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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 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

Samir Rasslan Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil 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´ Antonio Sanches 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 Milton de Arruda Martins Nelson de Luccia Noedir Antonio Groppo Stolf Olavo Pires de Camargo Paulo Andrade Lotufo

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

Paulo Hila´rio Nascimento Saldiva Paulo Marcelo Gehm Hoff Pedro Puech-Lea˜o Remo Susanna Ricardo Ferreira Bento Ricardo Nitrini 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 Assistants 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 CEP 05403-010 Sa˜o Paulo/SP Tel.: +55-11-2661-6235 Email: clinics.office@gmail.com Website: www.scielo.br/clinics Submission: http://mc04.manuscriptcentral.com/clinics-scielo 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.

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ISSN-1807-5932

CLINICS CONTENTS Clinics 2012 67(8):859–985

EDITORIAL

Pediatrics in Clinics: Highlights Mauricio Rocha e Silva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 859

CLINICAL SCIENCES

Carotid stenosis: what is the high-risk population? Jong Hun Park, Alvaro Razuk, Paulo Fernandes Saad, Gustavo Jose´ Politzer Telles, Walter Khegan Karakhanian, Alexandre Fioranelli, Alessandra Caivano Rodrigues, Giuliano Giova Volpiani, Pollyanna Campos, Roberto Massayoshi Yamada, Valter Castelli Jr., Roberto Augusto Caffaro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 865

Sexual function after anterior vaginal wall prolapse surgery Paulo Cezar Feldner Jr., Carlos Antonio Delroy, Se´rgio Brasileiro Martins, Rodrigo Aquino Castro, Marair Gracio Ferreira Sartori, Manoel Joa˜o Batista Castello Gira˜o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 871

The effects of angiotensin-converting enzyme inhibitors on peritoneal protein loss and solute transport in peritoneal dialysis patients Taner Basturk, Abdulkadir Unsal, Yener Koc, Eren Nezaket, Elbis Ahbap, Tamer Sakaci, Mustafa Sevinc . . . . . . . . 877

Skeletal muscle major histocompatibility complex class I and II expression differences in adult and juvenile dermatomyositis Samuel Katsuyuki Shinjo, Adriana Maluf Elias Sallum, Clovis Artur Silva, Suely Kazue Nagahashi Marie . . . . . . . . 885

Predicting dysthyroid optic neuropathy using computed tomography volumetric analyses of orbital structures Allan C. Pieroni Gonc¸alves, Lucas Nunes Silva, Eloı´sa M. M. S. Gebrim, Suzana Matayoshi, Ma´rio Luiz Ribeiro Monteiro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 891

Effects of arterial oxygen tension and cardiac output on venous saturation: a mathematical modeling approach Fernando Godinho Zampieri, Marcelo Park, Luciano Ce´sar Pontes Azevedo, Marcelo Britto Passos Amato, Eduardo Leite Vieira Costa. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 897

M2-polarized macrophages promote metastatic behavior of Lewis lung carcinoma cells by inducing vascular endothelial growth factor-C expression Bicheng Zhang, Yafei Zhang, Guoqing Yao, Juan Gao, Bo Yang, Yong Zhao, Zhiguo Rao, Jianfei Gao . . . . . . . . . 901


Laparoscopic nephrectomy for xanthogranulomatous pyelonephritis – Are there predictive factors for success? Marcelo Lima, Ricardo Miyaoka, Juliano Moro, Carlos D’Ancona . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 907

Serum testosterone, sex hormone-binding globulin and total calcium levels predict the calcaneal speed of sound in men Kok-Yong Chin, Ima-Nirwana Soelaiman, Isa Naina Mohamed, Wan Zurinah Wan Ngah . . . . . . . . . . . . . . . . . . . 911

A clinical follow-up of 35 Brazilian patients with Prader-Willi Syndrome Caio Robledo D’Angioli Costa Quaio, Tatiana Ferreira de Almeida, Lilian Maria Jose´ Albano, Israel Gomy, Debora Romeo Bertola, Monica Castro Varela, Celia P. Koiffmann, Chong Ae Kim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 917

BASIC RESEARCHES

Preconditioning of the response to ischemia/reperfusion-induced plasma leakage in hamster cheek pouch microcirculation Fabiana Gomes da Conceic¸a˜o, Cristiane Maria Simonato Conde, Erik Svensjo¨, Daniel Alexandre Bottino, Eliete Bouskela . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 923

Vitreous pharmacokinetics and electroretinographic findings after intravitreal injection of acyclovir in rabbits Francisco Max Damico, Mariana Ramos Scolari, Gabriela Lourenc¸on Ioshimoto, Beatriz Sayuri Takahashi, Armando da Silva Cunha Jr., Sı´lvia Ligo´rio Fialho, Daniela Maria Bonci, Fabio Gasparin, Dora Fix Ventura . . . . . . . . . . . . . . . 931

Upregulation of matrix synthesis in chondrocyteseeded agarose following sustained bi-axial cyclic loading Belinda Pingguan-Murphy, Illida Nawi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 939

REVIEWS

Thyroid nodule management: clinical, ultrasound and cytopathological parameters for predicting malignancy Frederico F. R. Maia, Denise Engelbrecht Zantut-Wittmann . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 945

Auditory stimulation and cardiac autonomic regulation Vitor E. Valenti, Heraldo L. Guida, Ana C. F. Frizzo, Ana C. V. Cardoso, Luiz Carlos M. Vanderlei, Luiz Carlos de Abreu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 955

RAPID COMMUNICATIONS

Correlation of disability and pain with postural balance among women with chronic low back pain Guilherme Carlos Brech, Silvia Ferreira Andrusaitis, Gabriela Faller Vitale, Ju´lia Maria D’Andre´a Greve . . . . . . . . . 959

Malignant peripheral nerve sheath tumors: clinicopathological aspects, expression of p53 and survival Karin S. G. Cunha, Anabela C. Caruso, Paulo A. S. de Faria, Licı´nio E. da Silva, Andre´a R. C. Pires, Mauro Geller, Vaˆnia S. Lopes, Rodrigo S. de Moura-Neto . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 963

The long-term impact of a program to prevent central line-associated bloodstream infections in a surgical intensive care unit Adriana P. Paula, Priscila R. Oliveira, Erique P. Miranda, Cassia S. Felix, Clara B. Lorigados, Arlete M. Giovani, Ana Lucia L. Lima . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 969


TECHNICAL NOTE

An innovative model of autologous anorectal transplantation with pudendal nerve reconstruction Flavio Henrique Ferreira Galvao, Victor Edmund Seid, Daniel Reis Waisberg, Ruy Jorge Cruz-Jr, Hugo Hirano, Sergio Catanozi, Eleazar Chaib, Luiz Augusto Carneiro D’Albuquerque . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 971

CASE REPORTS

Diagnosis of an ectopic adrenocorticotropic hormonesecreting bronchial carcinoid by somatostatin receptor scintigraphy I˙nan Anaforog˘lu, Kerem Ersoy, Mehmet As¸ık, Savas¸ Karyag˘ar, Ekrem Algu¨n . . . . . . . . . . . . . . . . . . . . . . . . . . . . 973

A possible case of Churg-Strauss syndrome in a 9-year-old child Jinling Liu, Yingchun Xu, Zhimin Chen, Xuefeng Xu, Meiping Lu, Yingshuo Wang, Yunlian Zhou, Weizhong Gu . . . . 977

MSX2 copy number increase and craniosynostosis: copy number variation detected by array comparative genomic hybridization Karla de Oliveira Pelegrino, Sofia Sugayama, Karina Lezirovitz, Ana Lu´cia Catelani, Fernando Kok, Maria de Lourdes Chauffaille . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 981


CLINICS 2012;67(8):859-864

DOI:10.6061/clinics/2012(08)01

EDITORIAL

Pediatrics in Clinics: Highlights Mauricio Rocha e Silva Hospital das Clı´nicas, Faculdade de Medicina, Universidade de Sa˜o Paulo, Sa˜o Paulo/SP, Brazil.

Turkey with KD included in Medline. Orlando et al. (5) analyzed the results of treating 39 deep venous malformations patients with low doses of ethanol. Patients were followed up prospectively over a median period of 18 months. All of the lesions affected limbs, and the main symptom reported was pain (97.4%). Each patient underwent fortnightly alcohol application sessions under local anesthesia on an outpatient basis. Symptoms completely disappeared in 14 patients (35.9%) and improved in 24 (61.5%). They conclude that deep venous malformation patients using ethanol at low doses was effective, with a low complication rate.

As a multidisciplinary medical journal CLINICS has covered virtually every medical area. We here highlight one of our strongest fields, Pediatrics, responsible for slightly over 10% of all our original research papers over 2010-11.

CARDIOVASCULAR Sellami et al. (1) examined the effect of the histocompatibility antigen HA-1 disparity on the incidence of both acute and chronic graft-versus-host disease in Tunisian recipients of hematopoietic stem cells in 60 patients and their 60 respective sibling hematopoietic stem cell donors. Their findings support previously reported data suggesting a significant association between HA-1 disparity and the risk of acute graft-versus-host disease following hematopoietic stem cell transplantation. Tedde et al. (2) compared bar displacement and complication rates in three retrospective series of mostly pediatric patients operated on by the same surgical team for pectus excavatus: (i) original, unmodified Nuss technique; (ii): ’’third point fixation’’ technique; and (iii) correction performed with modifications to the stabilizer and stabilizer position. With bar displacement and instability no longer significant postoperative risks, the Nuss technique should be considered among the available options for the surgical correction of pectus excavatum in pediatric patients. Dutra et al. (3) aimed to test five microsatellite markers (D7S1870, D7S489, D7S613, D7S2476, and D7S489) for the diagnosis of Williams-Beuren syndrome, to determine the size and parental origin of microdeletion, compare the clinical characteristics in 97 patients with different sizes of the deletion and parental origin. Microdeletions were present in 84 (86.6%) patients. Maternal deletions were found in 52.4%, paternal deletions in 47.6% of patients. Supraventricular aortic stenosis, ocular and urinary abnormalities were more frequent in the patients with a deletion. They conclude that the use of the five microsatellite markers was informative in all patients, and may thus be considered as an alternative method for molecular diagnosis in Williams-Beuren syndrome. Kayiran et al. (4) described the demographic, clinical, and laboratory features of 30 children, aged ,5 years. with Kawasaki disease managed at the American Hospital, Istanbul, Turkey. Coronary artery abnormalities were detected in nine patients. They claim that this study is the most comprehensive series of children from

INFECTIOUS DISEASES Contri et al. (6) described nutritional status, body composition and lipid profile in children and adolescents receiving protease inhibitors and conclude that the use of protease inhibitors, per se, does not seem to significantly interfere with anthropometric measures, body composition and food intake of HIV-infected children and adolescents. However, this antiretroviral therapy was associated with a significant increase in triglyceride and LDL-cholesterol in our subjects. Nakano et al. (7) examined the antimicrobial resistance profile and the prevalence of resistance genes in Bacteroides spp. and Parabacteroides distasonis strains isolated from children’s intestinal microbiota. Their results indicate an increase in the resistance to several antibiotics in intestinal Bacteroides spp. and Parabacteroides distasonis and demonstrate that these microorganisms harbor antimicrobial resistance genes that may be transferred to other susceptible intestinal strains. Hepatitis was the object of four studies. Carrilho et al. (8) performed a national survey to update hepatocellular carcinoma epidemiology in Brazil and determined the clinical and epidemiological profiles of patients in different Brazilian regions. They conclude that the epidemiology, classification, and therapy selection for hepatocellular carcinoma varied among Brazilian regions. Hepatitis C infection was the most common etiology of liver cirrhosis; chemoembolization was the most common therapy employed. Liver cirrhosis was the main risk factor for its development in Brazil. Because of its intimate relationship with the gastrointestinal tract four papers on Toxocara are included here. Simbalista et al. (9) described the evolution and outcome of children hospitalized with communityacquired pneumonia receiving penicillin (inclusion criteria: age .2 months; IV penicillin G: 200,000 IU/kg/day for .48 h; chest x-ray results). Of 154 studied cases, 123 (80%) and 40 (26%) had pulmonary infiltrate or pleural effusion, respectively. Penicillin G successfully treated 82% (126/154) of the study group and improvement was marked on the first day of treatment. Adenovirus studies are described in two articles. Nishiwaki-Dantas et al. (10) who sought to identify

Tel.: 55 11 2661 6235 Email: mrsilva36@hcnet.usp.br 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.

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Chlamydia trachomatis via polymerase chain reaction vs. direct fluorescent antibody assay in patients with vernal keratoconjunctivitis. The direct fluorescent antibody assay detected Chlamydia trachomatis in a 49.4% of vernal keratoconjunctivitis patients, while the polymerase chain reaction only succeeded in 20% of cases. Although the diagnosis of trachoma is essentially clinical, the disease may not be detected in vernal keratoconjunctivitis patients. Due to the high frequency of chlamydial infection detected in patients with vernal keratoconjunctivitis, we suggest considering routine laboratory tests to detect Chlamydia trachomatis in patients with severe and refractory allergic disease.

hospital stay with milk from a human milk bank according to the caloric-protein value vs. 10 fed milk from their own mothers and conclude that milk from the human bank allowed a satisfactory growth and good clinical evolution for these infants. Balaban et al. (17) investigated whether early weaning constitutes a risk factor for overweight at preschool age and identified other factors that affect this association through a case-control study of 366 children aged 2 to 6 years (176 boys and 190 girls) from three Brazilian cities. They conclude that the potential protective effect of breastfeeding against overweight among preschool children is weaker than genetic and other environmental factors. Nascimento et al. (18) analyzed in 409 preschool children (3.2¡0.3 years) the usefulness of the weight gain/ height gain ratio from birth to two and three years of age as a predictive risk indicator of excess weight at preschool age. Prevalence of excess weight was 28.8%, of overweight and obesity, 8.8%. The correlation coefficients between the body mass index z-scores of the preschool children and the birth weights or body mass indices at birth were low (0.09 and 0.10, respectively). They found that, regardless of weight/ height at birth, the mean ratio between the weight gain per g/cm of height growth from birth correlated strongly with body mass index of preschool children. This ratio may be a good indicator of the risk of excess weight and obesity in preschool-aged children. Vanderlei et al. (19) compared the autonomic function of 121 obese and eutrophic children (812 years) by analyzing heart rate variability. The obese children exhibited modifications in heart rate variability, characterized by a reduction in both sympathetic and parasympathetic activity. These findings stress the need for the early holistic care of obese children to avoid future complications.

NEUROLOGY Samelli et al. (11) developed and analyzed the efficacy of a low-cost screening tool to identify and classify hearing loss in children. A total of 214 (2- 10 years) children participated in this study. A conductive hearing loss was found in 39% of children, sensorineural hearing loss in 7.4%, and normal hearing in 53.3%. The results suggest that the questionnaire could be used as a screening tool to classify children with normal hearing or hearing loss and according to the type of hearing loss based on the total questionnaire score. Goncalves et al. (12) evaluated neurophysiological auditory brainstem responses to clicks and repeated speech stimuli to determine if they differ between typically developing children and children with phonological disorders. They conclude that the early stages of the auditory pathway processing of an acoustic stimulus are not similar in typically developing children and those with phonological disorders. These findings suggest that there are brainstem auditory pathway abnormalities in children with phonological disorders. Unlu et al. (13) described through a retrospective study the effect of multilevel botulin toxin-A injections in the lower extremities, focusing mainly on gross motor function and functional status in 72 cerebral palsy patients (mean age 6.7 years). They claim that a single multilevel BTX-A injection reduces spasticity and improves motor function in children with cerebral palsy. Bem et al. (14) evaluated a retrospective cohort of Wilson’s disease patients from southern Brazil during a 40-year follow-up period in what is claimed to be the first retrospective description of a population of Wilson’s disease patients of mainly European continental origin who live in southern Brazil. They find that Wilson’s disease is treatable if correctly diagnosed, and an adequate quality of life can be achieved, resulting in a long overall survival. Olandoski et al. (15) analyzed the evolution of renal function in patients with congenital neurogenic bladder by reviewing the records of 58 pediatric patients with respect to the following attributes: gender, age, etiology of neurogenic bladder, reason for referral, medical/surgical management, episodes of treated urinary tract infections, urodynamics, DMSA scintigraphy, weight, height, blood pressure, glomerular filtration rate, microalbuminuria and metabolic acidosis. The mean age at presentation was 4.2 +/- 3.5 years. They conclude that patient referral to a pediatric nephrologist was late. A reduction in the number of urinary tract infections was observed with adequate treatment, but microalbuminuria and metabolic acidosis occurred frequently despite adequate management.

PNEUMOLOGY Guimaraes et al. (20) assessed pulmonary function and the prevalence of atopy in 85 school-age children who were very low birth weight as infants and compared those who had bronchopulmonary dysplasia to those who did not. Their data showed no significant differences in lung function between bronchopulmonary dysplasia and no bronchopulmonary dysplasia patients at school age and no evidence of an association between atopy and bronchopulmonary dysplasia. Soeiro Ade et al. (21) described the demographic data, etiology, and pulmonary histopathological findings of different diseases in the autopsies of 4,710 patients (age range: 1-99 years) with acute respiratory failure. Bronchopneumonia was the most common diagnosis in these cases. The most prevalent pulmonary histopathological pattern was diffuse alveolar damage, which was associated with different inflammatory conditions. Further studies are necessary to elucidate the complete pathophysiological mechanisms involved with each disease and the development of acute respiratory failure. Stollar et al. (22) studied correlations between forced expiratory volume in one second (FEV1), chest radiography, chest computed tomography, 6-minute walk test, and Shwachman-Kulczycki score in 43 pediatric patients with cystic fibrosis and tested whether the Shwachman-Kulczycki score is still useful in monitoring the severity of the disease. They conclude that this score remains an useful tool for monitoring the severity of cystic fibrosis, adequately reflecting the functional impairment

NUTRITION Aprile et al. (16) described the growth and clinical evolution of 30 very low birth weight infants fed during

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death were multiple organ dysfunction syndrome, mechanical ventilation, use of vasoactive drugs, hospital-acquired infection, parenteral nutrition and duration of hospitalization. Fifty-four patients died; pediatric risk of mortality score was significantly lower in survivors. They conclude that the pediatric risk of mortality score showed adequate discriminatory capacity and thus constitutes a useful tool for the assessment of prognosis for pediatric patients admitted to a tertiary pediatric intensive care units. Shoroghi et al. (30) investigated and compared the efficacy of oral midazolam with two different dosages in orange juice on perioperative hemodynamics and behavioral changes in 90 children who underwent skin laser treatment in an academic educational Hospital. They were randomly assigned to 1 of 3 groups of 30 each: the placebo group received 0.1 ml/kg orange flavored juice, group 2 and 3 receiving 0.5 and 1 mg/kg of midazolam mixed in an equal volume of orange juice, respectively. The main outcome measures included the mask acceptance, patients’ behavioral scales and postoperative events. They conclude that the 1 mg.kg(-1) midazolam optimized the children’s behavior during skin laser treatment with no serious adverse effects, enhancing their parents’ satisfactions about the sedative protocol.

and chest radiography and tomography changes, especially in patients with greater impairment of lung function. Caution should be exercised when assessing patients with mild lung disease, because its limitations therein should be considered.

