3

3 0

3 3

3 3

3 3

3 3

3 3

3 3

3 3

2 3

3 3

3 3

4 5 4 4 4 4 4 4 3 4 5

4 5 4 5 4 5 5 5 3 4 5

4 5 5 5 5 5 5 4 3 4 5

5 5 4 5 4 5 5 4 3 4 5

5 5 5 4 4 5 4 4 3 4 4

4 4 0 5 4 5 5 4 3 4 4

4 5 4 5 5 4 5 4 3 5 5

5 4 5 5 5 5 5 4 3 5 5

5 4 4 5 4 4 4 4 3 5 4

5 5 4 4 4 4 5 4 3 4 4

5 5 4 5 5 5 5 4 3 5 5

5 5 4 5 5 5 5 5 3 5 5

4 5 4 5 4 4 5 4 3 4 5

4 4 4 4 4 4 4 4 2 5 4

4 3 3 4 2 3 3 4 3 4

3 4 4 2 2 3 3 4 3 4

3 3 3 2 2 3 3 3 3 4

2 3 3 3 2 3 3 3 0 4

3 3 3 4 2 3 3 2 4 3

3 3 3 4 2 3 3 4 3 4

3 4 3 4 2 3 4 3 3 3

4 4 3 3 2 3 3 4 3 3

2 3 3 3 2 3 3 3 3 4

3 3 3 3 2 3 3 3 3 4

3 4 3 3 2 3 3 3 3 4

4 4 3 2 3 3 3 4 3 4

3 3 2 4 2 3 3 3 4 3

3 3 4 3 5 4 4 4 3 3

3 3 2 2 3

2 2 2 2 4

3 3 2 2 4

2 3 3 3 3

2 3 2 2 3

2 3 2 4 3

2 4 3 3 4

2 4 2 2 2

0 3 2 2 3

2 3 3 2 3

3 3 2 3 3

2 3 4 3 3

2 3 2 2 4

4 4 3 2 4

4 3 4 4 4 4 4 4 4

5 4 5 4 4 4 5 5 4

4 4 4 4 5 4 5 5 4

4 3 5 4 5 3 5 5 2

4 3 4 4 4 4 4 5 2

4 3 5 3 3 3 5 5 4

4 4 4 4 5 4 5 5 5

5 3 5 5 5 5 5 5 5

3 3 0 3 4 3 5 4 5

4 4 4 3 4 3 4 4 4

4 4 5 5 4 4 5 5 4

4 3 5 5 5 4 5 5 5

4 4 4 4 4 4 5 4 4

3 3 3 2 3 4 3 3 5

DISCUSSION Most inter- and intra-observer BI-RADS concordance studies have examined mammography because BI-RADS has been used for mammography since 1993. Recent studies of interobserver agreement in BI-RADS ultrasound assessments have yielded kappa values ranging from 0.28 to 0.83, indicating a subjectively derived assessment of the morphological lesion characteristics (6-9,11,15-24).

One limitation of this study is the small number of cases (40) compared to other studies, which have had 55 to 267 cases (6-9,11,15-25). These cases did not consist of a random sample from the relevant female population. However, the cases were not selected according to pathological characteristics; therefore, no direct selection bias was apparent.

No previously published study has used 14 observers, although one used 10 radiologists (with only 10 patients) (8).

Additionally, most interobserver studies have used static image diagnosis (6-9,11,15-25). The exceptions include Berg et al. (8) and Bosch et al. (9), both of which were real-time analyses.

A second limitation of this study is the retrospective analysis of photographic records rather than real-time examination, which reflects the real clinical situation.

However, no images were rejected by the observers.

Another possible limitation is our not examining the possible correlations between clinical information and mammographic findings. The importance of these correlations may be seen in Skaane et al. (21), who concluded that the knowledge of previous mammography results is important for properly using BI-RADS in ultrasound. They measured kappa indices of 0.58 (range 0.52–0.66) for mammography, 0.48 (range 0.37–0.61) for ultrasound, and 0.71 (range 0.63–0.79) for both methods combined.

Berg et al. (8) measured a kappa value of 0.52 for the BIRADS categorizations of 11 radiologists, which was comparable to the results of mammography agreement studies.

After grouping BI-RADS categories 1, 2, and 4A together and categories 4B, 4C, and 5 together, Berg et al. (8) obtained

186

CLINICS 2012;67(2):185-189

Interobserver concordance in breast ultrasound Calas MJG et al.

Table 2 - The number of interobserver disagreements on cases, according to lesion type (histology) and years of experience, for the 14 observers using the BI-RADS classification system for breast ultrasonography.

Case types

Cyst 1 2 3 4 Fibrocystic Alteration Hematoma Fibroadenomas 1 2 3 4 5 6 7 8 9 Carcinoma 1 2 3 4 5 6 7 8 9 Total Benign Malignant

Discordances according to experience (years)

Lesion size (mm)

Concordance

,5

5-10

.10

1 2

1 3 6

2 3 1 1

24.9 14.2 25.4 13.3 16.5 18.2

13 13 09 11 13 05

1 2 1 -

1 1 1 4 1 1 2 3 2

1 2 1 1 2 -

6.4 24.0 13.6 22.1 11.4 10.1 19.2 13.9 8.2

11 09 12 09 13 13 12 08 12

1 1 -

1 4 1 1 1 1 3 1

1 3 3 1 1 1 -

17.9 26.9 22.5 15.6 7.9 12.4 11.1 19.5 14.7

12 06 13 10 13 12 12 10 13

7 2

26 13

14 10

a kappa value of 0.56. When the categories were dichotomized as BI-RADS 1, 2, 3 vs. 4A, 4B, 4C, 5, the kappa value was 0.48. These results differ from our current finding of an increase in the kappa value (from 0.3 to 0.6) after category grouping.

Using BI-RADS for mammography and ultrasound, Lazarus et al. (23) have identified a high concordance for highly suspicious lesions (k = 0.56, BI-RADS 5). Similar results were obtained in our study: 11 of the 16 (complete or partial) agreement cases were classified as BI-RADS 4 or 5.

Baker and Soo (3) analyzed 152 photographic records from 86 hospitals; in 23 cases (15.1% of the records), they noted a disagreement in interpretation. These disagreements were defined as classification differences that resulted in treatment changes, similar to the definition of disagreement used in this study. Their discrepancies included four false-negative cases, 14 false-positives, 3 cases that were described as cysts but which were found to be solid masses in biopsies, and two cases of differences between the sonographic and mammographic findings. We identified 24 cases of disagreement in our study, of which 15 were benign and nine were malignant lesions.

Eight of the 14 observers in our study classified a case of medullary carcinoma as benign. This result is similar to one reported in Rahbar et al. (19); the observers agreed on the criteria leading to a benign classification, even in one case of medullary carcinoma. These misclassifications are understandable because this type of carcinoma is characterized by a partially circumscribed contour and a discrete posterior acoustic enhancement that can be confused with a complicated cyst.

Shimamoto et al. (15) evaluated 54 lesions (30 benign and 24 malignant) and reported accuracies ranging from 53.7% to 61.1% in the junior observers group and from 64.8% to 72.2% in the senior group. The authors suggested that agreement was more dependent on case difficulty than on observer experience. Although our study was not designed to evaluate the accuracy of the individual observers or to correlate that accuracy with variables such as experience and lesion size, the lack of significant differences among our experience categories suggests similar results. At this point, it is important to note that the BI-RADS lexicon has been used by the observers in our study since 2005, and this familiarity may explain why experience was not statistically significant. Perhaps experience would have been more of an influence if the study had included lesion detection.