ONCOLOGY Estrozi and Bacchi (23) described some clinical pathological characteristics of neuroendocrine tumors of the gastroenteropancreatic tract in Brazilian patients. The study investigated clinical pathological features of 773 Brazilian gastroenteropancreatic neuroendocrine tumor cases from all the geographic regions of Brazil. They conclude that in this series, the proportion of NET cases in the total number of surgical pathology cases at their institution over the past 12 years is increasing. Gualco et al. (24) provide clinical pathological characteristics of 1,301 cases of pediatric/ adolescent lymphomas in patients from different geographic regions of Brazil and conclude that some of the results found in this study may reflect the heterogeneous socioeconomical status and environmental factors of the Brazilian population in different regions. Tamashiro et al. (25) assessed clinical and laboratory features that differentiate acute lymphoblastic leukemia from systemic juvenile idiopathic arthritis at disease onset in 102 systemic juvenile idiopathic arthritis patients retrospectively evaluated. Their study emphasizes the importance of investigating leukemia in patients presenting with musculoskeletal manifestations and, in particular, limb pain associated with thrombocytopenia. Vaisman et al. (26) designed a study to evaluate 65 patients (4-20 years of age) with differentiated thyroid carcinoma diagnosed before 20 years of age and to determine the factors associated with the response to the initial therapy. They conclude that metastases, both lymph nodal and distant, are important predictors of the persistence of disease after initial therapy in children and adolescents with differentiated thyroid cancer.

ORTHOPEDICS Chen et al. (31) determine the features of earthquakerelated pelvic crush fractures versus non-earthquake fractures with digital radiography and multidetector row computed tomography and conclude that earthquakerelated pelvic crush fractures can be characterized by a high incidence of pelvic fractures occurring in the pubis, comminuted fractures, and Type C fractures predominantly composed by subtype C3, despite a low incidence of multiple fractures. Frequency of occurrence in children was reported. Chu et al. (32) compared the features of head traumas caused by the Sichuan earthquake with those of other common head traumas using multidetector computed tomography. They focused the differences between fractures and intracranial injuries and the relationships between extracranial and intracranial injuries. As depicted with computed tomography, the severity of earthquake-related head traumas in survivors was milder, and isolated extracranial injuries were more common in earthquakerelated head traumas than in non-earthquake-related injuries, which may have been the result of different injury causes, mechanisms and settings. Occurrence of lesions in children is described.

GASTROENTEROLOGY Castro-Antunes et al. (27) evaluated the frequencies of the HLA genotypes DQ2 and DQ8 and the alleles A1*05, A1*0201, B1*0201 and B1*0302 in individuals with celiac disease in Recife, northeastern Brazil. They conclude that celiac disease was associated with the genotypes DQ2 and DQ8. DQ2 predominated, but the frequency distribution was different from what has been found in European populations and was closer to what has been found in the Americas. The high frequencies of the HLA genotypes DQ2 and DQ8 that were found in first-degree relatives would make it difficult to use these HLA genotypes for routine diagnosis of celiac disease in this group. Hong et al. (28) aimed to develop a decision model based on classification and regression tree analysis for the prediction of large esophageal varices in cirrhotic patients. They conclude that a decision tree model that consists of spleen width, portal vein diameter and prothrombin time may be useful for prediction of large esophageal varices in cirrhotic patients.

PSYCHIATRY Zappitelli et al. (33) endeavored to identify psychiatric diagnoses in a sample of 35 children (6-17 years) who had at least one parent with bipolar disorder type I. Their results support previous findings of an increased risk for developing psychopathology, predominantly mood and disruptive disorders, in the offspring of bipolar individuals. Prospective studies with larger samples are needed to confirm and expand these results. Lin et al. (34) evaluated the risks and rates of readmission and their predictors at 14 days, one year, and five years after discharge for the psychiatric population in Taiwan through a prospective study based on claims from 44,237 first-time hospitalized psychiatric patients discharged in 2000, who were followed for up to five years after

INTENSIVE CARE AND ANESTHESIA Costa et al. (29) endeavored to use the pediatric risk of mortality tool to determine mortality risk factors in a tertiary pediatric intensive care units: 359 patients were included; the variables that were found to be risk factors for

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discharge. The study found that the significant predictors for psychiatric readmission 14 days to five years after discharge were essentially the same except for patient’s age and hospital accreditation level. This study also highlighted the importance of socioeconomic factors in the prediction of readmission.

and immunological disorders because although frequency of DGS is high, few patients with a confirmed diagnosis are followed up. Noma et al. (40) endeavored to identify barriers to attendance for eye examination of schoolchildren. Facilities offered in this project were: examination arranged during weekends, free transportation, spectacle donation and two different opportunities for exam. A questionnaire was applied, by interview, to a sample consisting of parents seen in a community project. Those who missed the first call and attended the recall were screened to identify the reasons for non-attendance. The main causes of absenteeism were lack of awareness and work commitment. Absenteeism could be overcome via improved structuring of the first call. The recall increases attendance coverage of target population by only 15.2%. Wang et al. (41) reported the incidence and recurrence of acute otitis media in Taiwan’s pediatric population and that the annual incidence rate of was estimated to be 64.5 cases per 1,000 children. The overall one-year cumulative incidence rate of recurrence was 33.1%, and the incidence density rate was 33.5 cases per 100 personyears, with the highest figure (41.2 cases per 100 personyears) noted for children aged 0-2 years. They conclude that acute otitis media remains a major threat to children’s health in Taiwan. Male children and very young children require more aggressive preventive strategies to reduce the risk of recurrence.

OTHER Sole et al. (35), on behalf of the Online Latin American Survey of Anaphylaxis (OLASA) endeavored to identify the main clinical manifestations, triggers, and treatments of severe allergic reactions in patients who were seen by allergists from July 2008 to June 2010 in 15 Latin American countries and Portugal (n = 634): 31.5% 18 years old or less, 41.6% were male. Etiologic agents were identified in 87.4% of cases: drugs (31.2%), foods (23.3%), and insect stings (14.9%), but foods predominated in children. The main clinical manifestations were cutaneous. Treatment for acute anaphylactic reactions was not appropriate. They conclude that it is necessary to improve educational programs in order to enhance the knowledge on this potentially fatal emergency. Hua-Li et al. (36) explored the characteristics of seasonal distribution and the influences of meteorological factors including temperature and humidity on active systemic lupus erythematosus trough a retrospective analysis of 640 patients living in the city of Zhanjiang, China. In winter, when there are weaker ultraviolet (UV) rays, the ratio of patients with active SLE to total inpatients was 3.89%, which is significantly higher than in other seasons with stronger UV rays, including 2.17% in spring, 1.87% in summer and 2.12% in autumn. The number of patients with active SLE had significant negative correlation with mean temperature and was not significantly related to mean humidity. Active SLE has the characteristics of seasonal distribution and is associated with temperature. The mechanism remains to be further studied. Eren et al. (37) present special clinical and laboratory features of 294 cases of mushroom poisoning in a Turkish University Hospital (Cumhuriyet): of these 294 patients (age of 3-072, 173 were female, with 90 were under the age of 16 years). Mushrooms were consumed in the early summer by 173 patients. The onset of symptoms was within two hours for 101 patients and the most common first-noticed symptoms were gastrointestinal. Patients were discharged within one to ten days. Three patients suffering from poisoning caused by wild mushrooms died from fulminant hepatic failure. They conclude that education of the public about the consumption of mushrooms and education of health personnel working in health centers regarding early treatment and transfer to hospitals with appropriate facilities are important for decreasing the mortality. Ferrer et al. (38) described the causes of admission to the public health system for children from zero to nine years of age in the city of Sa˜o Paulo during the years 2002 to 2006 and compare these results to those from the national data. Their findings show a paradoxical increase in the number of hospitalizations during an expansion of primary attention, indicating that the rise was not associated with a significant improvement in the quality of service. Fomin et al. (39) described clinical and laboratorial data and phenotypic characteristics of patients with DiGeorge Syndrome and point out the need of suspecting the presence of the syndrome in all patient presenting with heart defects, facial abnormalities (associated or not with hypocalcemia),

DENTISTRY Areias et al. (42) characterized the environmental and host factors associated with dental caries in Portuguese children with and without Down syndrome through a sibling-matched, population-based, cross-sectional survey. They conclude that Portuguese children with Down syndrome have lower caries rates than children without Down syndrome. This claim that this reduced prevalence may be associated with the parents’ greater concern about oral health care in Down syndrome children, resulting in their taking them sooner to visit a dentist, as well as to a higher bruxism prevalence and delayed tooth eruption. Carrillo et al. (43) characterized the population seen at the dentistry unit of the hematology-oncology service of a tertiary medical center in Sa˜o Paulo and conclude that the characteristics of the studied population were similar to those of the general Brazilian and global populations, especially regarding gender and diagnosis distributions. The aim of implementation of the dentistry unit was to maintain good oral health and patients’ quality of life, which is critical to provide oral care and prevent future oral problems. Motta et al. (44) endeavored to determine whether there is a correlation between halitosis and mouth breathing in 55 children between 3 and 14 years of age divided into two groups (nasal and mouth breathing) for the assessment of halitosis. There were a significantly greater number of boys with the mouth-breathing pattern than girls. A total of 23.6% of the participants had no mouth odor, 12.7% had mild odor, 12.7% had moderate odor and 50.9% had strong odor. There was a statistically significant association between halitosis and mouth breathing. They conclude that the occurrence of halitosis was high among the children evaluated, and there was a statistically significant association between halitosis and mouth breathing. Perinetti et al. (45) investigated whether malocclusal traits correlate with body posture alterations in 122 young subjects to determine

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Pediatrics in Clinics Rocha-e-Silva M 18. Nascimento VG, Bertoli CJ, Leone C. Ratio of weight to height gain: a useful tool for identifying children at risk of becoming overweight or obese at preschool age. Clinics. 2011;66(7):1223-6, http://dx.doi.org/ 10.1590/S1807-59322011000700017. 19. Vanderlei LC, Pastre CM, Freitas Junior IF, Godoy MF. Analysis of cardiac autonomic modulation in obese and eutrophic children. Clinics. 2010 Jun;65(8):789-92. 20. Guimaraes H, Rocha G, Pissarra S, Guedes MB, Nunes T, Vitor B. Respiratory outcomes and atopy in school-age children who were preterm at birth, with and without bronchopulmonary dysplasia. Clinics. 2011;66(3):425-30, http://dx.doi.org/10.1590/S1807-59322011000300 011. 21. Soeiro Ade M, Ruppert AD, Canzian M, Parra ER, Farhat C, Capelozzi VL. Demographic, etiological, and histological pulmonary analysis of patients with acute respiratory failure: a study of 19 years of autopsies. Clinics. 2011;66(7):1193-7, http://dx.doi.org/10.1590/S1807-59322011000700012. 22. Stollar F, Adde FV, Cunha MT, Leone C, Rodrigues JC. ShwachmanKulczycki score still useful to monitor cystic fibrosis severity. Clinics. 2011;66(6):979-83, http://dx.doi.org/10.1590/S1807-59322011000600 010. 23. Estrozi B, Bacchi CE. Neuroendocrine tumors involving the gastroenteropancreatic tract: a clinicopathological evaluation of 773 cases. Clinics. 2011;66(10):1671-5. 24. Gualco G, Klumb CE, Barber GN, Weiss LM, Bacchi CE. Pediatric lymphomas in Brazil. Clinics. 2010;65(12):1267-77, http://dx.doi.org/ 10.1590/S1807-59322010001200008. 25. Tamashiro MS, Aikawa NE, Campos LM, Cristofani LM, Odone-Filho V, Silva CA. Discrimination of acute lymphoblastic leukemia from systemic-onset juvenile idiopathic arthritis at disease onset. Clinics. 2011;66(10):1665-9. 26. Vaisman F, Bulzico DA, Pessoa CH, Bordallo MA, Mendonca UB, Dias FL, et al. Prognostic factors of a good response to initial therapy in children and adolescents with differentiated thyroid cancer. Clinics. 2011;66(2):281-6, http://dx.doi.org/10.1590/S1807-59322011000200017. 27. Castro-Antunes MM, Crovella S, Brandao LA, Guimaraes RL, Motta ME, Silva GA. Frequency distribution of HLA DQ2 and DQ8 in celiac patients and first-degree relatives in Recife, northeastern Brazil. Clinics. 2011;66(2):227-31, http://dx.doi.org/10.1590/S1807-59322011000200008. 28. Hong WD, Dong LM, Jiang ZC, Zhu QH, Jin SQ. Prediction of large esophageal varices in cirrhotic patients using classification and regression tree analysis. Clinics. 2011;66(1):119-24, http://dx.doi.org/10.1590/ S1807-59322011000100021. 29. Costa GA, Delgado AF, Ferraro A, Okay TS. Application of the pediatric risk of mortality (PRISM) score and determination of mortality risk factors in a tertiary pediatric intensive care unit. Clinics. 2010;65(11):1087-92, http://dx.doi.org/10.1590/S1807-59322010001100005. 30. Shoroghi M, Arbabi S, Farahbakhsh F, Sheikhvatan M, Abbasi A. Perioperative effects of oral midazolam premedication in children undergoing skin laser treatment. A double-blinded randomized placebo-controlled trial. Acta Cir Bras. 2011 Aug;26(4):303-9, http:// dx.doi.org/10.1590/S0102-86502011000400010. 31. Chen TW, Yang ZG, Dong ZH, Tang SS, Chu ZG, Shao H. Earthquakerelated pelvic crush fracture vs. non-earthquake fracture on digital radiography and MDCT: a comparative study. Clinics. 2011;66(4):629-34, http://dx.doi.org/10.1590/S1807-59322011000400018. 32. Chu ZG, Yang ZG, Dong ZH, Chen TW, Zhu ZY, Shao H. Comparative study of earthquake-related and non-earthquake-related head traumas using multidetector computed tomography. Clinics. 2011;66(10):1735-42, http://dx.doi.org/10.1590/S1807-59322011001000011. 33. Zappitelli MC, Bordin IA, Hatch JP, Caetano SC, Zunta-Soares G, Olvera RL, et al. Lifetime psychopathology among the offspring of Bipolar I parents. Clinics. 2011;66(5):725-30, http://dx.doi.org/10.1590/S180759322011000500003. 34. Lin CH, Chen WL, Lin CM, Lee MD, Ko MC, Li CY. Predictors of psychiatric readmissions in the short- and long-term: a population-based study in Taiwan. Clinics. 2010 May;65(5):481-9. 35. Sole D, Ivancevich JC, Borges MS, Coelho MA, Rosario NA, Ardusso LR, et al. Anaphylaxis in Latin America: a report of the online Latin American survey on anaphylaxis (OLASA). Clinics. 2011;66(6):943-7, http://dx.doi.org/10.1590/S1807-59322011000600004. 36. Hua-Li Z, Shi-Chao X, De-Shen T, Dong L, Hua-Feng L. Seasonal distribution of active systemic lupus erythematosus and its correlation with meteorological factors. Clinics. 2011;66(6):1009-13, http://dx.doi. org/10.1590/S1807-59322011000600015. 37. Eren SH, Demirel Y, Ugurlu S, Korkmaz I, Aktas C, Guven FM. Mushroom poisoning: retrospective analysis of 294 cases. Clinics. 2010 May;65(5): 491-6. 38. Ferrer AP, Sucupira AC, Grisi SJ. Causes of hospitalization among children ages zero to nine years old in the city of Sao Paulo, Brazil. Clinics. 2010;65(1):35-44, http://dx.doi.org/10.1590/S1807-59322010000100007. 39. Fomin AB, Pastorino AC, Kim CA, Pereira CA, Carneiro-Sampaio M, Abe-Jacob CM. DiGeorge Syndrome: a not so rare disease. Clinics. 2010;65(9):865-9, http://dx.doi.org/10.1590/S1807-59322010000900009.

possible clinical applications. Their current findings, particularly with regard to the use of posturography as a diagnostic aid for subjects affected by dental malocclusion, do not support existence of clinically relevant correlations between malocclusal traits and body posture. Souza et al. (46) verified dental abnormalities and the oral health condition in pediatric patients suffering from hypophosphatemic rickets through a prospective study of oral conditions. This report employed a simple method to be easily reproducible: oral clinical exam and radiographic evaluation. They conclude that these patients frequently present dental alterations not completely recoverable with treatment (dental abscesses excepted) and need periodical oral examinations.

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teaching hospital. Clinics. 2010 Jun;65(6):569-73, http://dx.doi.org/ 10.1590/S1807-59322010000600002. 44. Motta LJ, Bachiega JC, Guedes CC, Laranja LT, Bussadori SK. Association between halitosis and mouth breathing in children. Clinics. 2011;66(6):93942, http://dx.doi.org/10.1590/S1807-59322011000600003. 45. Perinetti G, Contardo L, Biasati AS, Perdoni L, Castaldo A. Dental malocclusion and body posture in young subjects: a multiple regression study. Clinics. 2010 Jul;65(7):689-95. 46. Souza MA, Soares Junior LA, Santos MA, Vaisbich MH. Dental abnormalities and oral health in patients with Hypophosphatemic rickets. Clinics. 2010;65(10):1023-6, http://dx.doi.org/10.1590/S1807-59322010001000017.

40. Noma R, Carvalho Rde S, Kara-Jose N. Why are there defaulters in eye health projects? Clinics. 2011;66(9):1585-9. 41. Wang PC, Chang YH, Chuang LJ, Su HF, Li CY. Incidence and recurrence of acute otitis media in Taiwan’s pediatric population. Clinics. 2011; 66(3):395-9, http://dx.doi.org/10.1590/S1807-59322011000300005. 42. Areias CM, Sampaio-Maia B, Guimaraes H, Melo P, Andrade D. Caries in Portuguese children with Down syndrome. Clinics. 2011;66(7):1183-6, http://dx.doi.org/10.1590/S1807-59322011000700010. 43. Carrillo C, Vizeu H, Soares-Junior LA, Fava M, Filho VO. Dental approach in the pediatric oncology patient: characteristics of the population treated at the dentistry unit in a pediatric oncology brazilian

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DOI:10.6061/clinics/2012(08)02

CLINICAL SCIENCE

Carotid stenosis: what is the high-risk population? Jong Hun Park,I Alvaro Razuk,II Paulo Fernandes Saad,II Gustavo Jose´ Politzer Telles,II Walter Khegan Karakhanian,II Alexandre Fioranelli,II Alessandra Caivano Rodrigues,II Giuliano Giova Volpiani,II Pollyanna Campos,II Roberto Massayoshi Yamada,II Valter Castelli Jr.,II Roberto Augusto CaffaroII I

Federal University of Sa˜o Francisco Valley (UNIVASF), Petrolina/PE, Brazil. II Santa Casa Faculty of Medical Sciences, Sa˜o Paulo/SP, Brazil.

OBJECTIVE: Prevention is the best treatment for cerebrovascular disease, which is why early diagnosis and the immediate treatment of carotid stenosis contribute significantly to reducing the incidence of stroke. Given its silent nature, 80% of stroke cases occur in asymptomatic individuals, emphasizing the importance of screening individuals with carotid stenosis and identifying high-risk groups for the disease. The aim of this study was to determine the prevalence and the most frequent risk factors for carotid stenosis. METHODS: A transversal study was conducted in the form of a stroke prevention campaign held on three nonconsecutive Saturdays. During the sessions, carotid stenosis diagnostic procedures were performed for 500 individuals aged 60 years or older who had systemic arterial hypertension and/or diabetes mellitus and/or coronary heart disease and/or a family history of stroke. RESULTS: The prevalence of carotid stenosis in the population studied was 7.4%, and the most frequent risk factors identified were mean age of 70 years, carotid bruit, peripheral obstructive arterial disease, coronary insufficiency and smoking. Independent predictive factors of carotid stenosis include the presence of carotid bruit or peripheral obstructive heart disease and/or coronary insufficiency. CONCLUSIONS: The population with peripheral obstructive heart disease and carotid bruit should undergo routine screening for carotid stenosis. KEYWORDS: Carotid Stenosis; Risk Factors; Early Diagnosis. Park JH, Razuk A, Saad PF, Telles GJ, Karakhanian WK, Fioranelli A, et al. Carotid stenosis: what is the high-risk population? Clinics. 2012;67(8):865870. Received for publication on March 17, 2012; First review completed on March 23, 2012; Accepted for publication on April 1, 2012 E-mail: paulo.saad@univasf.edu.br Tel.: 55 87 38629394

However, this type of lesion is easily diagnosed with noninvasive methods and can be managed with effective therapeutic options (4). Barnett et al. (5) estimated that approximately two million North Americans and Europeans have treatable asymptomatic carotid artery stenosis. Roederer et al. (6) estimated the risk of stroke or total carotid occlusion at 46% per year in patients with asymptomatic carotid stenosis greater than or equal to 80%. According to Chambers et al. (7), individuals with carotid stenosis greater than or equal to 75% have a 26% risk of developing stroke in three years. Given its silent nature, 80% of stroke cases occur in asymptomatic individuals (8), hence the importance of screening individuals with carotid stenosis and identifying groups at high risk for the disease. Moreover, the annual progression of moderate to severe stenosis is 14% (9). Few studies have focused on determining the prevalence of carotid stenosis in the general population because the operational costs of routine screening are high. Nevertheless, the estimated prevalence of carotid stenosis greater than or equal to 50% in the general population ranges from 2 to 8%, and the estimated prevalence of stenosis greater than or equal to 80% ranges from 1 to 2% (10). Therefore,

INTRODUCTION In 2004, the Atlas of Heart Disease and Stroke published by the World Health Organization registered a total of 129.2 deaths as a result of stroke in Brazil (1). The same publication reported that approximately 15 million people suffer from stroke annually worldwide, and 5.5 million of them die as a result of the disease. Ischemic stroke accounts for 67% to 83% of all strokes (2). It can be caused by carotid artery disease, cardiac arrhythmia and arterial hypertension (3). Extracranial cerebrovascular disease stems from atherosclerotic lesions located predominantly in the carotid bifurcation and are responsible for up to 75% of ischemic stroke events (2). These lesions can cause cerebral ischemia via the embolization of atheromatous plaques, the formation of thrombi or arterial occlusion.

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.

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stenosis, according to the model proposed by Jacobowitz et al. (2,13). This scan consists of a modified ultrasonographic exam, which has been regularly validated and compared with angiography and angioresonance as part of an accreditation by the Intersocietal Commission for the Accreditation of Vascular Laboratories (ICAVL). The test consists of an examination of the distal common carotid artery and the bulb and proximal segments of the internal and external carotid arteries, including the determination of peak systolic velocities in the internal carotid artery. A velocity greater than or equal to 120 cm/s is considered to indicate the presence of .50% stenosis. In addition to this criterion, the common and internal carotid arteries underwent a morphological analysis that classified them as normal, narrowed, less than 50% stenosis, or greater than 50% stenosis. Ultrasound exams were conducted by three radiologists specializing in vascular ultrasonography. All positive results were subsequently confirmed by a second examiner. The volunteers were split into two groups based on the ultrasound exam findings:

studies have attempted to identify a high-risk population that would benefit from the early diagnosis of carotid stenosis. According to Yin and Carpenter (11), screening asymptomatic individuals is only cost-effective in populations presenting with the following characteristics: carotid stenosis prevalence greater than 4.5%; echo Doppler specificity greater than 91%; stroke incidence during clinical treatment greater than 3.3%; reduction in stroke risk greater than 37%; and occurrence of stroke associated with carotid endarterectomy consistent with the rates published by NASCET and ACAS. Furthermore, the operational cost per exam must be less than US$300. According to a committee of the American Society of Neuroimaging (12), cost-effective screening lowers the stroke risk when the carotid stenosis prevalence is between 5% and 20%, and screening does not reduce stroke risk and is not cost-effective when the carotid stenosis prevalence is lower than 5%. In Brazil, no screening studies in this patient group have been conducted for the general population or a selected population. This lack of studies informed the rationale for this study, which sought to contribute to a deeper understanding of the role of carotid stenosis screening in populations at risk for atherosclerosis, to determine the prevalence of the disease and to characterize the population at high risk of developing the disease.

N N

Positive: Internal carotid artery stenosis $50% Negative: Internal carotid artery stenosis ,50%

The groups were then analyzed for the following variables:

N N N N

METHODS This study was previously approved by the Research Ethics Committee under Project No. 469/07. The Department of Vascular and Endovascular Surgery conducted a transverse study in the form of a stroke prevention campaign in May and June 2004. The campaign was publicized through pamphlets, flyers displayed on newsstands and at pharmacies and bakeries and via radio broadcasts and advertisements in local newspapers in downtown Sa˜o Paulo city. The campaign was run on three non-consecutive Saturdays. Carotid stenosis diagnosis sessions were offered to interested individuals who were aged 60 years or older and who were diagnosed with one of the following conditions: systemic arterial hypertension, diabetes mellitus, coronary heart disease or familial history of stroke. These factors constituted the study’s inclusion criteria. Interested individuals were asked to call the telephone number provided and to schedule a day and time for an interview and exam. A total of 632 interviews were scheduled by phone. Of these, 582 individuals participated in the scheduled interviews. Eighty-two volunteers did not meet the campaign criteria and were excluded from the study. The final sample consisted of 500 individuals. The interviews were conducted by eight physicians from the Department of Vascular and Endovascular Surgery, who administered a predetermined questionnaire addressing the health history and familial morbid antecedents of each volunteer. A physical exam was then performed to check for carotid bruit, palpate the pulses of the extremities and take blood pressure readings. After the interview and physical exam, the volunteers were referred for an ultrasonography exam with Doppler (Prologic 7 device by GE - Waukesha, USA and SD800 device by Phillips - Amsterdam, Netherlands) to detect occult carotid

N N N N N N N N N

Age Gender Race Arterial hypertension (SAH): Systolic arterial pressure $140 mmHg and/or diastolic $90 mmHg (14) or chronic use of anti-hypertensive medication; Diabetes mellitus (DM) with a fasting glucose exam $126 mg/dL (15) or the use of oral hypoglycemics or insulin; Dyslipidemia: Cholesterol levels $200 mg/dL or LDL levels $130 mg/dL or triglyceride levels $150 mg/dL (16) or the use of statins or fibrates; Peripheral obstructive arterial disease (POAD): The absence of pulses in the extremities, confirmed by a second examiner; Coronary insufficiency (CI): The presence of stable or unstable angina, history of myocardial infarction, myocardial revascularization or coronary angioplasty; Other cardiopathies, such as arrhythmias, valvulopathies, congestive cardiac insufficiency or the use of a pacemaker or an internal cardioverter defibrillator; Smoking: Current smokers who have smoked at least 100 cigarettes throughout their lifetime (17); Signs and symptoms of a cerebral ischemic event: Asymptomatic, syncope, transient ischemic attack or stroke (amaurosis fugax, aphasia or dysphasia and motor deficit); Family history of stroke in first-degree relatives; Presence of carotid bruit based on auscultation of the cervical region at the medial border of the sternocleidomastoid muscle with a stethoscope.

A 5% level of significance was adopted. Values expressed in frequencies were compared using the chi-squared test and its variations (Fisher’s exact test and a generalization of

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Carotid stenosis: high risk-population Park JH et al.

variables PAOD, Smoking and CI are individual risk factors for internal carotid artery stenosis .50%. Regarding cerebral ischemia symptoms, no statistically significant differences were observed between the carotid stenosis-positive and negative groups in terms of reported syncope, motor sequelae or amaurosis fugax. Ten individuals (2%) tested positive for carotid bruit (Table 3). Carotid bruit was identified in 50% of the subjects in the positive group, while only 7.1% of the individuals without carotid bruit had significant stenosis of the carotid artery. No statistically significant association was observed between the sum of the risk factors and an increased prevalence of carotid stenosis (Table 4). Based on the model containing the six most significant variables that were identified during the univariate analysis, we concluded that the presences of bruit and POAD are predictors of carotid stenosis (p,0.05). The presence of cardiac bruit increased the probability of a positive exam result 12-fold (OR = 12.6) and that POAD increased the likelihood of a positive result 3-fold (OR = 3.7) compared to the chance of positivity when these factors were absent (Table 5). The variable ‘‘coronary insufficiency’’ had a descriptive level of approximately 5% (p = 0.045). Taking this level as significant, we can conclude that, on average, the likelihood of a positive ultrasound result is doubled in the presence of angioplasty, coronary artery bypass or angina compared to the absence of these conditions (OR = 2.3). Table 5 shows the most frequent variables associated with a positive ultrasound result.

Fisher’s exact test for tables with low frequencies). Special attention was given to the variable "carotid bruit", which was treated as a dependent variable. The Student’s t test, controlled by Levene’s test, was used to compare continuous values. A logistic regression analysis was used to determine the relationship between the study variables and exam results. The regression model was tested by selecting the variables that were significant at a level of 10% in a univariate analysis (p,0.100). The data were tabulated and analyzed using the SAS 9.1 software program.

RESULTS The characteristics of the study sample are shown in Table 1. Ultrasound examinations revealed positive results (i.e., $50% carotid stenosis) in 37 individuals, representing 7.4% of the population assessed. When positive and negative carotid stenosis results were compared by subjects’ ages, the mean age of the positive group (70.4¡6.8 years) was statistically significantly higher than that of the negative group (67.5¡6.3; p = 0.01). However, no statistical differences in gender or race were detected between the carotid stenosis-positive and negative groups. Table 2 presents a comparison of the carotid stenosispositive and negative groups in terms of the presence of risk factors for atherosclerosis. The results indicate that only the

Table 1 - Characterization of a sample of 500 participants in the Stroke Prevention Campaign who underwent carotid stenosis screening.

DISCUSSION The diagnostic method employed in this study was ultrasonography with Doppler using a modified version of the approach described by Jacobowitz et al. (8). The cut-off value adopted was close to the value recommended by Carsen III et al. (18), who observed optimal sensitivity (91%), specificity (95%), positive predictive value (89%) and negative predictive value (96%) with a cut-off of 115 cm/s in screening exams that took an average of 3.2 minutes to perform per patient. In this population-based study, the prevalence of .50% stenosis of the internal carotid artery was 7.4%. The prevalence of significant asymptomatic carotid stenosis ($50%) varies. Prevalences between 4 and 8% were found in the general population, with advanced age being the only independent predictor of significant asymptomatic carotid stenosis (8,9). In contrast, the values reported by Qureshi et al. (12) ranged from 7% to 35%, depending on the isolated or associated presence of such risk factors as age over 65 years, smoking, coronary insufficiency, and hypercholesterolemia. In this study, no statistical significances were identified for the variables gender, race, SAH, DM, dyslipidemia, cardiopathies, familial history of stroke, or cerebral ischemia symptoms. Similarly, the sum of the risk factors did not predict carotid stenosis. In terms of gender, the literature only reports a statistically significant difference when analyzing specific subgroups, such as candidates for myocardial revascularization (greater risk among women) (19) or individuals with POAD

Total Age (years) Mean¡S.D. Median Minimum-maximum Gender - n (%) Female Male Race - n (%) White Black Yellow Signs and symptoms of stroke - n (%) Asymptomatic Visual symptoms Syncope Sequelae Variables - n (%) SAH Dyslipidemia Family history of stroke Smoking CI (angina, angioplasty or coronary artery bypass) DM PAOD Cardiopathy Bruit Total

67.8¡6.4 66.0 60–89 279 221

(55.8%) (44.2%)

413 60 27

(82.6%) (12.0%) (5.4%)

255 57 53 18

(51.0%) (11.4%) (10.6%) (3.6%)

364 250 215 182 122

(72.8%) (50.0%) (43.0%) (36.4%) (24.4%)

116 107 90 10 500

(23.2%) (21.4%) (18.0%) (2.0%) (100.0%)

SAH: Arterial hypertension; CI: Coronary insufficiency; DM: Diabetes mellitus; PAOD: Peripheral obstructive arterial disease.

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Table 2 - Comparison of variables considered risk factors for atherosclerosis between the groups that were positive and negative for carotid stenosis on ultrasound. Ultrasound

Presence of risk factors for atherosclerosis

SAH No Yes DM No Yes Dyslipidemia No Yes PAOD No Yes CI No Yes Cardiopathy No Yes Smoking No Yes Family history of stroke No Yes

Positive

130 (96.3%) 333 (91.2%)

5 (3.7%) 32 (8.8%)

352 (91.7%) 111 (95.7%)

32 (8.3%) 5 (4.3%)

229 (91.6%) 234 (93.6%)

21 (8.4%) 16 (6.4%)

369 (94.4%) 91 (83.5%)

22 (5.6%) 18 (16.5%)

358 (94.7%) 105 (86.1%)

20 (5.3%) 17 (13.9%)

377 (92.0%) 86 (95.6%)

33 (8.0%) 4 (4.4%)

300 (94.3%) 163 (89.6%)

18 (5.7%) 19 (10.4%)

261 (91.6%) 202 (94.0%)

24 (8.4%) 13 (6.0%)

p = 0.054

p = 0.147

p = 0.393

p,0.001*

p = 0.002*

p = 0.237

p = 0.020*

p = 0.315

Smoking is considered one of the most important risk factors in the genesis of atherosclerosis. However, there is a lack of consensus in the literature regarding the predictive value of the association between smoking and carotid stenosis. Some authors have confirmed this link (2,9,10), while others have failed to observe a statistically significant association. In this study, smoking was reported in 51% of the carotid stenosis-positive group. While the univariate analysis revealed that smoking was a predictive factor for carotid stenosis, the association was not significant in the subsequent multivariate analysis. Consistent with our findings, several studies have identified both POAD (20,24,25) and CI (2,10,12,20) as independent predictors for significant asymptomatic carotid stenosis. In male populations with POAD, a carotid stenosis prevalence of 18% was detected in young adults (20), and a 20% prevalence was identified in elderly adults (25). CI was found to be a predictive factor for .50% carotid stenosis in both of these groups, especially the younger group (19,25). In a Chinese population, this prevalence

(greater risk among men) (20). No reports were found that described differences related to race (2,8,10). The percentage of patients with SAH was significantly higher in the group that tested positive for .50% carotid stenosis, although SAH was not an independent predictor. This finding contrasts with some authors’ results (2,8) but corroborates the observations of Qureshi et al. (12), indicating that isolated SAH should not be used as a marker for screening asymptomatic carotid stenosis. The presence of DM was not significantly correlated with carotid stenosis. This finding is consistent with the majority of previous scientific studies, although DM is regarded as a risk factor in the pathogenesis of atherosclerosis (9,10,21). The presence of dyslipidemia did not predict carotid stenosis in this study, corroborating the results of most studies in the literature (2,8,9,20,22). Univariate analysis showed that of the variables studied, advanced age, smoking, POAD, and CI were predictors of carotid stenosis. The direct relationship between age and carotid stenosis risk has been confirmed by several previous studies (9,10,21,23,24), and our results corroborated this finding.

Table 4 - Association between the number of risk factors and the presence of carotid stenosis on ultrasound.

Table 3 - Comparison of the presence of carotid bruit and carotid stenosis findings on ultrasound. Ultrasound Negative Bruit - n (%) No Yes Total patients

p-value

Negative

Total

Ultrasound Negative

p-value No. of risk factors – n (%) 1 2 3 or more Total patients

Positive p,0.001

455 (92.9%) 35 (7.1%) 5 (50.0%) 5 (50.0%) 460 (92.0%) 40 (8.0%)

490 (100.0%) 10 (100.0%) 500 (100.0%)

p-value: Fisher’s exact test.

p-value

p = 0.064 78 (97.5%) 122 (94.6%) 263 (90.4%) 463 (92.6%)

p-value: Fisher’s exact test.