Del Frate et al. (24) found that interobserver variation depended on the size of the lesion, with a better concordance (k = 0.71–0.83) for lesions .7 mm. In Abdullah et al. (25), five breast radiologists retrospectively evaluated 267 breast masses (113 benign and 154 malignant) using the BI-RADS lexicon. The reviewers had no access to any other patient data. The interobserver BI-RADS agreement was assessed with the Aickin revised eˆ statistic and varied considerably (k = 0.30). This result is similar to the value (k = 0.28) reported

by Lazarus et al. (23) and is slightly below those reported in this study; however, it is lower than the value (k = 0.53) reported by Lee et al. (11). This inconsistency is probably related to the subdivision of BI-RADS category 4 (i.e., 4A, 4B, 4C), which reduces the frequency of agreement. This consideration was also discussed by Lee et al. (11), who

187

Interobserver concordance in breast ultrasound Calas MJG et al.

CLINICS 2012;67(2):185-189

3. Baker JA, Soo MS. Breast US: assessment of technical quality and image interpretation. Radiology. 2002;223:229-38, http://dx.doi.org/10.1148/ radiol.2231011125.

4. Chen SC, Cheung YC, Su CH, Chen MF, Hwang TL, Hsueh S. Analysis of sonographic features for the differentiation of benign and malignant breast tumors of different sizes. Ultrasound Obstet. Gynecol.2004;23:18893, http://dx.doi.org/10.1002/uog.930.

5. Paulinelli RR, Freitas-Ju´nior R, Moreira MAR, Moraes VA, BernardesJu´nior JRM, Vidal CSR, et al. Risk of malignancy in solid breast nodules according to their sonographic features. J Ultrasound Med. 2005;24:63541.

6. Arger PH, Sehgal CM, Conant EF, Zuckerman J, Rowling SE, Patton JA. Interreader variability and predictive value of US descriptions of solid breast masses: pilot study. Acad Radiol. 2001;8(4):335-42, http:// dx.doi.org/10.1016/S1076-6332(03)80503-2.

7. Baker JA, Kornguth PJ, Soo MS, Walsh R, Mengoni P. Sonography of solid breast lesions: observer variability of lesion description and assessment. AJR. 1999;172(6):1621-25.

8. Berg WA, Blume JD, Cormack JB, Mendelsen EB. Operator dependence of physician-performed whole-breast US: lesion detection and characterization. Radiology. 2006;241(2):355-65, http://dx.doi.org/10.1148/ radiol.2412051710.

9. Bosch AM, Kessels AGH, Beets GL, Vranken KLCG, Borstlap AC, Von Meyenfeldt MF, et al. Interexamination variation of whole breast ultrasound. Br J Radiol. 2003;75:328-31, http://dx.doi.org/10.1259/bjr/17252624.

10. Heinig J, Witteler R, Schmitz R, Kiesel L, Steinhard J. Accuracy of classification of breast ultrasound findings based on criteria used for BIRADS. Ultrasound Obstet Gynecol. 2008;32:573-58, http://dx.doi.org/ 10.1002/uog.5191.

11. Lee H-J, Kim E-K, Kim MJ, Hyun Youk JI, Young Lee JI, Kang DR, et al.

Observer variability of Breast Imaging Reporting and Data System (BIRADS) for breast ultrasound. Eur J Radiol. 2008;65(2):293-98, http:// dx.doi.org/10.1016/j.ejrad.2007.04.008.

12. American College of Radiology. Illustrated Breast Imaging Reporting and Data System Atlas (BI-RADSH): Ultrasound. In: 4th ed, Reston, VA, American College of Radiology. (ACR); 2003.

13. Kundel HL, Polansky M. Measurement of observer agreement. Radiology.

2003;228:303-08, http://dx.doi.org/10.1148/radiol.2282011860.

14. R Development Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. 2004, Internet URL [http://www.R-project.org].

15. Shimamoto K, Sawaki A, Ikede M, Satake H, Naganawa S, Tadokoro M, et al. Interobserver agreement in sonographic diagnosis of breast tumors.

Eur J Ultrasound. 1998;8:25-33, http://dx.doi.org/10.1016/S0929-8266(98) 00047-0.

16. Calas MJG, RMVR Almeida, Gutfilen B. Pereira WCA (2009) Intraobserver interpretation of breast ultrasonography following the BI-RADS classification. Eur J Radiol. 2010;74(3):525-28, http://dx.doi.org/ 10.1016/j.ejrad.2009.04.015.

17. Costantini M, Belli P, Ierardi C, Franceschini G, La Torre G, Bonomo L. Solid breast mass characterization: use of the sonographic BI-RADS classification. Radiol Med. 2007;112(6):877-94, http://dx.doi.org/ 10.1007/s11547-007-0189-6.

18. Hong AS, Rosen EL, Soo MS, Baker JA. BI-RADS for sonography: positive and negative predictive values of sonographic features. AJR. 2005;184:1260-65.

19. Rahbar G, Sie AC, Hansen GC, Prince JS, Melany ML, Reynolds HE, et al.

Benign versus malignant solid breast masses: US differentiation.

Radiology. 1999;213(3):889-94.

20. Stavros AT, Thickman D, Rapp CL, Dennis MA, Parker SH, Sisney GA. Solid breast nodules: use of sonography to distinguish between benign and malignant lesions. Radiology. 1995;196(1):123-34.

21. Skaane P, Engedal K, Skjennald A. Interobserver variation in the interpretation of breast imaging. Comparison of mammography, ultrasonography, and both combined in the interpretation of palpable noncalcified breast masses. Acta Radiol. 1997;38:497-502.

22. Skaane P, Engedal K. Analysis of sonographic features in the differentiation of fibroadenoma and invasive ductal carcinoma. AJR. 1998;170:109-14.

23. Lazarus E, Mainiero MB, Schepps B, Koelliker SL, Livingston LS. BIRADS Lexicon for US and Mammography: interobserver variability and positive predictive value. Radiology. 2006;239(2):385-91, http://dx.doi. org/10.1148/radiol.2392042127.

24. Del Frate C, Bestagno A, Cerniato R, Soldano F, Isola M, Puglisi F, et al.

Sonographic criteria for differentiation of benign and malignant solid breast lesions: size is of value. Radiol Med. 2006;111:783-96, http:// dx.doi.org/10.1007/s11547-006-0072-x.

25. Abdullah N, Mesurolle B, El-Khoury M, Kao E. Breast Imaging Reporting and Data System Lexicon for US: interobserver agreement for assessment of breast masses. Radiology. 2009;252(3):665-72, http:// dx.doi.org/10.1148/radiol.2523080670.

26. Lai XJ, Zhu QL, Jiang YX, Dai Q, Xia Y, Liu H, Zhang J, You SS, Wang HY. Inter-observer variability in Breast Imaging Reporting and Data

noted a low percentage of 4B responses (4.8% vs. 19.4% in Abdullah et al.) (25).

The most recent study published by Lai et al. (26) used a methodology similar to ours. It evaluates 30 breast lesions that underwent resection surgeries and utilizes 12 observers with different amounts of experience using ultrasound with BIRADS for breast imaging. For experienced observers, the kappa values of categories 3, 4 and 5 were 0.72, 0.28 and 0.60, respectively. The authors concluded that diagnostic agreement decreases as the breast imaging experience of the radiologist decreases. Our study found that experience is not directly related to agreement. This difference is perhaps explained by the most-experienced group of professionals in Lai et al. having more than three years of experience, while the least-experienced professionals in our study had less than five years.

Several studies have proposed using diagnostic methodologies based on image parameter estimation to improve the consistency of image interpretation. These techniques aim to quantify the morphological characteristics of tumors, such as shape and texture, and to use the results for differentiating between benignancy and malignancy. These complex procedures, including computer-aided diagnosis (CAD) systems that may reduce discrepancies between observers and thus improve ultrasound accuracy, continue to be investigated (27-30).