868

Total

Positive

2 (2.5%) 7 (5.4%) 28 (9.6%) 37 (7.4%)

80 (100.0%) 129 (100.0%) 291 (100.0%) 500 (100.0%)


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between the number of risk factors and an increased prevalence of carotid stenosis. In the second study (2), 610 individuals were screened. A 10.8 % prevalence of carotid stenosis was found, indicating a strong correlation with CI. Our screening study revealed a lower prevalence (7.4%), but CI was also found to be an independent predictor of .50% carotid stenosis. Among the studied risk factors for atherosclerotic disease, the presence of carotid bruit, PAOD, advanced age, CI and smoking were significantly correlated with a diagnosis of .50% carotid stenosis in univariate analyses. However, a multivariate logistic regression analysis only identified correlations between carotid stenosis and carotid bruit, PAOD and CI, which were determined to be significant independent risk factors for carotid stenosis. The results of relative risk calculations confirmed the association between stenosis and carotid bruit. Conclusive studies to determine the cost-benefit of this screening are needed. Although the prevalence of asymptomatic carotid stenosis was significant in the population studied (7.4%) and was consistent with the recommendations by Yin and Carpenter (11), the cost of each confirmed diagnosis in this study was US$ 358.00. The acceptable recommended cost is approximately US$ 300.00 per positive diagnosis. The adoption of more specific criteria, such as those employed in this study, combined with the introduction of portable ultrasonography equipment may increase the costeffectiveness of carotid stenosis screening and may increase physicians’ awareness of the importance of identifying high-risk individuals for stroke prevention. The prevalence of carotid stenosis in the studied population was 7.4%, and the most frequent risk factors identified were a mean age of 70 years, carotid bruit, peripheral obstructive arterial disease, coronary insufficiency and smoking. Independent predictive factors of carotid stenosis include the presence of carotid bruit or peripheral obstructive heart disease and/or coronary insufficiency. Populations that meet these criteria should undergo routine screening for carotid stenosis.

Table 5 - Logistic regression analysis for the most frequently occurring variables and positive ultrasound findings.

Variables: Bruit PAOD CI SAH Smoking No. of risk factors

OR[1] (score estimate)

OR[1] - Confidence interval (95%)

p-value

12.6 3.7 2.3 2.4 1.4 0.9

[2.8; 55.6] [1.7; 8.1] [1.0; 5.0] [0.8; 6.8] [0.9; 2.2] [0.6; 1.4]

0.001* 0.001* 0.045* 0.116 0.133 0.671

SAH: Arterial hypertension; CI: Coronary insufficiency; DM: Diabetes mellitus; PAOD: Peripheral obstructive arterial disease. p-value (model) ,0.0001; N = 500; [1]OR = odds ratio.

reached 24.7%, and risk factors included advanced age, carotid bruit and smoking, with a particular emphasis on the number of cigarettes smoked per day (24). A study involving 168 patients with atherosclerotic aortoiliac disease identified a 28% prevalence of significant carotid stenosis; advanced age and the presence of carotid bruit were predictors of more severe stenoses in this population (26). The close relationship between carotid stenosis and coronary heart disease has been recognized, as the diseases share the same risk factors and pathogenic mechanisms. The literature indicates that the prevalence of CI in patients with extracranial cerebral disease ranges from 40% to 60%, and the carotid disease rate in candidates for myocardial revascularization ranges from 2 to 27%, based on a review of 12 studies published between 1983 and 1996 (27). The 13.9% prevalence of $50% carotid stenosis among individuals with CI in this study is consistent with the rate reported in the literature. Several studies have reported a positive relationship between cervical bruit and carotid stenosis (28,29). According to the ACAS, ipsilateral bruit was detected in 75% of individuals with $60% carotid stenosis, suggesting that 80% of patients with significant carotid disease had carotid bruit. Some screening programs have identified carotid bruit as a strong predictor of carotid stenosis, particularly among men with PAOD (24,25). Our results found that 50% of individuals with $50% carotid stenosis had carotid bruit; its sensitivity as a predictor was 50%. Lavenson Jr. et al. (30) screened 176 individuals to validate a stroke prevention protocol and found a carotid bruit rate similar to that identified in this study. Despite its low sensitivity, the presence of carotid bruit proved highly specific for the occurrence of carotid stenosis, yielding a negative predictive value of 0.99. Two other studies also aimed to determine the population that would benefit most from carotid stenosis screening. The first of these studies (8) included 394 individuals and found a carotid stenosis prevalence ranging between 1.8% (no risk factors) and 66.7% (four associated risk factors). Heart disease and arterial hypertension have been determined as predictors of significant asymptomatic carotid stenosis. However, this study failed to confirm this association

AUTHOR CONTRIBUTIONS Park JH contributed to the final revision of the manuscript, collected data, conceived the study project and conducted the literature review. Razuk A, Castelli Junior V and Caffaro RA contributed to the final revision of the manuscript and conceived the study project. Saad PF contributed to the final revision of the manuscript, collected data, and conducted the literature review. Telles GJ, Rodrigues AC, Volpiani GG, Campos P and Yamada RM contributed to the final revision of the manuscript and collected data. Karakhanian WK contributed to the final revision of the manuscript and conducted the literature review. Fioranelli A contributed to the final revision of the manuscript.

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17. Ministe´rio da Sau´de. Secretaria de Vigilaˆncia em Sau´de (SVS) e Instituto Nacional do Caˆncer (Inca): Inque´rito domiciliar de comportamentos de risco de morbidade referida de doenc¸as e agravos na˜o transmissı´veis. DATASUS – TABNET. Indicadores e Dados Ba´sicos. Brasil, 2006. Internet: www.datasus.gov.br. 18. Carsen CG, III, Elmore J, Franklin DP, Thomas DD, Mordan F, Wood GC. Use of limits color-flow duplex for a carotid screening project. Am J Surg. 1999;178(2):174-7. 19. Shirani S, Boroumand MA, Abbasi SH, Maghsoodi N, Shakiba M, Karimi A, et al. Preoperative carotid artery screening in patients undergoing coronary artery bypass graft surgery. Arch Med Res. 2006;37(8):987-90, http://dx.doi.org/10.1016/j.arcmed.2006.06.001. 20. Valentine RJ, Hagino RT, Boyd PI, Kakish HB, Clagett GP. Utility of carotid duplex in young adults with lower extremity atherosclerosis: How aggressive should we be in screening young patients? Cardiovasc Surg. 1997;5(4):408-13, http://dx.doi.org/10.1016/S0967-2109(97)000306. 21. Jackson VP, Bendick PJ. Duplex ultrasound screening for carotid arteriosclerotic disease in asymptomatic patients. J Ultrasound Med. 1985;4(8):411-5. 22. Klop RBJ, Eikelboom BC, Taks ACJM. Screening of the internal carotid arteries in patients with peripheral vascular disease by colour-flow duplex scanning. Eur J Vasc Surg. 1991;5(1):41-5, http://dx.doi.org/ 10.1016/S0950-821X(05)80925-9. 23. Ahn SS, Baker JD, Walden K, Moore WS. Which asymptomatic patients should undergo routine screening carotid duplex scan? Am J Surg. 1991;162(2):180-3, http://dx.doi.org/10.1016/0002-9610(91)90184-F. 24. Cheng SWK, Wu LLH, Ting ACW, Lau H, Wong J. Screening for asymptomatic carotid stenosis in patients with peripheral vascular disease: A propsective study and risk factor analysis. Cardiovasc Surg. 1999;7(3):303-9, http://dx.doi.org/10.1016/S0967-2109(98)00115-X. 25. de Virgilio C, Toosie K, Arnell T, Lewis RJ, Donayre CE, Baker JD, et al. Asymptomatic carotid artery stenosis screening in patients with lower extremity atherosclerosis in patients with lower extremity atherosclerosis: A prospective study. Ann Vasc Surg. 1997;11(4):374-7, http:// dx.doi.org/10.1007/s100169900063. 26. Miralles M, Corominas A, Cotillas J, Castro F, Clara A, Vidal-Barraquer F. Screening for carotid and renal artery stenoses in patients with aortoiliac disease. Ann Vasc Surg. 1998;12(1):17-22, http://dx.doi.org/ 10.1007/s100169900109. 27. Budaj A, Flasinska K, Gore JM, Anderson FA Jr, Dabbous OH, Spencer FA, et al. Magnitude of and risk factors for in-hospital and postdischarge stroke in patients with acute coronary syndromes: Findings from a Global Registry of Acute Coronary Events. Circulation. 2005;111(24):3242-7, http://dx.doi.org/10.1161/CIRCULATIONAHA.104.512806. 28. Sutton KC, Dai WS, Kuller LH. Asymptomatic carotid artery bruits in a population of elderly adults with isolated systolic hypertension. Stroke. 1985;16(5):781-4, http://dx.doi.org/10.1161/01.STR.16.5.781. 29. Sutton KC, Wolfson SK Jr, Kuller LH. Carotid and lower extremity arterial disease in elderly adults with isolated systolic hypertension. Stroke. 1987;18(5):817-22, http://dx.doi.org/10.1161/01.STR.18.5.817. 30. Lavenson Jr GS. A new accurate, rapid and cost-effective protocol for stroke-prevention screening. Cardiovasc Surg. 1998;6(6):590-3, http:// dx.doi.org/10.1016/S0967-2109(98)00083-0.

4. Zhu C, Norris JW. Role of carotid stenosis in ischemic stroke. Stroke. 1990;21(8):1131-4, http://dx.doi.org/10.1161/01.STR.21.8.1131. 5. Barnett HJ, Eliasziw M, Meldrum HE, Taylor DW. Do the facts and figures warrant a 10-fold increase in the performance of carotid endarterectomy on asymptomatic patients? Neurology. 1996;46(3):6038, http://dx.doi.org/10.1212/WNL.46.3.603. 6. Roederer GO, Langlois YE, Jager KA, Primozich JF, Beach KW, Phillips DJ, et al. The natural history of carotid arterial disease in asymptomatic patients with cervical bruits. Stroke. 1984;15(4):605-13, http:// dx.doi.org/10.1161/01.STR.15.4.605. 7. Chambers BR, Dinnan GA. Carotid endarterectomy for asymptomatic carotid stenosis. Cochrane Database Syst Rev. 2005;19(4):CD001923. 8. Jacobowitz GR, Rockman CB, Gagne PJ, Adelman MA, Lamparello PJ, Landis R, et al. A model for predicting occult carotid artery stenosis: Screening is justified in a selected population. J Vasc Surg. 2003; 38(4):705-9, http://dx.doi.org/10.1016/S0741-5214(03)00730-4. 9. Mineva PP, Manchev IC, Hadjev DI. Prevalence and outcome of asymptomatic carotid stenosis: A population-based ultrasonography study. Eur J Neurol. 2002;9(4):383-8, http://dx.doi.org/10.1046/j.14681331.2002.00423.x. 10. Qureshi AI, Janardhan V, Benneett SE, Luft AR, Hopkins LN, Guterman LR. Who should be screened for asymptomatic carotid artery stenosis? Experience from the Western New York stroke screening program. J Neuroimaging. 2001;11(2):105-11, http://dx.doi.org/10.1111/j.15526569.2001.tb00019.x. 11. Yin D, Carpenter JP. Cost-effectiveness of screening for asymptomatic carotid stenosis. J Vasc Surg. 1998;27(2):245-55, http://dx.doi.org/ 10.1016/S0741-5214(98)70355-6. 12. Qureshi AI, Alexandrov AV, Tegeler CH, Hobson RW, Baker JD, Hopkins LN. Guidelines for screening of extracranial carotid artery disease: A statement for healthcare professionals from the Multidisciplinary Practice Guidelines Committee of the American Society of Neuroimaging; Cosponsered by the Society of Vascular ans Interventional Neurology. J Neuroimaging. 2007;17(1):19-47, http:// dx.doi.org/10.1111/j.1552-6569.2006.00085.x. 13. Morales MM, Anacleto A, Buchdid MA, Simeoni PR, Ledesma S, Ceˆntola C, et al. Morphological and hemodynamic patterns of carotid stenosis treated by endarterectomy with patch closure versus stenting: a duplex ultrasound study. Clinics. 2010;65(12):1315-1323, http://dx.doi.org/ 10.1590/S1807-59322010001200015. 14. V Diretrizes Brasileiras de Hipertensa˜o Arterial. Arq. Bras. Cardiol. [serial on the Internet]. 2007 Sep [cited 2012 Mar 16]; 89(3):e24-e79. Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid= S0066782X2007001500012&lng=en. http://dx.doi.org/10.1590/S0066782X2007001500012. 15. Diretrizes da Sociedade Brasileira de Diabetes - 2008. [Internet data]. Sa˜o Paulo: Sociedade Brasileira de Diabetes. Access 2012, mar, 16. Available in: http://www.diabetes.org.br/attachments/diretrizes-sbd-2008-mar12.pdf. 16. III Diretrizes Brasileiras Sobre Dislipidemias e Diretriz de Prevenc¸a˜o da Aterosclerose do Departamento de Aterosclerose da Sociedade Brasileira de Cardiologia (2001). [Internet data]. Sa˜o Paulo: Sociedade Brasileira de Cardiologia. Access 2012, Mar, 16. Available in: http://publicacoes. cardiol.br/consenso/2001/77Supl-III/default.asp.

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DOI:10.6061/clinics/2012(08)03

CLINICAL SCIENCE

Sexual function after anterior vaginal wall prolapse surgery Paulo Cezar Feldner Jr., Carlos Antonio Delroy, Se´rgio Brasileiro Martins, Rodrigo Aquino Castro, Marair Gracio Ferreira Sartori, Manoel Joa˜o Batista Castello Gira˜o Universidade Federal de Sa˜o Paulo, Department of Gynecology, Section of Urogynecology and Pelvic Surgery, Sa˜o Paulo/SP, Brazil.

OBJECTIVE: The aim of this study was to compare female sexual function after surgical treatment of anterior vaginal prolapse with either small intestine submucosa grafting or traditional colporrhaphy. METHODS: Subjects were randomly assigned, preoperatively, to the small intestine submucosa graft (n = 29) or traditional colporrhaphy (n = 27) treatment group. Postoperative outcomes were analyzed at 12 months. The Female Sexual Function Index questionnaire was used to assess sexual function. Data were compared with independent samples or a paired Student’s t-test. ClinicalTrials.gov: NCT00827528. RESULTS: In the small intestine submucosa group, the total mean Female Sexual Function Index score increased from 15.5¡7.2 to 24.4¡7.5 (p,0.001). In the traditional colporrhaphy group, the total mean Female Sexual Function Index score increased from 15.3¡6.8 to 24.2¡7.0 (p,0.001). Improvements were noted in the domains of desire, arousal, lubrication, orgasm, satisfaction, and pain. There were no differences between the two groups at the 12month follow-up. CONCLUSIONS: Small intestine submucosa repair and traditional colporrhaphy both improved sexual function postoperatively. However, no differences were observed between the two techniques. KEYWORDS: Sexual Function; Pelvic Organ Prolapse; SIS Graft. Feldner Jr PC, Delroy CA, Martins SB, Castro RA, Sartori MG, Gira˜o MJ. Sexual function after anterior vaginal wall prolapse surgery. Clinics. 2012;67(8):871-875. Received for publication on March 30, 2012; First review completed on April 2, 2012; Accepted for publication on April 3, 2012 E-mail: pfeldner@alfa.epm.br Tel.: 55 115573-9228

repairs (10,12). The anterior compartment is the most common site of recurrence, with failure rates as high as 40% (12). Despite anatomical and functional improvements, Pauls et al. (13) showed that sexual function was unchanged after vaginal reconstructive surgery. They concluded that the lack of benefit may be attributable to postoperative dyspareunia. This is a controversial issue, as some authors have shown improvements (14,15) and others have not (16). Despite the widespread use of mesh in surgery to correct prolapse, few safety and efficacy studies have been published. Therefore, the use of mesh during vaginal repair procedures remains controversial. Uncontrolled studies have reported significant problems associated with the use of mesh for vaginal repair (17). Previous studies are difficult to assess and compare for several reasons, namely the inconsistent use of primarily unvalidated, self-made questionnaires; the lack of a definition for sexual function and dysfunction; and the failure to assess impacts on quality of life (18). The aim of this study was to assess sexual function using a validated sexual function questionnaire administered following vaginal surgery for anterior vaginal wall prolapse.

INTRODUCTION Female Sexual Dysfunction (FSD) is multifactorial and involves physical, social, and psychological dimensions. FSD is defined as disorders of desire, arousal, orgasm, or pain that could cause emotional distress (1). Earlier studies have shown that this problem affects 39-53% of women in the general population (2-4). Women with pelvic floor disorders (PFDs), such as pelvic organ prolapse (POP) and stress urinary incontinence (SUI), commonly have problems related to sexual function (5-7). More than 225,000 surgical procedures are performed for POP each year in the USA, and the estimated cost of these surgeries is over US $1 billion (8-10). The lifetime risk of prolapse surgery has been estimated to be approximately 11%, and 30% of patients undergo re-operation for recurrent prolapse (11). In the USA, approximately 150,000 surgeries are performed every year for cystocele and/or rectocele

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.

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This study was a prospective and randomized study that was approved by a local ethics committee and was registered at ClinicalTrials.gov (NCT00827528). The primary outcome was the anatomical success of the surgery, which was defined as the absence, at the 12-month follow-up, of POP-Q stage II or higher prolapse (point Ba). The secondary objective was to measure quality-of-life outcomes and sexual function. The two surgical techniques, the outcomes regarding anatomical cure, the P-QoL questionnaire and the complications have been previously reported (24). Similar to Pauls’ (13) retrospective analysis, a sample size of 18 women in each group was chosen to detect a difference of 5 points in the FSFI score with 90% power. All numerical data were expressed as the means¡standard deviations. To detect preoperative intergroup differences, we used a Mann-Whitney U test for continuous variables and a chi-square test for categorical variables. Data were normally distributed, which enabled the use of an independent samples t-test to assess the postoperative difference between the two groups and a paired Student’s t-test to assess the same group before and after the surgery. The level of significance was 0.05.