In practice, BI-RADS categorization is defined by a combination of the mammographic and sonographic features, but this generalization did not hold in our study. Although the sample used here included only 40 lesions, this study allowed identifying the critical issues that deserve attention and further inquiry. Our kappa value for the BI-RADS classification (0.389, fair) indicates the need for standardization. Our results also indicate the need for a more meticulous version of BI-RADS, the need for real-time quantitative lesion analysis to reduce observer variation and the need to improve the accuracy of ultrasound examinations.

AUTHOR CONTRIBUTIONS The four authors form an interdisciplinary research group (all from the Federal University of Rio de Janeiro) investigating the clinical and engineering aspects of radiology and have worked together since 2006 (with some publications in congresses and in a peer-reviewed journal).

The corresponding author (Maria Julia G. Calas, MD) is a PhD student, and the subject of this manuscript was taken from her thesis; therefore, she was responsible for most of the specific details described.

Nevertheless, each of the four authors contributed to all aspects of the study, from design to manuscript preparation, and all are familiar with the contents. Calas MJG conceived and designed the study, prepared and edited the manuscript, and was also responsible for the study integrity guarantee, literature research, clinical and experimental studies and data analysis. Gutfilen B was responsible for the manuscript preparation, study integrity guarantee, experimental studies and data analysis. Pereira WCA conceived and designed the study, prepared and edited the manuscript, and was also responsible for the experimental studies and data analysis. Almeida RMVR prepared the manuscript, and was also responsible for the study integrity guarantee and statistical analysis.

REFERENCES 1. Berg WA, Blume JD, Cormack JB, Mendelson EB, Lehrer D, Bo¨hm-Ve´lez M, et al. Combined screening with ultrasound and mammography vs mammography alone in women at elevated risk of breast cancer. JAMA. 2008;299(18):2151-63, http://dx.doi.org/10.1001/jama.299.18.2151.

2. Karssemeijer N, Otten JDM, Verbeek ALM, Groenewoud JH, Koning HJ, Hendriks JHCL, et al. Computer-aided detection versus independent double reading of masses on mammograms. Radiology. 2003;227:192200, http://dx.doi.org/10.1148/radiol.2271011962.

188

CLINICS 2012;67(2):185-189

Interobserver concordance in breast ultrasound Calas MJG et al.

29. Alvarenga AV, Pereira WCA, Infantosi AFC, Azevedo CM. Complexity curve and grey level co-occurrence matrix in the texture evaluation of breast tumour on ultrasound images. Medical Physics. 2007;34(2):379-87, http://dx.doi.org/10.1118/1.2401039.

30. Alvarenga AV, Pereira WCA, Infantosi AFC, Azevedo CM. Assessing the performance of morphological parameters in distinguishing breast tumors on ultrasound images. Medical Engineering & Physics.

2010;32(1):49-56, http://dx.doi.org/10.1016/j.medengphy.2009.10.007.

System (BI-RADS) ultrasound final assessments. Eur J Radiol 2011, http://dx.doi.org/10.1016/j.ejrad.2011.04.069.

27. Drukker K, Giger ML, Vyborny CJ, Mendelson EB. Computerized detection and classification of cancer on breast ultrasound. Acad Radiol.

2004;11:526-535, http://dx.doi.org/10.1016/S1076-6332(03)00723-2.

28. Horsch K, Giger ML, Venta LA, Vyborny CJ. Computerized diagnostic of breast lesions on ultrasound. Medical Physics. 2002;29(2):157-64, http:// dx.doi.org/10.1118/1.1429239.

189

CLINICS 2012;67(2):191-192

DOI:10.6061/clinics/2012(02)17

CASE REPORT

Paroxetine-induced severe sleep bruxism successfully treated with buspirone Aysel Milanlıoglu Van Medical Park Hospital, Clinic of Neurology, Van, Turkey.

Email: ayselmilanlioglu@yahoo.com Tel.: 90-530-8263565 / 90-432-2223222

eventually increased to 10 mg/nightly. Within 2 weeks of beginning buspirone treatment, the patient and her husband reported the disappearance of the jaw pain and stiffness, as well as the bruxism symptoms.

INTRODUCTION Sleep bruxism is characterized by the involuntary clenching or grinding of the teeth during sleep and can cause severe health problems, including the destruction of tooth structure, temporo-mandibular joint dysfunction, myofascial pain, and severe sleep disturbances (1). Iatrogenic sleep bruxism may be common during treatment with pyschotropic medications, such as antipsychotics and antidepressants, especially selective serotonin reuptake inhibitors (SSRIs) (2). This article reports the case of a depressive woman with paroxetine-induced sleep bruxism who was successfully treated with buspirone.

DISCUSSION Bruxism is a common movement disorder that affects 821% of the population. The majority of bruxism symptoms are mild or moderate, although rare but severe cases may lead to serious periodontal damage, temporo-mandibular dysfunction, sleep disturbances, jaw pain, and stiffness. As a result, such cases must be treated with medication (3).

The neurochemical mechanism underlying the development of bruxism remains unclear. However, recent investigations have suggested that the central dopaminergic system (especially within the meso-cortical tract), which controls muscular or motor activity, may be involved in the pathophysiology of bruxism (4). It has been hypothesized that the mechanism for SSRI-induced bruxism may involve excessive serotonergic action on the meso-cortical neurons arising from the ventral tegmental area. This action leads to a dopaminergic deficit, which causes a specific form of akathisia and akathisia-like movement of the jaw muscles, thereby leading to bruxism (5).

Kishi described an old, depressive Japanese man who displayed paroxetine-induced bruxism symptoms that were effectively treated with the 5-HT1A receptor tandospirone (6). To the best of our knowledge, the case described here is the first reported case of paroxetine-induced sleep bruxism to be treated successfully with buspirone.

Drug-induced movement disorders typically respond to a reduction in drug dosage, whereas our patient exhibited no improvement following the dose reduction or discontinuation of the drug in question. As a result, these symptoms may have been prevented through the use of buspirone alone.

Buspirone is an agonist of the 5-HT1A receptor that increases dopaminergic neuron firing in the ventral tegmental area and increases the synaptic release of dopamine in the prefrontal cortex. These effects ameliorate druginduced bruxism. Buspirone can also ameliorate extrapyramidal side effects, such as akathisia and tardive dyskinesia, and this property may be an additional explanation for the bruxism-related effects of the drug (5).

Thus, the present case describes rare but important side effects associated with the use of SSRIs. In light of our findings, we emphasize that all clinicians should be aware that antidepressants may cause bruxism. Furthermore, buspirone may be an effective treatment for the bruxism associated with the use of these medications.

CASE DESCRIPTION A 38-year-old married woman who was suffering from severe headaches, sadness, anhedonia, and feelings of worthlessness and decreased energy and concentration visited our outpatient clinic. This patient was diagnosed with a depressive mood and muscle contraction headaches, and a regimen of paroxetine was initiated with a bedtime dose of 10 mg/day. The patient had no previous personal or family history of any movement disorder, substance abuse or mental illness. After 7 days at this dosage without adverse effects, the dose was increased to 20 mg/day. At the 3-week follow-up visit, the patient showed marked improvement in her headaches and depressive symptoms, and she was pleased with this treatment. However, her husband noted his wife’s severe teeth clenching and associated loud grinding noises during sleep. All laboratory results were within the normal ranges, and all potential organic contributors to depression and bruxism were excluded. The dental examination also did not reveal any abnormalities.

A few days later, the patient started to complain of jaw pain and stiffness in the morning. As this problem caused her distress, the dosage was reduced to 10 mg/day. Due to the patient’s lack of improvement over the next week, paroxetine treatment was discontinued, although the bruxism did not resolve after the therapy had been discontinued.