MATERIALS AND METHODS We included pre- and post-menopausal women who were referred for vaginal surgery and who had at least stage II anterior vaginal wall prolapse (point Ba$+1). Patients were randomly assigned to one of two groups: SIS graft or traditional anterior colporrhaphy. Subjects were excluded if they had undergone pelvic radiotherapy or if they had pelvic sepsis, gynecologic cancer, vulvovaginal infections, a current history of smoking or alcoholism, any chronic disabling diseases, or hypertension. All patients underwent a standard physical examination that included pelvic organ prolapse quantification (POP-Q) (19). The examination included POP-Q in the gynecological position with an empty bladder. A Valsalva maneuver or cough demonstrated the maximum descent of the involved pelvic organ. Preoperatively and at 12 months postoperatively, patients were asked to complete the Female Sexual Function Index (FSFI) questionnaire (20), which was translated to Portuguese and validated for Brazilian women (21,22). The FSFI is used to investigate problems with sexual function in the previous four weeks. The FSFI is a comprehensive, 19-item tool that assesses six domains of sexual function, namely desire, arousal, lubrication, orgasm, satisfaction, and pain. The questionnaire provides a comprehensive assessment of baseline and postoperative changes. To calculate each domain score, the scores of the related items are added, and the result is multiplied by a coefficient. Within the domains, a score of zero indicates that the patient reported no sexual intercourse in the previous four weeks. Consequently, the maximum domain score is 6, and the lowest score is 0 for four domains and 0.8 and 1.2, respectively, for two domains. The total FSFI score is calculated by adding the mean scores of all six domains. Higher scores indicate better sexual function. Total FSFI scores range from a low of 2 to a high of 36. The FSFI is self-administered. Multichannel urodynamics were performed on subjects who had urinary incontinence prior to the surgery. We used clinical patterns to evaluate menopausal status; namely, women who reported current and regular periods were defined as premenopausal, while women who reported more than 12 months of amenorrhea were defined as postmenopausal. We used methods and definitions from the International Continence Society subcommittee for the standardization of terminology (23). Figure 1 shows a flow chart of the progression of patients through the study. All eligible women who agreed to participate in the study and who provided written informed consent were enrolled. After they were enrolled by a physician investigator, subjects were assigned to the two treatment groups: SIS and traditional repair. One week before surgery, individuals were randomized by a computer-generated list that was prepared by the Biostatistics Center at the Federal University of Sa˜o Paulo. The list was centrally maintained. There were no drop-outs in the follow-up, and the data were analyzed with an intentionto-treat analysis. The preoperative assessments were conducted by one investigator. After the surgery, the examiners were blinded to the surgical intervention that each subject received. The postoperative follow-ups were performed by three investigators who were also blinded to the group assignments.

RESULTS Preoperatively, the groups were homogeneous with respect to age, body mass index, parity, hormonal status, stage of anterior prolapse, previous surgery for prolapse, previous stress urinary incontinence and previous hysterectomy (Table 1). Scores in the FSFI domains were also homogeneous preoperatively (Table 2). There were 23 (79.3%) sexually active patients in the SIS group and 22 (81.4%) in the TC group. All of the patients were in heterosexual relationships. At the 12-month follow-up, both groups had significantly improved sexual quality of life. In the SIS group, the total mean FSFI score increased from 15.5¡7.2 to 24.9¡7.5 (p,0.001). In the TC group, the total mean FSFI score increased from 15.3¡6.8 to 24.2¡7.0 (p,0.001). Statistically significant improvements were noted in all domains, including desire, arousal, vaginal lubrication, ability to achieve orgasm, sexual satisfaction, and pain. However, there were no differences between the groups (Table 2). Women were also questioned, preoperatively and at 12 months, about dyspareunia. At the 12-month follow-up, 5 of the 29 (17.2%) patients in the SIS group and 4 of the 27 (14.8%) patients in the traditional repair group reported dyspareunia (p = 0.90). There were no infections or graft erosions that could have caused the dyspareunia.

DISCUSSION FSD has become a popular research area because of its importance to quality of life. However, the routine identification of FSD is still lacking (25). Surgery for prolapse and urinary incontinence does not necessarily ensure optimal sexual function, and conflicting results have been reported (13-16). Using the FSFI at 12 months after the operation, our results showed that SIS graft repair and traditional colphorraphy resulted in improvements in sexual quality of life; however, no differences were observed between the two techniques.

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Prolapse surgery and sexual function Feldner Jr PC et al.

Figure 1 - Flow chart of patient progression through the study.

There are few studies on the sexual outcomes of anterior colporrhaphy compared with graft-augmented repair. Some authors have agreed that cystocele repair is not associated with dyspareunia or impaired sexual function, but this issue is controversial in the literature (26). Anterior repair appears to have a negative effect on sexual function only when it is combined with another procedure. Colombo et al. assessed 23 women who had an anterior repair and found that 56% had mild-to-severe postoperative dyspareunia; however, the patients also had a posterior repair and perineorrhaphy (26,27). Data from one trial suggested a reduction in dyspareunia, from 30% preoperatively to 22% postoperatively, in a comparison of three surgical techniques of anterior colporrhaphy (28). Another trial compared anterior colporrhaphy with and without porcine dermis inlay (Pelvicol). At the one-year follow-up, there were no differences in dyspareunia between the groups (29). Graft anterior repair exposure has ranged from 0 to 30%; however, the data regarding dyspareunia and sexual dysfunction are insufficient (17). Jia et al. reported that 7.1-12.8% of patients had de novo dyspareunia after surgical

Table 1 - Baseline data. Values are presented as the means (¡standard deviation) or the number of subjects (n).

Age (y) Parity Body mass index Hormonal status Postmenopausal Premenopausal POP-Q Stage II III IV Prior POP surgery Prior SUI surgery Prior hysterectomy

SIS (n = 29)

Traditional repair (n = 27)

p-value

53.8¡9.7 4.3¡1.8 27.3¡4.9

56.3¡13.0 4.0¡2.1 27.5¡4.5

0.42 0.68 0.89

19 (65.5%) 10 (34.5%)

13 (48.2%) 14 (51.8%)

0.44

9 (31.0%) 19 (65.5%) 1 (3.5%) 7 (24.1%) 5 (17.2%) 3 (10.3%)

13 (48.2%) 12 (44.4%) 2 (7.4%) 7 (25.9%) 3 (11.1%) 1 (3.7%)

0.27 0.87 0.78 0.65

POP-Q: pelvic organ prolapse quantification. SUI: stress urinary incontinence.

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not specifically designed to assess changes in sexual health that are caused by PFDs. Therefore, it may not be sensitive to meaningful changes in sexual function in our population. Additionally, the questionnaires do not screen for sexual activity, which may underestimate the impact of PFDs on sexual function, as women with severe dysfunction may elect to become sexually inactive (18). Rigorous RCTs are required to determine the comparative efficacy of grafts and their optimal place in clinical practice. Functional results are important as outcome measures of anatomical results in the assessment of pelvic floor surgery. Following pelvic floor surgery, sexual function assessment tools are needed to define clear outcomes for sexual function.

Table 2 - Preoperative and postoperative FSFI scores of women who underwent SIS grafting or traditional anterior repair. Values are given as the means (¡ standard deviation).

Desire Pre-op Post-op p-value** Arousal Pre-op Post-op p-value** Lubrication Pre-op Post-op p-value** Orgasm Pre-op Post-op p-value** Satisfaction Pre-op Post-op p-value** Pain Pre-op Post-op p-value** Total Pre-op Post-op p-value** *

SIS graft

Traditional repair

p-value*

2.5¡1.2 4.1¡1.5 ,0.001

2.6¡1.1 3.9¡1.6 ,0.001

0.38 0.65

2.7¡0.9 4.3¡1.1 ,0.001

2.8¡0.7 4.2¡1.0 ,0.001

0.48 0.67

2.8¡0.8 4.1¡1.0 ,0.001

2.8¡1.0 4.2¡1.0 ,0.001

0.35 0.88

ACKNOWLEDGMENTS

2.3¡1.2 4.0¡0.9 ,0.001

2.2¡1.1 4.1¡1.0 ,0.001

0.55 0.74

AUTHOR CONTRIBUTIONS

2.1¡0.9 4.0¡1.0 ,0.001

2.0¡0.8 4.0¡1.0 ,0.001

0.25 0.92

3.1¡1.1 3.9¡1.1 ,0.001

2.9¡1.0 3.8¡1.2 ,0.001

0.90 0.85

15.5¡7.2 24.4¡7.5 ,0.001

15.3¡6.8 24.2¡7.0 ,0.001

0.76 0.65

The authors thank Handle Cook for providing the SIS grafts.

All of the authors qualify for authorship, as they all contributed substantially to the work by contributing to the conception and design, acquisition, analysis and interpretation of the data, draft of the manuscript, critical revision for important content and approval of the final version of the manuscript.

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Independent samples t-test. Paired Student’s t-test.

**

repair of POP using graft augmentation (30). Foon et al. (31) concluded that erosions and dyspareunia were common adverse events in the surgical treatment of anterior vaginal wall prolapse with adjuvant materials. In a cross-sectional study using the FSFI questionnaire, Najafabady et al. (32) assessed the prevalence and associated factors of anorgasmia among 1,200 reproductive-age Iranian women. Compared with the normal orgasm group, the authors found that 26.3% of the anorgasmic and most of the anorgasmic women were highly unsatisfied with their sexual relationship. There were several limitations to the present study. The study was initially intended for a 12-month follow-up. A longer follow-up could potentially identify changes in sexual function and could demonstrate a possible degradation of SIS, which has been noted in previous SIS slings used for stress urinary incontinence. The sample size was calculated based on the primary objective of achieving an anatomical cure. The sample size may have been inadequate to detect small differences between the groups, and the power calculation was suboptimal, especially for the FSFI scores. Although the surgical procedure was standardized, there may have been variations in the surgical technique that was employed by different surgeons. However, this would more closely represent expected outcomes in actual clinical practice compared with trials employing only one surgeon. Another important matter is that the FSFI questionnaire that was utilized to assess the sexual function in women with PFDs has some limitations. The FSFI questionnaire is

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Prolapse surgery and sexual function Feldner Jr PC et al.

15. Weber AM, Walters MD, Piedmonte MR. Sexual function and vaginal anatomy in women before and after surgery for pelvic organ prolapse and urinary incontinence. Am J Obstet Gynecol. 2000;182(6):1610-5, http://dx.doi.org/10.1067/mob.2000.107436. 16. Helstrom L, Nilsson B. Impact of vaginal surgery on sexuality and quality of life in women with urinary incontinence or genital descensus. Acta obstetricia et gynecologica Scandinavica. 2005;84(1):79-84. 17. Maher C, Baessler K, Glazener CMA, Adams EJ, Hagen S. Surgical management of pelvic organ prolapse in women: A short version Cochrane review. Neurourol Urodyn. 2008;27(1):3-12, http:// dx.doi.org/10.1002/nau.20542. 18. Pons ME. Sexual health in women with pelvic floor disorders: measuring the sexual activity and function with questionnaires-a summary. Int Urogynecol J. 2009;20:S65-S71, http://dx.doi.org/10.1007/s00192-009-0828-7. 19. Bump RC, Mattiasson A, Bo K, Brubaker LP, DeLancey JOL, Klarskov P, et al. The standardization of terminology of female pelvic organ prolapse and pelvic floor dysfunction. Am J Obstet Gynecol. 1996;175(1):10-7, http://dx.doi.org/10.1016/S0002-9378(96)70243-0. 20. Rosen R, Brown C, Heiman J, Leiblum S, Meston C, Shabsigh R, et al. The Female Sexual Function Index (FSFI): A multidimensional self-report instrument for the assessment of female sexual function. J Sex Marital Ther. 2000;26(2):191-208. 21. Thiel Rdo R, Dambros M, Palma PC, Thiel M, Riccetto CL, Ramos Mde F. [Translation into Portuguese, cross-national adaptation and validation of the Female Sexual Function Index]. Rev Bras Ginecol Obstet. 2008;30(10): 504-10, http://dx.doi.org/10.1590/S0100-72032008001000005. 22. Hentschel H, Alberton DL, Capp E, Goldim JR, Passos EP. Validac¸a˜o do Female Sexual Function Index (FSFI) para uso em portugueˆs. Rev HCPA. 2007;27(1):10-4. 23. Haylen BT, de Ridder D, Freeman RM, Swift SE, Berghmans B, Lee J, et al. An International Urogynecological Association (IUGA)/International Continence Society (ICS) joint report on the terminology for female pelvic floor dysfunction. Int Urogynecol J. 2010;21(1):5-26, http:// dx.doi.org/10.1007/s00192-009-0976-9. 24. Feldner PC, Castro RA, Cipolotti LA, Delroy CA, Sartori MGF, Girao MJBC. Anterior vaginal wall prolapse: a randomized controlled trial of

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SIS graft versus traditional colporrhaphy. Int Urogynecol J. 2010;21(9):1057-63, http://dx.doi.org/10.1007/s00192-010-1163-8. Bekker M, Beck J, Putter H, van Driel M, Pelger R, Nijeholt ALA, et al. The Place of Female Sexual Dysfunction in the Urological Practice: Results of a Dutch Survey. J Sex Med. 2009;6(11):2979-87, http:// dx.doi.org/10.1111/j.1743-6109.2009.01460.x. Achtari C, Dwyer PL. Sexual function and pelvic floor disorders. Best Pract Res Cl Ob. 2005;19(6):993-1008, http://dx.doi.org/10.1016/ j.bpobgyn.2005.08.012. Colombo M, Vitobello D, Proietti F, Milani R. Randomised comparison of Burch colposuspension versus anterior colporrhaphy in women with stress urinary incontinence and anterior vaginal wall prolapse. Brit J Obstet Gynaec. 2000;107(4):544-51, http://dx.doi.org/10.1111/ j.1471-0528.2000.tb13276.x. Weber AM, Walters MD, Piedmonte MR, Ballard LA. Anterior colporrhaphy: A randomized trial of three surgical techniques. Am J Obstet Gynecol. 2001;185(6):1299-304, http://dx.doi.org/10.1067/ mob.2001.119081. Meschia M, Pifarotti P, Bernasconi F, Magatti F, Riva D, Kocjancic E. Porcine skin collagen implants to prevent anterior vaginal wall prolapse recurrence: A multicenter, randomized study. J Urology. 2007;177(1):1925, http://dx.doi.org/10.1016/j.juro.2006.08.100. Jia X, Glazener C, Mowatt G, MacLennan G, Bain C, Fraser C, et al. Efficacy and safety of using mesh or grafts in surgery for anterior and/or posterior vaginal wall prolapse: systematic review and meta-analysis. Bjog-Int J Obstet Gy. 2008;115(11):1350-61, http://dx.doi.org/10.1111/ j.1471-0528.2008.01845.x. Foon R, Toozs-Hobson P, Latthe PM. Adjuvant materials in anterior vaginal wall prolapse surgery: a systematic review of effectiveness and complications. Int Urogynecol J. 2008;19(12):1697-706, http:// dx.doi.org/10.1007/s00192-008-0668-x. Najafabady MT, Salmani Z, Abedi P. Prevalence and related factors for anorgasmia among reproductive aged women in Hesarak, Iran. Clinics. 2011;66(1):83-6, http://dx.doi.org/10.1590/S1807-59322011 000100015.


CLINICS 2012;67(8):877-883

DOI:10.6061/clinics/2012(08)04

CLINICAL SCIENCE

The effects of angiotensin-converting enzyme inhibitors on peritoneal protein loss and solute transport in peritoneal dialysis patients Taner Basturk,I Abdulkadir Unsal,I Yener Koc,I Eren Nezaket,II Elbis Ahbap,I Tamer Sakaci,I Mustafa SevincI I

Department of Nephrology, Sisli Etfal Research and Education Hospital, Istanbul, Turkey. II Department of Biochemistry, Sisli Etfal Research and Education Hospital, Istanbul, Turkey.

OBJECTIVE: The objective of this study was to examine the effects of angiotensin-converting enzyme inhibitors on peritoneal membrane transport, peritoneal protein loss, and proteinuria in peritoneal dialysis patients. METHODS: Fifty-four peritoneal dialysis patients were included in the study. The patients were divided into two groups. Group 1 (n = 34) was treated with angiotensin-converting enzyme inhibitors. Group 2 (n = 20) did not receive any antihypertensive drugs during the entire follow-up. Eleven patients were excluded from the study thereafter. Thus, a total of 30 patients in Group 1 and 13 patients in Group 2 completed the study. We observed the patients for six months. Group 1 patients received maximal doses of angiotensin-converting enzyme inhibitors for six months. Parameters at the beginning of study and at the end of six months were evaluated. ClinicalTrial.gov: NCT01575652. RESULTS: At the end of six months, total peritoneal protein loss in 24-hour dialysate effluent was significantly decreased in Group 1, whereas it was increased in Group 2. Compared to the baseline level, peritoneal albumin loss in 24-hour dialysate effluent and 4-hour D/P creatinine were significantly increased in Group 2 but were not significantly changed in Group 1. A covariance analysis between the groups revealed a significant difference only in the decreased amount of total protein loss in 24-hour dialysate. Proteinuria was decreased significantly in Group 1. CONCLUSION: This study suggests that angiotensin-converting enzyme inhibitors reduce peritoneal protein loss and small-solute transport and effectively protect peritoneal membrane transport in peritoneal dialysis patients. KEYWORDS: Angiotensin-converting enzyme inhibitors, peritoneal protein loss, peritoneal transport. Basturk T, Unsal A, Koc Y, Nezaket E, Ahbap E, Sakaci T, et al. The effects of angiotensin-converting enzyme inhibitors on peritoneal protein loss and solute transport in peritoneal dialysis patients. Clinics. 2012;67(8):877-883. Received for publication on November 28, 2011; First review completed March 5, 2012; Accepted for publication on April 2, 2012 E-mail: tanerbast@yahoo.com Tel.: 90 5059271171

blood vessels, and reduplication of the vascular basement membrane (4). The use of angiotensin-converting enzyme inhibitors (ACE-Is) in kidney disease has been demonstrated to be effective in reducing proteinuria and slowing the progression of kidney disease. The main mechanism of the antiproteinuric action of ACEIs is the reduction of the negative effects of angiotensin II on kidney hemodynamics (5). ACE-Is can preserve peritoneal histology, peritoneal function, and mesothelial cell remodeling (6). Based on these beneficial effects of ACE-Is on proteinuria, we aimed to investigate the effects of ACE-Is on peritoneal membrane transport, peritoneal protein loss, and proteinuria in patients being treated with PD.

INTRODUCTION Ever since peritoneal dialysis (PD) has been used in the treatment of chronic kidney disease (CKD), high peritoneal protein loss has been observed after each PD exchange. In adult patients, the loss has been estimated at 6 to 13 g daily (1). PD patients lose significant quantities of protein and albumin during the dialysis procedure (2). Low serum albumin has been associated with high peritoneal membrane transport status and also correlates with mortality (3). Loss of peritoneal function is a major complication associated with long-term peritoneal dialysis. Changes observed include loss and degeneration of the mesothelium, submesothelial thickening, alterations in the structure and number of

PATIENTS AND METHODS Study design and patients

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.

This prospective cohort study was conducted at the Unit of Nephrology of Sisli Etfal Education and Research Hospital, Istanbul, Turkey. Prior to subject recruitment, the study protocol was reviewed and approved by the local

No potential conflict of interest was reported.

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protein, and salt-restricted diet. Patients did not use essential amino acids and peritoneal dialysis solutions containing amino acids. Serum urea, creatinine, and albumin levels were assessed by enzymatic colorimetric assay. Dialysate adequacy (Kt/V urea: dialysis and residual) and peritoneal transport (4-hour D/PCr) were measured using standard procedures (PD Adequest 1.4, 1994: Baxter Healthcare Corporation, Deerfield, IL, USA). Dialysate albumin loss was measured with the Bromo CresolGreen (BCG) method. Dialysate total protein loss was measured with the Biuret method. Urine protein concentration was determined with an immunoturbidimetric method.

ethics committee in accordance with the ethical principles for human investigations, and written informed consents were obtained from all patients. Between June 2008 and January 2009, 54 age- and gender-matched continuous ambulatory peritoneal dialysis (CAPD) patients were included in the study consecutively. Patients were divided into two groups according to decision of the physician: Group 1 (n = 34) consisted of patients treated with ACE-Is, and Group 2 (n = 20) consisted of patients not treated with ACE-Is. The inclusion criteria were chronic PD patients between 18 and 85 years old who had not received any antihypertensive drugs within the previous 12 months. All patients were on a standard CAPD program (2-2.5 L; 4 exchanges/day) without the use of icodextrin. The exclusion criteria were as follows: patients who had a history of antihypertensive treatment with ACE-Is or angiotensin-receptor blockers or aldosterone antagonists for the 12 months prior to the study time, intolerance to ACEIs, CAPD-related peritonitis within six months prior to or during the study period, history of malignant hypertension or hypertensive encephalopathy, or cerebrovascular accident within the six months prior to the study, chronic liver disease, and recent acute illness and/or history of any overt chronic inflammatory disease. We excluded four patients from Group 1. One patient developed peritonitis during the study; two patients could not tolerate the medication because of hypotension and cough; one patient underwent renal transplantation. In Group 2, seven patients were excluded. Two patients developed peritonitis; two patients could not tolerate the medication due to hypotension and cough; two patients had uncontrolled hypertension; one patient did not attend follow-up visits. Thus, 30 patients in Group 1 and 13 patients in Group 2 completed the six-month study. The type of ACE-I prescribed to the patients was chosen randomly. Group 1 patients received the maximum tolerated doses of ACE-Is (such as lisinopril and perindopril) for six months.

Statistical analysis Statistical analysis was performed using the SPSS 13.0 software package (SPSS Inc., Chicago, IL, USA). KolmogorovSmirnov tests were used to test the normality of data distribution. The data were expressed as arithmetic means and standard deviations. The chi-squared test was used to compare the categorical variables between groups. Independent sample T-tests and Mann-Whitney U tests were used between groups for normally and abnormally distributed continuous variables, respectively. Paired t-tests and Wilcoxon signed-rank tests were used to analyze changes within each group. A two-sided p-value ,0.05 was considered to be statistically significant.

RESULTS The baseline clinical, laboratory, and demographic characteristics of patients are presented in Table 1. There were Table 1 - Patient Characteristics.

Gender (Male/Female) Age (years) Mean duration of PD (months) Weekly Kt/V urea Weekly CCr * Systolic BP (mmHg)* Diastolic BP (mmHg)* Serum albumin (g/dl) 4-hour D/P creatinine* 24-hour peritoneal UF (ml) 4-hour peritoneal UF (ml) Proteinuria (mg/day) Diuresis (ml/day)

Baseline definitions, measurements, and biochemical analysis Demographic variables, including the etiology of CKD, age, and gender, were obtained from patients’ clinical charts. All blood samples were taken after 10 hours of overnight fasting. Serum urea, creatinine, and albumin levels were analyzed. Creatinine clearance [(CCr) dialysate, urine, and total] and Kt/V (dialysate, urine, and total) were calculated weekly. Daily volumes (UF), 24-hour protein, and albumin losses (dialysate, urine) were also recorded. Parameters at the beginning of study and at the end of the sixth month were evaluated. During the study, the dialysis regime remained the same for all patients. In both groups, we analyzed blood, 24-hour urine (in patients with residual diuresis .100 mL daily), and peritoneal effluent fluid at 4and 24-hour dwell times. Peritoneal effluent fluid at the 24hour dwell time was used to determine total protein, albumin, urea and creatinine. The urea kinetic test in closest proximity to the time of the peritoneal equilibration test (PET) was used in the analysis. After the subject had rested in the supine position for at least 15 minutes, blood pressure was measured with a standard mercury sphygmomanometer three times with the cuff around the right arm. Patients’ blood pressure measurements were taken on a regular basis every month. The mean values were calculated. All patients were on a standard 35-cal/kg/day carbohydrate, 1-2 g/kg/day

Primary kidney disease Diabetic nephropathy Chronic glomerulonephritis Chronic pyelonephritis Polycystic renal disease Obstructive nephropathy Drug use Amyloidosis Unknown

Group 1 (n = 30)

Group 2 (n = 13)

18/12 38.4¡20 21.3¡15.6

6/7 42¡16.4 18.2¡15.8

2.15¡0.43 64.97¡13.6 125¡18.7 76¡10.3 3.7¡0.3 0.66¡0.07 1330¡483

2.21¡0.32 52.3¡14.2 110.7¡21.7 64.6¡15.6 3.8¡0.34 0.54¡0.09 1298¡477

455¡185

465¡97.4

470¡662 453¡539

129.7¡187.8 211¡270

Number of patients Ratio, %

Number of patients Ratio, %

5 3

16.7 10

2 3

15.38 23.07

3 3 2 2 1 11

10 10 6.7 6.7 3.3 36.6

2 3 3

15.38 23.07 23.07

All values are given as the means ¡ standard deviations. NS: nonsignificant, CCr: creatinine clearance, BP: blood pressure, D/P: dialysate/ plasma, UF: daily volumes. * p,0.05.

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Effects of ACE-Is on peritoneal membrane in PD Basturk T et al.

in Group 2. Only the decrease in peritoneal total protein loss at 24 hours of dwell time was significant following ACE-Is treatment (p,0.001). Statistically significant differences were not identified in comparisons of the other studied parameters (p.0.05) (Table 2, Figures 1A, 1B, 3A). In Group 2, at the end of six months, 4-hour D/P creatinine and peritoneal albumin losses at 24 hours of dwell time were increased significantly. Other parameters did not change significantly in Group 2 (p.0.05) (Table 3, Figures 2A, 2B, 3B). After ACE-Is were added to the treatment of the PD patients in Group 1, proteinuria levels were reduced significantly (p = 0.011). Three patients became anuric; thus, the total number of anuric patients increased to twelve at the end of study. Residual renal function was surprisingly increased in eight patients at the end of six months and was decreased in ten patients. In total, the residual renal function decreased in Group 1, but this difference was not statistically significant (p.0.05) (Table 2). In Group 2, proteinuria levels did not change significantly during the study period (p.0.05). Three patients became anuric,

Table 2 - The effects of ACE-I treatment on the measured parameters in Group 1. Parameter Weekly Kt/V urea Weekly CCr Systolic BP (mmHg) Diastolic BP (mmHg) Serum albumin (g/dl) 24-hour peritoneal UF (ml) 4-hour peritoneal UF (ml) Proteinuria (mg/day) Diuresis (ml/day)

Baseline

At the end of six months

p-value

2.15¡0.43 64.97¡13.6 125¡18.7 76¡10.3 3.7¡0.3 1330¡483 455¡185 470¡662 453¡539

2.16¡0.46 69.5¡30. 111¡23.74 70¡11.4 3.74¡0.4 1413¡689 468¡178 224¡362 392¡574

NS NS 0.001 0.003 NS NS NS 0.011 NS

All values are given as the means ¡ standard deviations. NS: nonsignificant, CCr: creatinine clearance, BP: blood pressure, UF: daily volumes.

no significant differences in gender, age, or mean duration of PD between groups (p.0.05 for all) (Table 1). After six months, the decreases in systolic and diastolic blood pressures were statistically significant in Group 1 but not

Figure 1 - A. Total Loss of Protein in Group 1. B. Total Loss of Albumin in Group 1.

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CLINICS 2012;67(8):877-883

decreased, but this difference was not statistically significant (p.0.05) (Table 3). Covariance analysis between groups revealed a significant difference only in the amount of total protein loss in 24hour dialysate in favor of reduction (p = 0.048) in Group 1. No adverse effects, including hyperkalemia, were observed.

Table 3 - The parameters of untreated patients in Group 2. Parameter Weekly Kt/V urea Weekly CCr Systolic BP (mmHg) Diastolic BP (mmHg) Serum albumin (g/dl) 24-hour peritoneal UF (ml) 4-hour peritoneal UF (ml) Proteinuria (mg/day) Diuresis (ml/day)

Baseline

At the end of six months

p-value

2.21¡0.32 52.3¡14.2 110.7¡21.7 64.6¡15.6 3.8¡0.34 1298¡477 465¡97.4 129.7¡187.8 211¡270

2.32¡0.55 59.8¡16.05 111.5¡21.5 67.6¡15.3 3.79¡0.43 1303¡500 465¡129 113¡225 79.1¡218

NS NS NS NS NS NS NS NS NS

DISCUSSION This study demonstrates that treatment with ACE-Is may preserve peritoneal membrane transport and may reduce peritoneal total protein loss and proteinuria in patients with PD. ACE-Is affect the peritoneal membrane by increasing convective transport and decreasing diffusive transport— although in a limited way—and significantly reducing peritoneal protein losses at both 4-hour and 24-hour dwell times. The mechanisms of action of these effects have not been clearly described; however, they may be related to an effect on the permeability of the peritoneal membrane capillaries, where ACE-Is may act directly or indirectly by blocking the renin–angiotensin–aldosterone system (7).

All values are given as the means ¡ standard deviations. NS: nonsignificant, CCr: creatinine clearance, BP: blood pressure, UF: daily volumes.

increasing the number of anuric patients to ten in Group 2 at the end of six months. In addition, residual renal function was decreased in two patients, whereas it was increased in one patient. Overall, the residual renal function was also

Figure 2 - A. Total Loss of Protein in Group 2. B. Total Loss of Albumin in Group 2.

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Effects of ACE-Is on peritoneal membrane in PD Basturk T et al.

Figure 3 - A. 4-hours D/P Creatinine of Group 1. B. 4-hours D/P Creatinine of Group 2.

correlation has not been confirmed in a number of other studies (14,15). Plasma volume expansion should cause the dilution of plasma proteins and a reduction in serum levels. Volume expansion commonly occurs in chronic renal failure, and the degree of fluid overload is associated with the serum albumin concentration (16). In this study, serum albumin concentrations were not significantly changed at the end of six months of treatment with ACE-Is. Although we did not evaluate the volume statuses of our patients, the reason for the stable plasma albumin levels may have been the increased plasma volume with no change in total albumin mass in PD patients. Long-term PD causes morphological changes in the peritoneal membrane, including interstitial fibrosis, loss of the mesothelial cell layer and vasculopathy. One of the potential mechanisms of this damage is the presence of a local renin-angiotensin-aldosterone system (RAAS) by which injured peritoneal mesothelial cell-derived angiotensin-II (AII) causes activations in TGF-b and VEGF expression and epithelial-to-mesenchymal transition (EMT), which contribute to extracellular matrix accumulation and neoangiogenesis in submesothelial tissues. Duman et al. (17) reported that by inhibiting the overexpression of cytokines (i.e., TGFb1 and VEGF), renin–angiotensin system blockade ameliorates the peritoneal injury induced by the hypertonic

Coronel et al. (8) demonstrated that captopril reduced peritoneal albumin loss without a significant change in systemic blood pressure. This effect likely decreases capillary permeability, either by a direct action of the drug or indirectly, mediated by AII, prostaglandins, or kinins. Jearnsujitwimol et al. (9) demonstrated that candesartan could provide a nutritional benefit by attenuating peritoneal loss of albumin and mediates an effective antihypertensive action. In addition, Agraharkar et al. (10) investigated the effect of RAS blockage on peritoneal protein loss in PD patients. These authors concluded that RAS blockage did not reduce protein loss into the peritoneal fluid during dialysis. In our study, peritoneal total protein loss was significantly reduced in patients treated with ACE-Is. However, peritoneal albumin loss did not change significantly. Additionally, peritoneal albumin loss increased significantly in Group 2. Serum albumin is correlated with dietary protein intake in patients with renal disease. Peritoneal dialysis is associated with albumin and amino acid losses in the spent dialysate, which can reach 5–15 g/day. These losses may represent ,15% of the net daily protein intake (11). Kaysen GA et al. (12) reported a strong correlation between serum albumin level and peritoneal protein loss in 18 patients. Pollock CA et al. (13) reported a weak correlation in 134 patients. This

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PD solution. In addition, these authors found that ACEIs or AR blockers (or both) may preserve the viability of the peritoneum in continuous ambulatory PD patients over long periods. The oral administration of enalapril ameliorates changes in peritoneal function and morphology (18), and the oral administration of lisinopril (an ACEI) and valsartan (an AR blocker) has similar beneficial effects on peritoneal function and morphology (19). Multiple studies have shown that ACE-Is prevent peritoneal protein loss (8,9). As a result, ACE-Is have a potential beneficial effect on the prevention of peritoneal fibrosis. Based on these findings, ACE-Is may preserve the viability of the peritoneum in PD patients over the long term. In PD, both in vivo and in vitro studies involving the effects of ACE-Is on peritoneal membrane transporters have yielded different results. Kolesnyk et al. (20) have shown that treatment with ACEI/ARB in PD patients may prevent or retard the increase in D/P creatinine that occurs during long-term PD. In contrast, Jearnsujitwimol et al. (9) demonstrated that candesartan at a dose of 816 mg/day could effectively control blood pressure but caused no changes in peritoneal transport characteristics. We showed that peritoneal transport was not significantly decreased in Group 1 and was significantly increased in Group 2. The results of this study indicate that treatment with ACE-I in PD patients is likely to have a membraneprotective effect by preventing the increase in small-solute transport. Long-term treatment with ACE-Is attenuates the peritoneal alterations that can develop in long-term PD patients. Ultrafiltration failure in patients undergoing PD is a condition with an incidence that increases over time. This complication is related to increased cardiovascular morbidity and mortality and is a major cause of the abandonment of the treatment technique (21). Duman et al. (17) demonstrated that an ACE-I, enalapril, improved ultrafiltration capacity when administered intraperitoneally. This ACE-I appeared to have a slower rate of decline in ultrafiltration, effectively protecting against peritoneal fibrosis in long-term peritoneal dialysis (17). ACE-Is may have an important role in PD by affecting the rate of decline of residual renal function in PD patients. Li et al. (22) investigated the effect of ACE-Is on residual renal function. Those authors concluded that the ACE-I ramipril might reduce the rate of decline of residual renal function in PD patients. At 12 months, 14 patients in the ramipril group (n = 30) and 22 in the control group (n = 30) became anuric. Two randomized controlled trials showed positive effects of A-II inhibitors on residual glomerular filtration rate (rGFR) in peritoneal dialysis patients (22,23). In another study, Kolesnyk et al. (24) found no difference with respect to the rate of decline of rGFR and time of the development of anuria. In our study, there were no significant differences in ultrafiltration volume and residual diuresis between groups, although proteinuria was significantly decreased in Group 1 (p = 0.011). Therefore, the effect of ACE-Is may be important in preserving peritoneal function and in preventing protein losses, such as those experienced by PD patients, in whom considerable quantities of proteins are lost through the peritoneum and diuresis. There are limitations in our study that must be considered. The study groups were not large or well matched due to a limited number of patients. The follow-up period was

also short. The types and dosages of ACE-Is given to the patients were not strictly standardized. Our findings suggest that ACE inhibitors support a decline in peritoneal protein losses and small-solute transport and effectively protect peritoneal membrane transport in long-term peritoneal dialysis.

AUTHOR CONTRIBUTIONS Basturk T was responsible for the project and data collection. Unsal A was responsible for the project and editing the manuscript. Koc Y, Ahbap E, and Sakaci T collected the data and followed up with the patients. Nezaket E collected the data and performed the biochemical analyses. Sevinc M collected the data, followed up with the patients and edited the manuscript.

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Effects of ACE-Is on peritoneal membrane in PD Basturk T et al. 22. Li PKT, Chow KM, Wong TYH, Leung CB, Szeto CC. Effects of an angiotensin-converting renal function in patients receiving enzyme inhibitor on residual peritoneal dialysis - A randomized, controlled study. Annals of internal medicine. 2003;139(2):105-12. 23. Suzuki H, Kanno Y, Sugahara S, Okada H, Nakamoto H. Effects of an angiotensin II receptor blocker, valsartan, on residual renal function in patients on CAPD. Am J Kidney Dis. 2004;43(6):1056-64, http://dx.doi. org/10.1053/j.ajkd.2004.01.019. 24. Kolesnyk I, Noordzij M, Dekker FW, Boeschoten EW, Krediet RT. Treatment with angiotensin II inhibitors and residual renal function in peritoneal dialysis patients. Perit Dial Int. 2011;31(1):53-9, http://dx.doi. org/10.3747/pdi.2009.00088.