As buspirone has been shown to be useful for the treatment of bruxism, we began to administer buspirone to the patient at a dose of 5 mg/nightly. This dose was

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

191

Drug-induced bruxism Milanl覺oglu A

CLINICS 2012;67(2):191-192

4. Lavigne GJ, Kato T, Kolta A, Sessle BJ. Neurobiological mechanisms involved in sleep bruxism. Crit Rev Oral Biol Med. 2003;14:30-46, http:// dx.doi.org/10.1177/154411130301400104.

5. Jaffee MS, Bostwick JM. Buspirone as an antidote to venlefaxine-induced bruxism. Psychosomatics. 2000;41:535-36, http://dx.doi.org/10.1176/ appi.psy.41.6.535.

6. Kishi Y. Paroxetine-induced bruxism effectively treated with tandospirone. J Neuropsychiatry Clin Neurosci. 2007;19:90-1, http://dx.doi.org/ 10.1176/appi.neuropsych.19.1.90.

REFERENCES 1. Kuloglu M, Ekinci O, Caykoylu A. Venlafaxine-associated nocturnal bruxism in a depressive patient successfully treated with buspirone.

J Psychopharmacol. 2010;24:627-8, http://dx.doi.org/10.1177/0269881 109102612.

2. Winocur E, Gavish A, Voikovitch M, Emodi-Perlman A, Eli I. Drugs and bruxism: a critical review. J Orofac Pain. 2003;17:99-111.

3. Attansio R. An overview of bruxism and its management. Dent Clin North Am. 1997;41:229-41.

192

CLINICS 2012;67(2):193-194

DOI:10.6061/clinics/2012(02)18

CASE REPORT

Acupuncture and exercise capacity: A case report Reginaldo Ceolin do Nascimento,I Carlos Alberto Cordeiro Hossri,I Otavio Berwanger,II Vitor Oliveira CarvalhoII,III I Hospital do Corac¸a˜o (HCor), Servic¸o de Reabilitac¸a˜o Cardı´aca, Pulmonar e Metabo´lica, Sa˜o Paulo/SP, Brazil. II Hospital do Corac¸a˜o (IEP-HCor), Instituto de Ensino e Pesquisa, Sa˜o Paulo/SP, Brazil. III Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo (InCor HCFMUSP), Unidade de Cirurgia Cardı´aca Pedia´trica, Sa˜o Paulo/SP, Brazil.

Email: rceolin@hotmail.com Tel.: 55 11 3053-6611

vulgaris) was performed on four points: Zusanli (E36), Guanyuan (VC4), Shenshu (B23), and Zhishi (B52 or 47).

INTRODUCTION Although acupuncture was once a purely empirical technique; it is now used as a valuable tool in scientific research and health maintenance (1).

The physiological effects of inserting needles into specific body points have inspired studies on the prevention and treatment of various diseases and syndromes. Nevertheless, the effects of acupuncture on sedentary, healthy individuals have not yet been explored.

Acupuncture has been reported to improve endurance during a range of activities, from everyday tasks to several types of sports (1). Many benefits of acupuncture appear to be related to the release of endorphins, decreased sympathetic activity and an increased tolerance to fatigue (1,2).

Nevertheless, acupuncture as an adjunct technique for improving physical capacity remains poorly understood.

This case report aims to describe the cardiorespiratory parameters before and after acupuncture/moxibustion in a healthy, sedentary individual.

DISCUSSION In the present report, the patient exhibited a large improvement in his exercise capacity after the acupuncture therapy. The patient improved in peak VO2 by 38%, peak ventilation by 13% and O2 pulse by 38%. The hemodynamic variables did not indicate an expressive increase, except for in the peak systolic blood pressure (SBP) (increase of 12%). After acupuncture, the patient exhibited a 3% increase in resting systolic blood pressure (SBP), a 2% increase in peak diastolic blood pressure (DBP), a 4% increase in resting heart rate (HR), and a 2% increase in peak heart rate (Table 1).

Several acupuncture points have been proposed to act on the cardiovascular, respiratory and metabolic systems (3), with and without the aim of improving exercise capacity. The most commonly used points are Zusanli (ST36) and Neiguan (PC6) (3). However, other acupuncture points, such as LieQue (LU7) and XiMen (PC4), have also been reported in the literature to act on the cardiovascular system (4).

For the patient in the present case, the original combination of acupuncture points, in association with moxibustion, appeared to result in the large improvement in his exercise capacity. Ling et al. (3) evaluated the relationship between electroacupuncture and cardiopulmonary function in three groups of healthy male volunteers: 1) acupuncture at the Neiguan (PC6) and Zusanli (ST36) points; 2) acupuncture at non-acupoints; and 3) a control group. Ling et al. (3) concluded that acupuncture can decrease the resting heart rate and the production of carbon dioxide without causing a significant change in the resting metabolic rate.

CASE DESCRIPTION A 30-year-old sedentary, non-smoking male patient without risk factors for cardiovascular disease was referred to our hospital for cardiovascular assessment before an exercise-training program (a program to improve health and well-being). The patient was invited to attend one hour of acupuncture sessions for ten consecutive days. The patient did not participate in exercise training during the time period of these ten acupuncture sessions. To assess his exercise capacity, the patient underwent a treadmill cardiopulmonary exercise test (MedGraphics CardiO2, Medical Graphics Corp., St. Paul, MN, USA) with a ramp protocol before and after each session of acupuncture.

In this patient, we placed 25630 mm needles in the following points: Zusanli (ST36), Neiguan (PC6), Yanglingquan (GB34), Zhongfu (LU1), Guanyuan (CV4), Shangzhong (CV17), Geshu (BL17), Shenshu (BL23), and Zhishi (BL52 or BL47).

Moxibustion (a technique that involves heating the acupuncture points by burning herbs, usually dry Artemisia - Artemisia

Table 1 - Exercise and hemodynamic variables before and after acupuncture.

Peak VO2 (mL/kg/min) Peak Ventilation (L/min) O2 Pulse (mL/beat) Resting SBP (mmHg) Peak SBP (mmHg) Resting HR (bpm) Peak HR (bpm) Resting DBP (mmHg) Peak DBP (mmHg)

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Before

After

26 124 13 121 195 69 190 86 86

36 141 18 125 219 72 194 80 88

VO2, oxygen consumption; SBP, systolic blood pressure; DBP, diastolic blood pressure; HR, heart rate.

No potential conflict of interest was reported.

193

Acupuncture and cardiopulmonary function tests Ceolin R et al.

CLINICS 2012;67(2):193-194

Figure 1 - Acupuncture/moxibustion points used for the present case report. ST36: 3 inches below the kneecap; PC6: 2 inches above the wrist crease; GB34: the depression in the anterior-inferior head of the fibula; LU1: the space between the first and second rib, 6 inches from the midline of the body; CV4: 3 inches below the navel along the midline of the abdomen; CV17: the midline of the sternum at the level of the nipples; BL17: 1.5 inches from the midline, next to the bottom of the spinous process of seventh thoracic vertebra; BL23: 1.5 inches from the bottom edge of the spinous process of second lumbar vertebra; BL52 or BL47: 3 inches from the midline, next to the bottom edge of the second lumbar vertebra.

The body of literature lacks studies on acupuncture and exercise capacity gain in healthy and sedentary subjects.

Therefore, the most efficient combination of acupuncture points for achieving better exercise performance is still unknown and needs to be investigated as it is of great interest to exercise physiologists.

In the present report, acupuncture was demonstrated to be an efficient tool for improving the exercise capacity of a healthy, sedentary patient. Further studies are needed to better elucidate the benefits and indications of acupuncture, the optimal combination of acupoints to produce these effects and acupuncture’s role in exercise training.