18. Duman S, Gunal AI, Sen S, Asci G, Ozkahya M, Terzioglu E, et al. Does enalapril prevent peritoneal fibrosis induced by hypertonic (3.86%) peritoneal dialysis solution? Peritoneal Dialysis International. 2001;21(2):219-24. 19. Duman S, Sen S, Duman C, Oreopoulos DG. Effect of valsartan versus lisinopril on peritoneal sclerosis in rats. International Journal of Artificial Organs. 2005;28(2):156-63. 20. Kolesnyk I, Noordzij M, Dekker FW, Boeschoten EW, Krediet RT. A positive effect of AII inhibitors on peritoneal membrane function in longterm PD patients. Nephrol Dial Transpl. 2009;24(1):272-7. 21. Aguirre AR, Abensur H. Protective measures against ultrafiltration failure in peritoneal dialysis patients. Clinics. 2011;66(12):2151-7, http:// dx.doi.org/10.1590/S1807-59322011001200023.

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DOI:10.6061/clinics/2012(08)05

CLINICAL SCIENCE

Skeletal muscle major histocompatibility complex class I and II expression differences in adult and juvenile dermatomyositis Samuel Katsuyuki Shinjo,I Adriana Maluf Elias Sallum,II Clovis Artur Silva,II Suely Kazue Nagahashi MarieIII I

Faculdade de Medicina da Universidade de Sa˜o Paulo, Division of Rheumatology, Sa˜o Paulo/SP, Brazil. II Faculdade de Medicina da Universidade de Sa˜o Paulo, Pediatric Rheumatology Unit, Sa˜o Paulo/SP, Brazil. III Faculdade de Medicina da Universidade de Sa˜o Paulo, Department of Neurology, Sa˜o Paulo/SP, Brazil.

OBJECTIVE: To analyze major histocompatibility complex expression in the muscle fibers of juvenile and adult dermatomyositis. METHOD: In total, 28 untreated adult dermatomyositis patients, 28 juvenile dermatomyositis patients (Bohan and Peter’s criteria) and a control group consisting of four dystrophic and five Pompe’s disease patients were analyzed. Routine histological and immunohistochemical (major histocompatibility complex I and II, StreptoABComplex/HRP, Dakopatts) analyses were performed on serial frozen muscle sections. Inflammatory cells, fiber damage, perifascicular atrophy and increased connective tissue were analyzed relative to the expression of major histocompatibility complexes I and II, which were assessed as negatively or positively stained fibers in 10 fields (200X). RESULTS: The mean ages at disease onset were 42.0¡15.9 and 7.3¡3.4 years in adult and juvenile dermatomyositis, respectively, and the symptom durations before muscle biopsy were similar in both groups. No significant differences were observed regarding gender, ethnicity and frequency of organ involvement, except for higher creatine kinase and lactate dehydrogenase levels in adult dermatomyositis (p,0.050). Moreover, a significantly higher frequency of major histocompatibility complex I (96.4% vs. 50.0%, p,0.001) compared with major histocompatibility complex II expression (14.3% vs. 53.6%, p = 0.004) was observed in juvenile dermatomyositis. Fiber damage (p = 0.006) and increased connective tissue (p,0.001) were significantly higher in adult dermatomyositis compared with the presence of perifascicular atrophy (p,0.001). The results of the histochemical and histological data did not correlate with the demographic data or with the clinical and laboratory features. CONCLUSION: The overexpression of major histocompatibility complex I was an important finding for the diagnosis of both groups, particularly for juvenile dermatomyositis, whereas there was lower levels of expression of major histocompatibility complex II than major histocompatibility complex I. This finding was particularly apparent in juvenile dermatomyositis. KEYWORDS: Adult Dermatomyositis; Idiopathic Inflammatory Myopathies; Juvenile Dermatomyositis; Major Histocompatibility Complex; Muscle Biopsy. Shinjo SK, Sallum AM, Silva CA, Marie SK. Skeletal muscle major histocompatibility complex class I and II expression differences in adult and juvenile dermatomyositis. Clinics. 2012;67(8):885-890. Received for publication on December 29, 2011; First review completed on February 7, 2012; Accepted for publication on April 2, 2012 E-mail: Samuel.shinjo@gmail.com Tel.: 55 11 3061-7176

The incidence of DM is 5-10 cases per million per year, with the adult form of the disease primarily affecting individuals between 45 and 55 years old, whereas the pediatric form affects children and adolescents between 5 and 10 years old. DM is twice as common in women as in men, with no ethnic predilection (4). With respect to the immunopathological analysis, the expression of major histocompatibility complex (MHC) I on target cells is a prerequisite for antigen-specific T cellmediated cytotoxicity (5). Normal muscle fibers do not express this complex (6,7), but it is overexpressed in inflammatory myopathies (8-11). MHC I overexpression in muscle fibers in early juvenile DM (JDM) without therapy and even in the absence of lymphocytic infiltration and muscle damage has been

INTRODUCTION Idiopathic inflammatory myopathies are a heterogeneous group of autoimmune diseases and include dermatomyositis (DM), polymyositis, and inclusion body myositis. These myopathies differ in regard to their clinical, histological, and immunopathological features (1-3).

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.

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Figure 1 - Major histocompatibility complex I and II expression in muscle fibre membrane surface of juvenile and adult dermatomyositis. Notes: The immunohistochemistry analysis demonstrates significantly higher major histocompatibility complex I expression in fibres of juvenile dermatomyositis compared to adult dermatomyositis, in contrast to major histocompatibility complex II expression.

reported (12). Moreover, we have recently shown that MHC I overexpression in muscle fibers is both a premature and late marker of JDM regardless of the evolution time of disease, glucocorticosteroid therapy and the grading of inflammation by clinical, laboratory and histological parameters (13). Similarly, MHC I overexpression, mainly in the perifascicular area, has been described in adult DM (ADM), even in muscle biopsies without typical features, as inflammatory infiltrates and perifascicular atrophy. In contrast, MHC II is expressed on antigen-presenting cells and is necessary to activate T helper lymphocytes and to initiate an immune response. It is not usually expressed on normal myofibers (7) but is expressed on myoblasts in culture (14) that behave as antigen-presenting cells (15). However, the actual function of MHC II in inflammatory myopathies remains controversial in the literature. MHC I and II have been studied in ADM and JDM patients, although not systematically and simultaneously. Therefore, the aim of this study was to assess the expression of this class of proteins comparatively in ADM and JDM muscle biopsies and to describe the possible MHC expression mechanism(s) involved in both groups.

None of the ADM and JDM patients had a malignancy. The demographics, clinical manifestations at disease onset (cutaneous involvement: heliotrope and Gottron’s papule; heart involvement: myocarditis and acute myocardial infarct; gastrointestinal tract involvement: dysphagia; articular involvement: arthralgia and/or arthritis; and pulmonary involvement: pulmonary alterations in computer tomography and symptoms such as dyspnea) and laboratory data were obtained through a systematic review of the patient records. Limb muscle strength was graded according to the Medical Research Council as the following: grade 0, absence of muscle contraction; grade I, slight signs of contractility; grade II, movements of normal amplitude but not against the action of gravity; grade III, normal range of motion against gravity; grade IV, full mobility against gravity and degree of resistance; and grade V, complete mobility and strong resistance against the action of gravity (16). The laboratory data corresponded to information collected at disease onset. Creatine kinase (normal range: 24173 IU/L), lactate dehydrogenase (20-350 IU/L), aminotransferase alanine (10-36 IU/L), aminotransferase aspartate (10-36 IU/L) and aldolase (1.0-7.5 IU/L) were determined by the automated kinetic method. Muscle biopsies of the brachial biceps were routinely performed on the same day as the laboratory exams. Sequential frozen sections were first stained for hematoxylin-eosin (HE) and then by immunohistochemistry. Each muscle biopsy specimen was coded and analyzed separately by two investigators (SKS and AMES), who were blinded to the diagnosis and clinical status. When any discrepancy was noted, the sample was reviewed concomitantly. Fiber damage, inflammatory infiltrate, perifascicular atrophy and increased connective tissue were assessed semi-quantitatively as absent, minimal, moderate or intense. A visual analog scale (VAS) was also included to score the global degree of abnormality from 0 (no abnormality) to 10 (most abnormal). Monoclonal antibodies (Dakopatts, Glostrup, Denmark) against MHC I and II were used at dilutions of 1:100. The StreptABComplex/HRP immunohistochemical procedure was performed as follows. Serial frozen sections of 5 mm thickness were fixed for 10 minutes in acetone at 4 ˚C.

MATERIALS AND METHODS In total, 28 JDM and 28 ADM consecutive patients fulfilling Bohan & Peter’s criteria (3) were included in this study. The patients were followed between 1990 and 2010 in the Pediatric Rheumatology Unit of the Children’ Institute and the Inflammatory Myopathies Unit of the Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo. As a control group, we included nine muscle biopsies of the following patients: two with limb-girdle muscular dystrophy (27 and 40 years-old, with immunohistochemical studies), two with facio-scapular-humeral dystrophy (20 and 51 years-old, with molecular confirmations) and five with Pompe’s disease (ages of 2 years 1 month, 2 years 11 months, 11 years 10 months, 19 years 6 months and 43 years, with molecular diagnoses). No patients or controls had evidence of an associated malignancy. The study was approved by the local research ethics committee of our university hospital.

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of the percentages was calculated using a binomial distribution. The age- and disease duration-adjusted odds ratios (OR) and 95% CI were calculated based on an unconditional logistic model. These calculations were performed using the STATA version 7.0 (STATA, College Station, TX, USA) computer program. A p-value,0.05 was considered to be statistically significant.

Endogenous peroxidase was blocked with 1% H2O2 in absolute methanol three times for 10 minutes. After a rinse in phosphate-buffered saline (PBS 0.01 M, pH 7.4) for 5 minutes, the specimen was incubated in fetal bovine serum in a wet chamber for 1 hour at 37 ˚C. The primary antibody, diluted in PBS and 1% BSA, was applied in a wet chamber at 37 ˚C overnight. The slides were then washed in PBS, and the prepared secondary mouse biotinylated antibody (StreptAB Complex/HRP) was applied for 30 minutes at 37 ˚C, followed by rinsing in PBS. Subsequently, the prepared StreptAB Complex/HRP complex at a 1:100 dilution was applied for 30 minutes at 37 ˚C, and after rinsing in PBS, the slides were incubated with a chromogenic substrate solution for peroxidase 3,39-diaminobenzidine tetrahydrochloride solution, a chromogenic substrate for peroxidase. After a final rinse, hematoxylin counterstaining was performed. The slides were mounted and cover-slipped with an aqueous-based mounting medium. The preparations of all muscle specimens were performed at the same time as a batch. Under 200X magnification, 10 random fields, representing almost the entire area of the specimen and including an average of 5001000 muscle fibers, were analyzed. The immunohistochemical preparations were analyzed for the expression levels of MHC I and II using a semi-quantitative method: (-) = positively stained muscle fibers were absent; (+) = 1 to 10% positively stained fibers; (++) = 11 to 25% positively stained fibers; (+++) = 26 to 50% positively stained fibers; and (++++) = 51 to 100% positively stained fibers. The results were expressed as means¡standard deviations (SD) or percentages. The chi-square and Student‘s t-tests were employed for non-parametric and parametric values, respectively. The 95% confidence interval (95% CI)

RESULTS The demographic, clinical and laboratory features are shown in Table 1. The ages at disease onset were 42.0¡15.9 and 7.3¡3.4 years of age in ADM and JDM, respectively, and the disease times before the muscle biopsy were similar in both groups (9.4¡11.1 vs. 11.3¡16.0 months, p = 0.616). There were no differences between the groups with respect to the clinical and laboratory data, except for the creatine kinase and lactate dehydrogenase levels, which were higher in ADM than in JDM (Table 1). In general, the immunohistochemistry analysis revealed significantly higher MHC I-positive expression in the JDM group compared with the ADM group (96.4% vs. 50.0%, p,0.001). Analyzed individually, MHC I expression was observed in more than 50% (4+) of the fibers in four (14.3%) JDM cases, in up to 25% (2+) of the fibers in five (17.8%) cases, and in up to 50% (3+) of fibers in three (10.7%) cases. In 15 (53.6%) cases, MHC I was expressed in less than 10% (1+) of the fibers (Table 2). Of note, negative MHC I expression was observed in only one patient. In contrast, MHC I was positive in only half of the ADM cases, with 10 (35.7%) having more than 50% (4+) positive fibers. Scores of 1+ and 3+ were observed in one (3.6%) case each, whereas a

Table 1 - Clinical and laboratory features of adult and juvenile dermatomyositis. Features Age at disease onset¡SD (years) Disease duration¡SD (months) Gender: female (%) Constitutional symptoms (%) Cutaneous involvement Heliotrope (%) Gottron‘s papule (%) Heart involvement (%) Gastrointestinal tract involvement (%) Articular involvement (%) Pulmonary involvement (%) Muscle strength Upper limbs Grade I (%) Grade II (%) Grade III (%) Grade IV (%) Grade V (%) Lower limbs Grade I (%) Grade II (%) Grade III (%) Grade IV (%) Grade V (%) Laboratory alterations Creatine kinase¡SD (IU/L) Lactate dehydrogenase¡SD (IU/L) Aspartate aminotransferase¡SD (IU/L) Alanine aminotransferase¡SD (IU/L) Aldolase¡SD (IU/L)

ADM (n = 28)

JDM (n = 28)

p-value

42.0¡15.9 9.4¡11.1 24 (85.7) 14 (50.0)

7.3¡3.4 11.3¡16.0 20 (71.4) 11 (39.3)

0.616 0.193 0.420

26 (92.9) 28 (100.0) 0 0 11 (39.3) 7 (25.0)

21 (75.0) 22 (78.6) 3(10.7) 2 (7.1) 8 (28.6) 3 (10.7)

0.127 0.313 0.075 0.150 0.397 0.163

0 3 (10.7) 11 (39.3) 12 (42.9) 2 (7.1)

0 3 (10.7) 12 (42.9) 12 (42.9) 1 (3.5)

0.945

0 4 (14.3) 11 (39.3) 12 (42.9) 1 (3.5)

0 5 (17.8) 11 (29.3) 12 (42.9) 0

0.774

3820.9¡5332.3 1105.7¡ 1321.6 0.020 1665.0¡1311.2 937.8¡552.1

0.030 200.7¡254.4 174.0¡366.1 0.807 192.5¡305.1

171.4¡400.8 0.863 56.5¡103.6 16.7¡7.7 0.329

ADM: adult dermatomyositis; JDM: juvenile dermatomyositis; SD: standard deviation.

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Compared with JDM, inflammatory cell infiltration was observed in 22 (78.6%) ADM muscle specimens (p = 0.384). In contrast, fiber damage and increased connective tissue was observed in 23 (82.1%) ADM cases for both parameters, with significant differences between JDM and ADM (p = 0.006 and p,0.001, respectively). Interestingly, perifascicular atrophy was observed in only 14 (50.0%) ADM patients (p,0.001). The overview of the histopathological findings was graded using a VAS and was similar between the two groups (p = 0.748). Following the result pattern of the analyses of the individual histological parameters, the VAS was not correlated with the clinical parameters measuring disability, including limb muscle strength. Neither MHC I nor MHC II expression correlated with the interval of disease evolution to the biopsy, the age at disease onset, the grading of muscle strength, the creatine kinase level or any other clinical manifestation in either ADM and JDM (Table 3). Moreover, these proteins were not correlated with any of the histological parameters (inflammatory cell infiltration, fiber damage, perifascicular atrophy and connective tissue proliferation) in either group (p.0.050).

Table 2 - Immunohistological and histological analyses of muscle biopsies from untreated adult and juvenile dermatomyositis patients.

MHC in muscle fibers Class I expression (%) + ++ +++ ++++ Class II expression (%) + ++ +++ ++++ Histological analysis in muscle fibers Perifascicular atrophy Inflammatory infiltration (%) Absent Minimum Moderate Intense Fiber damage (%) Absent Minimum Moderate Intense Increased connective tissue (%) Absent Minimum Moderate Intense VAS Median (range)

ADM

JDM

(n = 28)

(n = 28)

p-value

14 1 2 1 10

(50.0) (3.6) (7.1) (3.6) (35.7)

1 (3.6) 15 (53.6) 5 (17.8) 3 (10.7) 4 (14.3)

,0.001

13 1 2 1 10

(46.4) (3.6) (7.1) (3.6) (35.7)

24 (85.7) 2 (7.1) 1 (3.6) 0 1 (3.6)

0.004

14 (50.0)

27 (96.4)

,0.001

6 (21.4)

2 (7.1)

14 (50.0) 6 (21.4) 2 (7.2)

17 (60.7) 5 (17.9) 4 (14.3)

0.006

5 (17.9) 14 (50.0) 4 (14.2) 5 (17.9)

13 (46.4) 15 (53.6) 0 0

,0.001

5 (17.9)

26 (92.9)

15 (53.6) 6 (21.4) 2 (7.1)

2 (7.1) 0 0

3.0 (0-9)

3.5 (1-8)

0.384

DISCUSSION Our study showed that MHC I and II were expressed in the muscle fibers of JDM and ADM, two idiopathic inflammatory myopathies, with a distinct pattern: there was greater MHC I expression in JDM and greater MHC II expression in ADM. These findings were observed regardless of the clinical, laboratory or histological features. The major strengths of this study were the stringent inclusion of untreated patients who fulfilled Bohan & Peter‘s criteria (3) for DM, the analysis of only one type of skeletal muscle, the use of standardized histological, histochemical and immunohistochemical processing methods, and the double-blind analyses by two independent investigators, which assured the homogeneity of the studied parameters and consistency of the results. The presence of inflammatory cells in the skeletal muscle biopsy in conjunction with the clinical and laboratory features resulted in the diagnosis of an inflammatory myopathy. However, the muscle biopsy findings may be non-specific or even normal, and in such cases, the diagnosis of inflammatory myopathy and, consequently,

0.748

ADM: adult dermatomyositis; JDM: juvenile dermatomyositis; MHC: major histocompatibility complex; VAS: visual analog score. Fiber damage, inflammatory infiltrate and increased connective tissue were assessed semi-quantitatively as absent, minimal, moderate or intense. The expression levels of MHC I and II were assessed by a semi-quantitative method: (-) = positively stained endothelial cells only; (+) = 1 to 10% positively stained fibers; (++) = 11 to 25% positively stained fibers; (+++) = 26 to 50% positively stained fibers; and (++++) = 51 to 100% positively stained fibers.