Berwanger O participated in the manuscript review. Carvalho VO performed the literature and manuscript review and participated in the manuscript writing. All the authors have read and approves the final manuscript.

REFERENCES 1. Ahmedov S. Ergogenic effect of acupuncture in sport and exercise: a brief review. J Strength Cond Res. 2010;24:1421-7, http://dx.doi.org/ 10.1519/JSC.0b013e3181d156b1.

2. Toma K, Walkowski S, Metzler-Wilson K, Wilson TE. Acupuncture attenuates exercise-induced increases in skin sympathetic nerve activity. Auton Neurosci. 2011;162:84-8, http://dx.doi.org/10.1016/ j.autneu.2011.04.004.

3. Lin JG, Ho SJ, Lin JC. Effect of acupuncture on cardiopulmonary function. Chin Med J (Engl). 1996;109:482-5.

4. Zhou J, Chi H, Cheng TO, Chen TY, Wu YY, Zhou WX, et al.

Acupuncture anesthesia for open heart surgery in contemporary China. Int J Cardiol. 2011;150:12-6, http://dx.doi.org/10.1016/j.ijcard.

2011.04.002.

AUTHOR CONTRIBUTIONS Nascimento RC collected the data, performed the literature review and paticipated in the manuscript writing. Hossri CAC collected the data.

194

CLINICS 2011;67(2):195-197

DOI:10.6061/clinics/2012(02)19

CASE REPORT

A combined minimally invasive approach for the treatment of aortoesophageal fistula caused by the ingestion of a chicken bone: case report and literature review Xiaodong Chen,I Jianwen Li,I Jie Chen,II Yu Zhou,III Yuanqi Zhang,I,IV Hongfei Ding,I Shuichuan Huang,I Zhi ZhangI I

Affiliated Hospital of Guangdong Medical College, Department of Vascular Surgery, Zhanjiang, Guangdong province, People9s Republic of China.

Affiliated Hospital of Guangdong Medical College, Department of Cardiothoracic Surgery, Zhanjiang, Guangdong province, People9s Republic of China.

III Affiliated Hospital of Guangdong Medical College, Department of Gastroenterology, Zhanjiang, Guangdong province, People’s Republic of China.

IV First Affiliated Hospital of Sun Yat-sen University, Research Center of Vascular Surgery, Department of Vascular Surgery, the Guangzhou, People’s Republic of China, Guangdong, China.

II

Email: gdyfywjk@163.com Tel.: 86 759-2369322

lower edge of thoracic vertebrate 5 (Figure 1). A diagnosis of AEF was highly suspected. Angiography was performed on March 31, 2011 and showed a 0.7 cm60.4 cm pseudoaneurysm of the descending aorta approximately 4.5 cm below the origin of the left subclavian artery, which was consistent with the results of the CTA. This finding confirmed the diagnosis of AEF. After an immediate multidisciplinary discussion, a plan for definitive treatment was established. An endovascular stent-graft (catalog number: TF2424C200X, Medtronic, Inc., USA) was placed inside the descending aorta to isolate the pseudoaneurysm. Postoperative angiography showed satisfactory exclusion results, and the patient was transferred to the operating room for thoracoscopic exploration. Swelling in the esophageal bed and inflammatory exudates were observed under the thoracoscope. Immediate mediastinal debridement and irrigation was performed using the thoracoscope. Two F16 silicone tubes were placed in the pleural cavity and the mediastinum to facilitate postoperative drainage. The patient underwent an esophageal endoscopy, and an esophageal fistula approximately 1.0 cm in diameter was found 25 cm away from the incisors (Figure 2A). A single-use retrievable esophageal stent-graft (Micro-Tech Co., Ltd. Nanjing China, length 8.0 cm, diameter 2.0 cm) was implanted to isolate the esophageal perforation followed by the placement of a three-lumen gastrojejunal tube for additional enteral nutrition. After spending three days in the intensive care unit (ICU), the patient was transferred back to our department. He had no melena and no fever or vomiting, and the mediastinal and pleural drainage gradually became clear. On the 7th day after the operation, esophageal imaging with meglumine diatrizoate showed no leakage, and the three-lumen gastrojejunal tube was removed. The patient was provided with a liquid diet. Both of the drainage tubes in the pleural cavity and the mediastinum were removed 25 days after the operation. Repeat CTA confirmed successful tamponade of the fistula 40 days after the operation (Figure 3).

Antibiotic treatment was administered during hospitalization. Laboratory findings showed that the patient’s WBC count was 23.06109/mL with 79.1% neutrophils (NEs) on the second day after the operation. The WBC count and

INTRODUCTION Aortoesophageal fistula (AEF) is a rare, catastrophic complication that can be caused by the ingestion of a foreign body (1,2). In the past, AEF was treated with surgical repair using thoracic esophagectomy and esophagogastrostomy.

However, the mortality rate for patients with AEF remains extremely high (2-4). Recently, we successfully treated a patient with AEF that was caused by the ingestion of a chicken bone. We placed an aortic endovascular stent graft followed by thoracoscopic mediastinal debridement and drainage. A retrievable esophageal stent was placed to isolate the fistula, which was removed after 80 days. In this report, we present our experience using a combined minimally invasive therapeutic approach for the treatment of AEF.

CASE DESCRIPTION A 22-year-old man was admitted to our hospital on March 28, 2011 complaining of seven days of retrosternal discomfort and a single episode of vomiting of approximately 50 mL of fresh red blood. The patient had swallowed a chicken bone prior to the onset of the discomfort. His vital signs were stable during physical examination, and there was no fever on arrival at the hospital. Laboratory findings revealed a white blood cell (WBC) count of 19.96109/L and hemoglobin (Hb) levels of 84 g/L. He vomited approximately 300 ml of bright red blood and produced a large amount of tarry stool on March 29, 2011. His Hb levels subsequently decreased to 73 g/L. CT angiography (CTA) demonstrated a mediastinal esophageal fistula with inflammatory exudate surrounding the descending aorta. An aortic pseudoaneurysm at the anterior wall of the descending aorta was also observed at the

Copyright ß 2011 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

195

An approach for treating aortoesophageal fistula Chen X et al.

CLINICS 2011;67(2):195-197

Figure 1 - CT angiography images. A) Mediastinal esophageal fistula with inflammatory exudate surrounding the descending aorta (arrow). B) An aortic pseudoaneurysm at the anterior wall of the descending aorta at the lower edge of thoracic vertebrae 5 (arrow).

NE returned to normal after 14 consecutive days of administration of the antibiotic meropenem based on cultures of the mediastinal exudates, which were positive for Pseudomonas aeruginosa seven days after the operation. The patient was switched to oral antibiotics and discharged on May 18, 2011. He was re-admitted to remove the esophageal stent 80 days after the operation. Esophageal endoscopy revealed a complete healing of the original fistula after removal of the esophageal stent (Figure 2B). No fever was observed during the treatment follow-up. After continuous administration of oral antibiotics for three months, the patient stopped the medication when blood laboratory findings and chest X-ray results were normal. The patient lived a normal life during six months of follow-up after the operation.

Although several successful cases have been documented since the first AEF operation reported by Ctercteko (6) in 1980, the survival rate of patients with AEF has remained dismal.

Zhang et al. (2) reported 32 cases of esophageal foreign body and/or aortoesophageal fistula between 1963 and 2010. Of 19 patients who underwent surgical interventions, three patients were completely cured and 16 patients died of fatal hemorrhage. The other 13 patients who were treated nonsurgically all died as a result of various treatments. The optimal management of AEFs remains controversial.

With the rapid development of new endovascular techniques, endovascular aortic stent grafts have shown significant advantages over traditional open surgery for the treatment of aorta fistulas (1). Endovascular stent placement for AEF has been described with good results (3,7).

Verhoeven et al. (1) suggested that endovascular aortic stent grafts should be considered as a first-choice lifesaving procedure. These grafts prevent fatal exsanguination and provide the valuable time necessary to optimize the patient’s condition before proceeding with additional treatments.

Postoperative infections due to the placement of aortic stents depends on the treatment of the esophageal fistula, mediastinal drainage and anti-infection treatments. Esophageal exclusion with mediastinal drainage followed by

DISCUSSION The widely accepted approach for treating AEFs caused by foreign bodies is a thoracotomy with aortic stent-graft interposition followed by reconstruction of the esophagus.

However, this procedure has extremely high mortality and morbidity rates (4,5).

The formidable challenges in treating these injuries are rupture of the AEF, mediastinal infection caused by esophageal perforation and endovascular stent-graft infection.

Figure 2 - Esophageal endoscopy. A) An esophageal fistula approximately 1.0 cm in diameter was found 25 cm away from the incisors prior to implantation of the esophageal stent (arrow). B) Complete healing of the original fistula after the removal of the esophageal stent (arrow).

196

CLINICS 2011;67(2):195-197

An approach for treating aortoesophageal fistula Chen X et al.

Figure 3A and B - CT angiography confirmed successful tamponade of the fistula.

secondary esophageal reconstruction was previously a standard surgical approach for the treatment of esophageal fistulas. However, this procedure is time consuming and can cause significant surgical trauma. Thoracoscopy is a minimally invasive technique for managing mediastinal and thoracic diseases (8,9). Esophageal stents are universally applied to treat stenosis of the esophagus and esophageal perforation (10,11). We used thoracoscopy instead of mediastinal debridement, irrigation and drainage. We implanted retrievable esophageal stents and placed a three-lumen gastrojejunal tube. Esophageal stents avoid saliva and/or exudates passing through the esophageal fistula into the mediastinum to prevent mediastinal infection. In this case, we chose a removable esophageal stent because the patient was a young man with benign esophageal disease. The threelumen gastrojejunal tube ensured early postoperative intake of nutrients. After removal of the three-lumen gastrojejunal tube on postoperative day 7, the patient was provided with a liquid diet for nutritional support.

This report represents the first case in which the minimally invasive approaches described were combined to successfully treat a patient with AEF. Based on our limited experiences, we suggest performing aortic endovascular repair by stent-graft to ultimately reduce the risk of aortic rupture, followed by thoracoscopy to treat mediastinal infections and placement of a retrievable esophageal stent in patients with AEF who have slight inflammation or perforation of the esophageal region and surrounding tissues. The combined approach resulted in less trauma and a more rapid recovery compared to conventional thoracotomy. This minimally invasive approach may be a safe and feasible method for treating patients with AEF and has the potential for improved treatment options for AEFs. A larger sample size and long-term follow-up of patients with AEFs will be required to further evaluate the feasibility of this novel combined approach.

AUTHOR CONTRIBUTIONS Chen XD and Li JW inserted the endovascular stent, drafted the document, reviewed the literature, revised and critically edited the drafts and contributed equally to this work. Zhang YQ assisted in drafting the document and participated as a surgeon in the case. Ding HF, Huang SC, and Zhang Z participated as surgeons in the case. Chen J performed the thoracoscopic operation. Zhou Y inserted and removed the esophageal stent.

REFERENCES 1. Verhoeven EL, Vourliotakis G. Thoracic endovascular aortic repair for aortobronchial or aortoesophageal fistulas: permanent or temporary salvage or not an option at all? J Endovasc Ther. 2009;16(4):441–42, http://dx.doi.org/10.1583/09-2741C.1.

2. Zhang X, Liu J, Li J, Hu J, Yu F, Li S, et al. Diagnosis and treatment of 32 cases with aortoesophageal fistula due to esophageal foreign body.

Laryngoscope. 2011;121(2):267-72, http://dx.doi.org/10.1002/lary.21366.

3. Jonker FH, Heijmen R, Trimarchi S, Verhagen HJ, Moll FL, Muhs BE. Acute management of aortobronchial and aortoesophageal fistulas using thoracic endovascular aortic repair. J Vasc Surg. 2009;50(5):999-1004, http://dx.doi.org/10.1016/j.jvs.2009.04.043.

4. Flores J, Shiiya N, Kunihara T, Yoshimoto K, Yasuda K. Aortoesophageal fistula: alternatives of treatment case report and literature review. Ann Thorac Cardiovasc Surg. 2004;10(4):241–46.

5. da Silva ES, Tozzi FL, Otochi JP, de Tolosa EM, Neves CR, Fortes F. Aortoesophageal fistula caused by aneurysm of the thoracic aorta: successful surgical treatment, case report and literature review. J Vasc Surg. 1999;30(6):1150–57, http://dx.doi.org/10.1016/S0741-5214(99) 70056-X. 6. Ctercteko G, Mok CK. Aorta-esophageal fistula induced by a foreign body: the first recorded survival. J Thorac Cardiovasc Surg. 1980;80(2): 233-35.

7. Metz R, Kimmings AN, Verhagen HJ, Rinkes IH, van Hillegersberg R. Aortoesophageal fistula successfully treated by endovascular stent-graft.

Ann Thorac Surg. 2006;82(3):1117–19, http://dx.doi.org/10.1016/ j.athoracsur.2006.01.091.

8. Kozuki A, Shinozaki H, Tajima A, Kase K. Successful treatment for descending necrotizing mediastinitis with severe thoracic emphysema using video-assisted thoracoscopic surgery. Gen Thorac Cardiovasc Surg. 2010;58(11):584-87, http://dx.doi.org/10.1007/s11748-009-0553-7.

9. Melo CB, Sarmento PA, Imaeda CJ, Daud DF, Hasimoto FN, Lea˜o LE. Descending necrotizing mediastinitis: minimally invasive thoracic surgical treatment. J Bras Pneumol. 2010;36(6):812-18, http:// dx.doi.org/10.1590/S1806-37132010000600019.

10. Bakken JC, Wong Kee Song LM, de Groen PC, Baron TH. Ues of a fully covered self-expandable metal stent for the treatment of benign esophageal diseases.Gastrointest Endosc. 2010;72(4):712-20, http:// dx.doi.org/10.1016/j.gie.2010.06.028.

11. Petersen JM. The use of a self-expandable plastic stent for an iatrogenic esophageal perforation. Gastroenterol Hepatol. 2010;6(6):389-91.

ACKNOWLEDGMENTS We would like to thank Prof. Wenjian Wang and Xun Hou, PhD of the First Affiliated Hospital of Sun Yat-sen University for editing the manuscript.

197

CLINICS 2012;67(2):199-201

DOI:10.6061/clinics/2012(02)20

CASE REPORT

Laparoscopic repair of an incarcerated bochdalek hernia in an elderly man Toygar Toydemir,I Hakan Akıncı,I Mehmet Tekinel,I Erdog˘an Su¨leyman,II Bu¨lent Acunas¸,III Mehmet Ali YerdelIII I

Istanbul Surgery Hospital, General Surgery Department, Nis¸antas¸ı-Istanbul/Turkey. II Istanbul Surgery Hospital, Radiology Department, Nis¸antas¸ı-Istanbul/ Turkey. III Istanbul Surgery Hospital, Head of General Surgery Department, Nis¸antas¸ı-Istanbul/Turkey.

Email: toygartoydemir@hotmail.com Tel.: 90 505 3743368

rectal examination revealed no masses or bleeding. The patient had a temperature of 37.5 ˚C, a heart rate of 100/min, and a blood pressure of 160/90 mmHg. Laboratory analysis revealed an elevated white blood cell count of 19,000/mm3.

The other laboratory results were normal. These signs were interpreted as a partial bowel obstruction of unknown origin because the patient was passing stool and flatus. An urgent computerized tomography (CT) scan with oral/ rectal and intravenous contrast was performed; the scan revealed a left-sided BH, with a large segment of incarcerated transverse colon (Figures 1-2).

The patient was taken to the operating room for laparoscopic repair of an incarcerated BH. The patient was placed in a modified lithotomy position, and five trocars were used.

The intra-abdominal pressure was maintained at 10 mmHg after the pneumoperitoneum was achieved via the open technique. The head and the left side of the patient were raised to 30 ˚. The omentum and the distal part of the transverse colon were found to be attached to the left-sided BH (Figure 3). The diaphragm was divided by a few centimeters to enable the reduction of the bowel. The dimensions of the defect were measured to be approximately 666 cm after the reduction of the omentum and colon into the abdomen. No signs of perforation or ischemia were present.

The defect was repaired with non-absorbable ‘‘09’ polypropylene sutures using a round-bodied 30-mm needle (Prolene; Ethicon, Bracknell, UK). An increased tidal volume was used before the last suture was placed to decrease any residual pneumothorax. A monofilament polypropylene (Prolene; Ethicon, Bracknell, UK) mesh was used to reinforce the repair, and a laparoscopic tacker was used for fixation of mesh.

The patient had an uneventful recovery, and he was discharged on postoperative day two. He remained well at his six-month follow-up, without evidence of clinical or radiological recurrence.

INTRODUCTION Congenital posterolateral diaphragmatic defects, such as Bochdalek hernias (BHs), usually present during the neonatal period with respiratory symptoms and are associated with significant mortality (1). However, a subset of patients with BHs may remain asymptomatic during childhood, and the condition may present as a surgical emergency in adulthood (2). Surgical repair of the defect is the recommended therapy for all patients with BHs, regardless of the presence of symptoms (3). Traditionally, the repair of diaphragmatic defects has been performed via laparotomy or thoracotomy, but the use of laparoscopy has challenged the use of these traditional procedures.

However, the laparoscopic management of incarcerated BH is rarely reported. In the present paper, we report the case of a patient who presented with a partial colonic obstruction that was caused by an incarcerated transverse colon through a Bochdalek defect and treated using a laparoscopic approach.

CASE DESCRIPTION A 77-year-old Caucasian man was admitted to our emergency department with abdominal pain and nausea that had occurred for the previous 24 hours; the patient had a one-year history of intermittent abdominal pain and chest tightness during physical effort. He had been evaluated for these chest problems approximately one year ago at another center and was determined to have no cardiac issues. The plain chest X-rays that were taken at that time revealed consolidated areas in the left basal lung segments, and the patient was treated for a diagnosis of pneumonia. The patient did not have a significant history of trauma.

When the patient presented at our hospital, his abdomen was mildly distended and hyperactive bowel sounds were heard. A physical examination revealed muscular rigidity in the left upper quadrant of the patient’s abdomen without rebound tenderness or signs of peritoneal irritation. The

DISCUSSION The initial reports of congenital diaphragmatic hernia (CDH) date back to 1679, which was first described by Lazarus Riverius. Riverius incidentally observed a CDH in a 24-year-old man during a postmortem examination (4). In addition, an improper or absent migration of the diaphragmatic muscle was first described by Victor Alexander Bochdalek in 1948. Embryologically, the canal between the septum transversarum and the esophagus closes during the

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

199

An unusual cause of a bowel obstruction Toydemir T et al.

CLINICS 2012;67(2):199-201

Figure 1 - Axial CT scan shows 6-cm opening at the posterior segment of the diaphragm. Transvers colon and surrounding fat tissues enter thorax through the diaphragmatic defect. The entering segment of the colon is narrowed. These changes displace diaphragm, spleen and surrounding fat tissues anteriorly.

Figure 3 - The view of the diaphragmatic defect after the bowel segment was reduced.

more likely to produce symptoms than right-sided defects because of the protective effect of the liver on the right side of the body.

The diagnosis of a BH in adulthood is problematic because of the rarity of this disease and the variety of the presenting symptoms. The presence of bowel sounds within the chest and the absence of breath sounds are typical findings associated with a BH. Misdiagnosis of a BH is common, as reported in the current case, and may be fatal.

A misdiagnosis rate of 38% has been reported by Thomas & Kapur (8). Misdiagnosis of BHs may lead to inappropriate interventions, such as tube thoracostomies (9). The patient that we described in the present case was treated for a pulmonary infection approximately one year prior to the writing of this case because of pulmonary symptoms and chest X-ray findings.

Clinicians should take great care during the management of patients with persistent pulmonary symptoms and abnormal chest X-ray findings. CT scans are the only way to directly visualize the focal defect in the diaphragm, and the presence of a soft tissue contour in the chest CT, in addition to opaque, filled, dilated bowel segments above the diaphragm, establishes a definitive diagnosis (10).

Because of the potential for life threatening complications, patients who have congenital or traumatic diaphragmatic hernias should undergo surgical repair regardless of the presence or absence of symptoms. Traditionally, the surgical management of diaphragmatic defects has been performed via laparotomy and/or thoracotomy. During the last two decades, successful laparoscopic repairs have also been reported. However, laparoscopic management of incarcerated BHs has been rarely reported (11-13). Several authors have suggested an abdominal approach for left-sided defects and a thoracic approach for right-sided hernias (14,15). The improved ability of separating adhesions between the hernia sac and pleura is the main advantage of transthoracic repair. In contrast, the management of a possible strangulation and the recognition of a possible malrotation, which is well documented with BHs, are better suited with a transabdominal approach. The ideal technique is not yet determined because of the lack of randomized trials comparing the two procedures. In addition, the procedure of choice depends on the surgeon’s experience.

eighth week of gestation, and the failure of this closure results in a BH. Although BHs present most often during the neonatal period, with life-threatening cardiopulmonary distress, many patients may remain asymptomatic until adulthood.

A recently published study, which reviewed all cases and case series from 1955 to 2011, identified a total of 124 articles comprising a total of 173 adult patients with BHs (5).

Because of the wide variation of presenting symptoms, the true incidence of the condition remains unknown. Mullins et al. reported an incidence of 0.17% after reviewing 13,138 CT scans. This previous study provides the best data on the incidence of asymptomatic BHs (6,7). Left-sided defects are

Figure 2 - Sagittaly reformated CT scan shows dilated proximal transvers colon segment filled with oral contrast material. The entering segment of the colon is narrowed. Thoracic segment of the colon contains air. These changes displace diaphragm, spleen and surrounding fat tissues anteriorly and inferiorly.

200

CLINICS 2012;67(2):199-201

An unusual cause of a bowel obstruction Toydemir T et al.

E collected the data in the literature. Toydemir T, Yerdel MAY, Akıncı H, and Acunas¸ B authored the paper.

Regardless of the type of surgical procedure, controversy exists for parts of the management of BHs, such as the dissection of the hernia sac and the use of prosthetic grafts.

Because the dissection of the sac is associated with a high risk of pleural injury, most surgeons prefer to leave the hernia sac in place (16). The development of a seroma in the remnant sac is a potential risk; however, it has been reported that the remnant sac completely disappears 30 days following surgery (60). We did not make any attempts to reduce the sac and did not observe any residual problems. Suturing the defect is likely important for the restoration of the anatomy between the thoracic and abdominal cavities. However, many surgeons prefer to construct a repair that is reinforced with a prosthetic graft because of the continuing stress on the diaphragm that results from respiratory movements and cardiac motions. Nevertheless, a tensionless type of repair has been validated as an option for BHs, which is similar to the type of repair used for all other hernia repairs. In addition, many types of meshes are available for use in these types of repairs. Although polypropylene mesh has the benefit of providing indefinite support and excellent tissue growth, erosion of the mesh into the gastrointestinal organs represents a theoretical risk. The decreased tendency for adhesion formation of polytetrafluoroethylene and other dual prostheses makes them more desirable. Surgeons should take great care during the fixation of grafts with a laparoscopic tacker where the diaphragm is relatively thin. Although a laparoscopic hernia stapler has the similar risks, such as pericardial injury, this method may be more useful near the pericardium because of the controlled firing advantage.

In conclusion, BHs are uncommon clinical findings in the adult population, and cases presenting with incarceration are even less common. CT is the preferred diagnostic tool for BHs. Laparoscopic repair of an incarcerated BH is an excellent option because of the advantages of its unique, minimally invasive nature.

REFERENCES 1. Adelman S, Benson CD. Bochdalek hernias in infant: Factors determining mortality. J Pediatr Surg. 1976:11(4):569-73, http://dx.doi.org/10.1016/ S0022-3468(76)80015-2.

2. Kocakusak A, Arikan S, Senturk O, Yucel AF. Bochdalek’s hernia in an adult with colon necrosis. Hernia. 2005:9(3):284–7, http://dx.doi.org/ 10.1007/s10029-004-0302-x.

3. Ketonen P, Mattila SP, Harjola PT, Jarvinen A, Mattila T. Congenital posterolateral diaphragmatic hernia in the adult. Acta Chir Scand.

1975:141(7):628–32.

4. Al-Emadi M, Helmy I, Nada MA, Al- Jaber H. Laparoscopic repair of Bochdalec hernia in an adult. Surg laparosc Endosc Percutan Tech.

1999;9:423-5 5. Brown SR, Horton JD, Trivette E, Hofmann LJ, Johnson JM. Bochdalek hernia in the adult: demographics, presentation and surgical management. Hernia. 2011:15:23-30, http://dx.doi.org/10.1007/s10029-0100699-3.

6. Mullins ME, Saini S. Imaging of incidental Bochdalek hernia. Semin Ultrasound CT MR. 2005:26(1):28–36.

7. Mullins ME, Stein J, Saini SS, Mueller PR. Prevalence of incidental Bochdalek’s hernia in a large adult population. Am J Roentgenol.

2001:177(2):363–6.

8. Thomas S, Kapur B. Adult Bochdalek hernia–clinical features, management and results of treatment. Jpn J Surg. 1999:21(1):114–9.

9. Chai Y, Zhang G, Shen G. Adult Bochdalek hernia complicated with a perforated colon. J Thorac Cardiovasc Surg. 2005:130(6):1729–30.

10. Wilbur AC, Gorodetsky A, Hibblen JF. Imaging findings of adult Bochdalek hernias. Clin Imaging. 1994:18:224–9, http://dx.doi.org/ 10.1016/0899-7071(94)90088-4.

11. McDonnell CO, Naughton P, Aziz A, Walsh TN. Laparoscopic repair of a strangulated Bochdalek hernia. Ir J Med Sci. 2003:172(3):145–6.

12. Harinath G, Senapati PS, Pollitt MJ, Ammori BJ. Laparoscopic reduction of an acute gastric volvulus and repair of a hernia of Bochdalek. Surg Laparosc Endosc Percutan Tech. 2002:12(3):180–3, http://dx.doi.org/ 10.1097/00129689-200206000-00009.

13. Esmer D, Alvarez-Tostado J, Alfaro A, Carmona R, Salas M. Thoracoscopic and laparoscopic repair of complicated Bochdalek hernia in adult. Hernia. 2007:12:307–9, http://dx.doi.org/10.1007/s10029-0070293-5.

14. Lindsey I, Woods SDS, Nottle PD. Laparoscopic management of blunt diaphragmatic injury. Aust N Z J Surg. 1997:67:619–21, http://dx.doi. org/10.1111/j.1445-2197.1997.tb04609.x.

15. Martin I, O’Rourke N, Gotley D, Shimthers M. Laparoscopy in the management of diaphragmatic rupture due to blunt trauma.

Aust N Z J Surg. 1998:68:584–6, http://dx.doi.org/10.1111/j.14452197.1998.tb02105.x.

16. Liem NT. Thoracoscopic surgery for congenital diapragmatic hernia: a report of nine cases. Asian J Surg. 2003:26:210-2, http://dx.doi.org/ 10.1016/S1015-9584(09)60305-5.

AUTHOR CONTRIBUTIONS Toydemir T, Akıncı H, and Tekinel M performed the surgery. Su¨leyman E and Acunas¸ B made the radiological diagnosis. Toydemir T and Su¨leyman

201

CLINICS 2012;67(2):203

DOI:10.6061/clinics/2012(02)21

ERRATA CLINICS 2011;66(12):2043-48 Page 2043 Replace Clarissa B. Oliveira,I,II ´Italo R. T. Medeiros,III Mario G. Greters,III Norberto A. F. Frota,IV Leandro Tavares Lucato,II Milberto Scaff,I Adriana B. ConfortoI For Clarissa B. Oliveira,I,II ´Italo R. T. Medeiros,III Mario G. Greters,III Norberto A. F. Frota,IV Leandro Tavares Lucato,VI Milberto Scaff,II Adriana B. ConfortoII,V Replace IAACD – Adult Physiotherapy, Sa˜o Paulo/SP, Brazil. IIHospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Neurology, Sa˜o Paulo/SP, Brazil. IIIHospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Otorhinolaryngology Department, Sa˜o Paulo/SP, Brazil. IVFortaleza University – School of Medicine, Fortaleza/CE, Brazil.

For IAACD – Adult Physiotherapy, Sa˜o Paulo/SP, Brazil. IIHospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Neurology Department, Sa˜o Paulo/SP, Brazil. IIIHospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Otorhinolaryngology Department, Sa˜o Paulo/SP, Brazil. IVFortaleza University – School of Medicine, Fortaleza/CE, Brazil. VInstituto Israelita de Ensino e Pesquisa Albert Einstein, Sa˜o Paulo/SP, Brazil. VIHospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Radiology Department, Sa˜o Paulo/SP, Brazil.

CLINICS 2011;66(09):1563-68 Page 1567 Include

ACKNOWLEDGMENTS The authors would like to thank FAPESP.

CLINICS 2011;66(04):691-700 Page 691 Replace Esteves SC, Miyaoka R, Agarwal A. 2011;66(4):PAGES An update on the clinical assessment of the infertile male.

Clinics. 2011;66(4):691-700.

For Esteves SC, Miyaoka R, Agarwal A. An update on the clinical assessment of the infertile male. Clinics. 2011;66(4):691700.

203

No Rebouças, pequenos eventos

tornam-se grandes.

Seja pequeno, médio ou grande, o Rebouças tem o espaço ideal para todo o tipo de evento. A melhor logística e infraestrutura de apoio maximizam os recursos operacionais e potencializam os encontros científicos, promocionais, institucionais ou culturais.

O Centro de Convenções Rebouças é isso: um dos mais bem planejados e tradicionais espaços receptivos do País, dimensionado para sediar congressos, convenções, exposições, seminários, simpósios, cursos, lançamento de produtos, entre outros, bem no coração da cidade de São Paulo.

Atualmente possui 8 ambientes climatizados e modernizados que atendem até 1.200 participantes, além de uma equipe preparada e empenhada em acompanhar cerca de 280 eventos por ano.

EM BREVE, estará ampliando suas instalações, que permitirá o dobro da capacidade de público.

Av. Rebouças, 600 - 05402-000 - São Paulo - Brasil - Tel.: 55 11 3898-7850 / Fax: 55 11 3898-7878 - reboucas@hcnet.usp.br

www.convencoesreboucas.com.br