Table 3 - Relationship among various myositis parameters and the MHC I / II presence in dermatomyositis.

score of 2+ was detected in two cases (7.1%) (Table 2). Figure 1 shows comparative representatives of the DMJ and DM biopsies. In the control group, only one patient with facioscapular humeral dystrophy was positive for MHC I, with less than 10% expression (1+). The percentage of MHC II expression was significantly higher in ADM than in JDM (50.0% vs. 14.3%, p = 0.004). In contrast, all patients in the control group were negative for MHC II. Inflammatory cell infiltration was detected in 26 out of 28 (92.9%) JDM muscle biopsies and in the majority of perifascicular atrophy patients (27, 96.4%) (Table 2). Additionally, minimal alterations in muscle fiber damage were observed in 15 (53.6%) patients, and only a slight increase in connective tissue was observed in two (7.1%) patients. Rimmed vacuoles were not detected in any biopsy.

Parameters

Interval of disease to biopsy Age at onset Degree of muscle strength Creatine kinase level Other clinical manifestations Perifascicular atrophy Fiber damage Increased connective tissue Inflammatory cell infiltration

ADM (n = 28)

JDM (n = 28)

MHC I

MHC II

MHC I

MHC II

0.807

0.807

0.787

0.515

0.086 0.251 0.362 .0.050

0.086 0.0251 0.362 .0.050

0.296 0.179 1.000 .0.050

0.767 0.484 0.141 .0.050

0.257 0.045 0.537 0.512

0.257 0.045 0.537 0.512

1.000 0.464 1.000 1.000

1.000 1.000 1.000 0.253

ADM: adult dermatomyositis; JDM: juvenile dermatomyositis; MHC: major histocompatibility complex. Data indicate p-values.

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mechanism. The results of this study combined with our previous observation of differential positive ICAM-1 expression in the muscle fibers of ADM, in contrast to the increased expression of ICAM-1 in the muscle vessels of JDM (29), corroborates the myocytotoxic mechanism in adult inflammatory myopathies, in contrast to muscle lesions through the involvement of microvasculature in JDM. The limitation of this study is inherent to the applied immunohistochemistry method, in which absolute quantitative data are difficult to obtain. However, the semiquantitative analysis performed by two independent investigators and based on microscopic fields chosen with uniform criteria, along with the staining of specimens performed in a single batch, were the strategies adopted to minimize bias. The analyses of these expression profiles in the context of the clinical evolution, therapy response, and the results of other complementary exams (such as capillaroscopy), along with comparisons to other inflammatory myopathies (such as polymyositis and inclusion body myositis), may improve our understanding of the pathophysiology of these diseases. In summary, JDM patients presented more MHC I positivity and perifascicular atrophy and less fiber damage, connective tissue proliferation and increased creatine kinase / lactate dehydrogenase levels compared with ADM patients. Immunohistochemical analysis of MHC I and II in muscle biopsies is confirmed as a complementary diagnostic tool for inflammatory myopathy, particularly with respect to the overexpression of MHC I in JDM cases.

the therapeutic approach might be delayed. MHC protein expression analysis by immunohistochemistry has proven to be extremely helpful in these cases as an additional diagnostic tool (2,11,17). In fact, in this study, MHC I expression was present in all JDM cases except for in one patient, who had a muscle biopsy showing perifascicular atrophy and minimal inflammatory cell infiltration without fiber damage. The overexpression of MHC I has been previously described by others (11,12,18-20), even in muscle biopsies reported as normal by conventional histology. In our study, 66.7% of the MHC I-positive JDM cases presented no (7.4%) or very low (59.3%) inflammatory cell infiltration in the analyzed muscle specimen. In contrast, MHC I overexpression was identified in only half of the ADM patients in our series. The previously reported frequency of MHC I positivity has been quite variable, ranging from 53.3% to 100% (11,1719,21,22). The smaller number and heterogeneity of previously studied cases may partially explain these discrepancies. Similarly, MHC I expression was detected in half of the ADM cases, including cases presenting no or only scattered inflammatory cell infiltration in the muscle biopsy (14.3% and 42.9% of the cases, respectively). Therefore, the positive MHC I expression in such histologically nearnormal cases proved to be a powerful tool to establish the diagnosis of inflammatory myopathy in combination with the compatible clinical and laboratorial findings, mainly in the JDM cases. The MHC II expression was present in fewer of our cases; it was observed in 50.0% and 14.3% of the ADM and JDM cases, respectively. Variable frequencies, ranging from 0% to 28.6%, have been reported in other studies (17,19,21). Whether the MHC complex up-regulation in DM results from a nonspecific response to muscle degeneration and regeneration or from a specific disease process is not fully understood. Highly expressed pro-inflammatory cytokines in DM (21,23), such as INFc and TNFa, have been reported to induce MHC I expression in in vitro assays (24,25). The overexpression of the MHC complex might also be secondary to the diffusion of secreted cytokines by inflammatory cells near the biopsied muscle area, as up-regulation of interleukin-1 might even reflect a stress response secondary to the early ischemic injury of the muscle fibers (18,21,26). The overexpression of MHC I and II in the muscle biopsies proved to be an independent factor of not only the duration of the disease and the degree of muscle damage (as determined by either the degree of muscle strength recorded during clinical examination or the muscle enzymatic measurements) but also the muscle histological findings. In fact, despite the absence of inflammatory cell infiltrates and/or perifascicular atrophy, MHC I and/or II overexpression was clearly detected in the muscle biopsies from patients with clinical features of DM, as demonstrated in this study and in other studies (11,18,20-22,27). Moreover, in our previous study, MHC I expression was independent of even corticosteroid therapy administered prior to the muscle biopsy (13). Our results indicated that MHC I is overexpressed in the muscle biopsies of JDM and ADM patients. MHC I- and IIdependent processes require the expression of ICAM-1. The ICAM-1 molecule is necessary to stabilize the interactions between the cell receptor and the MHC-peptide complex in antigen recognition (28). The ICAM-1 molecule, which is essential for cell adhesion, also triggers the cytotoxic

ACKNOWLEDGMENTS Sponsored by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico - CNPQ (grant 300248/2008-3 to CAS) and the Federico Foundation to SKS and CAS.

AUTHOR CONTRIBUTIONS Shinjo SK participated in the collection of the data, performed the statistical analysis and wrote the manuscript. Sallum AM participated in the collection of the data and performed the data analysis. Silva CA revised the manuscript. Marie SK contributed to the study design, wrote the manuscript and revised the manuscript.

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Proc Natl Acad Sci USA. 2000;97(16):9209-14, http://dx.doi.org/ 10.1073/pnas.97.16.9209. Figarella-Branger D, Lacroix C, Coquet M, Gherardi R, Pellissier JF. Idiopathic inflammatory myopathies. Ann Pathol. 2001;21(3):279-84. Pavlath GK. Regulation of class I MHC expression in skeletal muscle: deleterious effect of aberrant expression on myogenesis. J Neuroimmunol. 2002;125(1-2):42-50, http://dx.doi.org/10.1016/S0165-5728(02)00026-7. Civatte M, Schleinitz N, Krammer P, Fernandez C, Guis S, Veit V, et al. Class I MHC detection as a diagnostic tool in noninformative muscle biopsies of patients suffering from dermatomyositis (DM). Neuropathol Appl Neurobiol. 2003;29(6):546-52, http://dx.doi.org/10.1046/j.13652990.2003.00471.x. Li CK, Varsani H, Holton JL, Gao B, Woo P, Wedderburn LR. Juvenile dermatomyositis research group. MHC class I overexpression on muscles in early juvenile dermatomyositis. J Rheumatol. 2004;31(3):605-9. Sallum AME, Kiss MHB, Silva CAA, Wakamatsu A, Sachetti S, Lotufo S, et al. MHC class I and II expression in juvenile dermatomyositis skeletal muscle. Clin Exp Rheumatol. 2009;27(3):519-526. Cifuentes-Diaz C, Delaporteg C, Dautreaux B, Charron D, Fardeau M. Class II MHC antigens in normal human skeletal muscle. Muscle Nerve. 1992;15(3):295-302, http://dx.doi.org/10.1002/mus.880150307. Goebels N, Michaelis D, Wekerle H, Hohlfeld R. Human myoblasts as antigen-presenting cells. J Immunol. 1992;149(2):661-7. Hanissian AS, Masi AT, Pitner SE, Cape CC, Medsger TA Jr. Polymyositis and dermatomyositis in children: an epidemiologic and clinical comparative analysis. J Rheumatol. 1982;9(3):390-4. Jain A, Sharma MC, Sarkar C, Bhatia R, Singh S, Handa R. Major histocompatibility complex class I and II detection as a diagnostic tool in idiopathic inflammatory myopathies. Pathol Lab Med. 2007;131(7):1070-6. Emslie-Smith AM, Arahata K, Engel AG. Major histocompatibility complex class I antigen expression, immunolocalization of interferon subtypes, and T-cell mediated cytotoxicity in myopathies. Hum Pathol. 1989;20(3):224-31, http://dx.doi.org/10.1016/0046-8177(89)90128-7. Bartoccioni E, Gallucci S, Scuderi F, Ricci E, Servidei S, Broccolini A, et al. MHC Class I, II and Intercellular adhesion molecule-I (ICAM-I) expression in inflammatory myopathies. Clin Exp Immunol. 1994;95(1):166-72.

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DOI:10.6061/clinics/2012(08)06

CLINICAL SCIENCE

Predicting dysthyroid optic neuropathy using computed tomography volumetric analyses of orbital structures Allan C. Pieroni Gonc¸alves,I Lucas Nunes Silva,II Eloı´sa M. M. S. Gebrim,II Suzana Matayoshi,I Ma´rio Luiz Ribeiro MonteiroI I Faculdade de Medicina da Universidade de Sa˜o Paulo, Division of Ophthalmology, Sa˜o Paulo/SP, Brazil. II Faculdade de Medicina da Universidade de Sa˜o Paulo, Department of Radiology, Sa˜o Paulo/SP, Brazil.

OBJECTIVE: To evaluate the ability of orbital apex crowding volume measurements calculated with multidetectorcomputed tomography to detect dysthyroid optic neuropathy. METHODS: Ninety-three patients with Graves’ orbitopathy were studied prospectively. All of the patients underwent a complete neuro-ophthalmic examination and computed tomography scanning. Volumetric measurements were calculated from axial and coronal contiguous sections using a dedicated workstation. Orbital fat and muscle volume were estimated on the basis of their attenuation values (in Hounsfield units) using measurements from the anterior orbital rim to the optic foramen. Two indexes of orbital muscle crowding were calculated: i) the volumetric crowding index, which is the ratio between soft tissue (mainly extraocular muscles) and orbital fat volume and is based on axial scans of the entire orbit; and ii) the volumetric orbital apex crowding index, which is the ratio between the extraocular muscles and orbital fat volume and is based on coronal scans of the orbital apex. Two groups of orbits (with and without dysthyroid optic neuropathy) were compared. RESULTS: One hundred and two orbits of 61 patients with Graves’ orbitopathy met the inclusion criteria and were analyzed. Forty-one orbits were diagnosed with Graves’ orbitopathy, and 61 orbits did not have optic neuropathy. The two groups of orbits differed significantly with regard to both of the volumetric indexes (p,0.001). Although both indexes had good discrimination ability, the volumetric orbital apex crowding index yielded the best results with 92% sensitivity, 86% specificity, 81%/94% positive/negative predictive value and 88% accuracy at a cutoff of 4.14. CONCLUSION: This study found that the orbital volumetric crowding index was a more effective predictor of dysthyroid optic neuropathy than previously described computed tomography indexes were. KEYWORDS: Dysthyroid optic neuropathy; Multidetector computed tomography; Graves’ orbitopathy; Volume CT. Gonc¸alves AC, Silva LN, Gebrim EM, Matayoshi S, Monteiro ML. Predicting dysthyroid optic neuropathy using computed tomography volumetric analyses of orbital structures. Clinics. 2012;67(8):891-896. Received for publication on February 7, 2012; First review completed on March 19, 2012; Accepted for publication on April 7, 2012 E-mail: mlrmonteiro@terra.com.br Tel.: 55 11 3661-7582

glycosaminoglycans and increased fat content (1). Clinical signs include proptosis, congestive signs, strabismus, and dysthyroid optic neuropathy (DON) (2-5). DON is the most significant complication of GO. It affects 3.4 to 8% of GO patients (6-9) and requires prompt treatment to avoid permanent visual loss. Almost all instances of DON result from optic nerve compression at the orbital apex by enlarged extraocular muscles. The diagnosis of DON includes the following clinical features: decreased visual acuity (VA), abnormal visual fields (VF), impaired color and brightness perception, delayed visual evoked potentials, afferent pupillary defects and edema or atrophy of the optic nerve head (6). Unfortunately, some of these tests require the full cooperation of an alert and motivated patient and often render false positive results, particularly in patients with congestive GO. Furthermore, alternative causes of visual impairment secondary to GO,

INTRODUCTION Graves’ orbitopathy (GO) is the most common extrathyroid manifestation of Graves’ disease (GD). It occurs before, during, or after the onset of hyperthyroidism and, less frequently, in euthyroid or hypothyroid patients. The course of GO can be divided into active (congestive) and inactive (fibrotic) phases. Tissue expansion occurs within the relatively fixed volume imposed by the bony orbit and results in inflammation, the accumulation of hydrophilic

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.

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such as exposure keratopathy or secondary glaucoma, and coincidental conditions, such as amblyopia or cataracts, may also contribute to the considerable difficulty of detecting DON (10). Because diagnosing DON can be clinically difficult and because its prognosis improves significantly with early diagnosis and treatment, it is important to design objective tests that can identify patients at risk for developing DON (11). Computed tomography (CT) is the most frequently used imaging modality in patients with GO because of its capacity to visualize bone and soft tissue in the orbit. Previous studies have shown that a number of CT scan parameters can help to detect and facilitate the diagnosis of DON, primarily those parameters based on the detection of orbital apical crowding by enlarged extraocular muscles (46,12-16). Crowding has been successfully estimated with CT parameters, such as linear measurements of extraocular muscles (4,5,11,13), the subjective assessment of apical crowding (on single coronal images) (4,11,16,17) and measurements of the area (11) and volume (18,19) of the orbital muscles. Although linear and area measurements have proven to be helpful in the diagnosis of DON, the assessment of orbital apex crowding using volumetric estimates of structures could potentially improve the ability to detect and diagnose DON. In fact, Feldon et al. (2,18) used volumetric estimates of orbital contents to investigate the risk of developing DON in GO patients; however, those authors’ measurements required cumbersome manual calculations of the orbital muscles and other orbital structures. The recent introduction of multidetector-row computed tomography (MDCT) has resulted in shorter scanning times and higher resolution and has made it possible to reformat images from any plane, making orbital measurements easier to take and more precise. Using software for three-dimension orbital structure analysis, volumetric measurements can be easily calculated at a workstation (20). The purpose of this study was therefore to quantify muscle crowding and use it to test the ability of two volumetric indexes of muscle crowding based on volume measurements obtained with multidetector-computed tomography to predict DON in the orbits of GO patients.

appropriate near correction was used. To qualify as an abnormal VF on SAP, three adjacent abnormal points at the p,0.05 level or two adjacent points with one abnormal point at the p,0.01 level on the pattern deviation plot were required (22). Exclusion criteria included the following: age under 20 years, inability to cooperate with VF testing, spherical refraction over¡5 D, cylinder correction over¡3 D and unreliable VF test results. An unreliable Humphrey VF test was defined as one with more than 25% fixation losses, falsepositive or false-negative responses. Patients with clinical signs of glaucomatous optic neuropathy, optic disc anomaly, optic media opacities, previous orbital or strabismus surgery, and other types of neuropathies were also excluded. All patients were scanned with a 16-slice MDCT scanner (Brilliance 16; Philips Medical Systems, Nederland B.V., The Netherlands) without the use of sedation or intravenous contrast. The CT scans were obtained using contiguous axial slices, with the patient’s head positioned parallel to the Frankfurt plane. The patients were instructed to keep their eyes closed and steady in the primary gaze position. The scanning parameters were as follows: 120 Kv, 200 mAs, 16x0.75 mm detector configuration, 1.5 mm slice thickness and 0.7 mm slice increment. The images were postprocessed at a dedicated workstation and read by a single head-andneck radiologist (L.N.S) who was blinded to the patients’ clinical condition. Two volumetric indexes were calculated, one based on axial scans of the entire orbit content (up to the orbital rim) and one based on coronal scans of the orbital content from the midpoint of the orbital segment of the optic nerve to the orbital entrance of the optic canal.

Volume measurements of the entire orbit from axial scans To calculate volumes for the whole orbital content, contiguous axial scans were analyzed. The different tissues were color-coded according to their attenuation in Hounsfield units (HU). Tissue HU thresholds were defined using different window levels (WL) and window widths (WW) to separate the soft tissue (which mainly includes muscles, nerves and vessels) from other orbital tissues (mainly fatty content). Orbital soft tissue thresholds were set at 40/100 HU (WL/WW), and the fat tissue threshold was set at -160/230 HU (WL/WW). For the tissue volume measurements, each axial slice was traced along the bony orbit limits. The anterior boundary of the orbit was delineated by a straight line connecting the lateral and the medial orbital rim (Figure 1). The marking began at the topmost axial slice containing the orbit and continued downwards, slice by slice, until all of the orbit content was included. The volume of the muscles (including vessels and nerves), soft tissues and fat within the marked space was then estimated with the software (Figure 1). The volumetric orbital muscle crowding index (VCI) was calculated as the ratio between the soft tissue volume and the fat tissue volume.

PATIENTS AND METHODS The study followed the tenets of the Declaration of Helsinki and was approved by an institutional ethics committee. Ninety-three new patients with GO who were admitted to the orbit service of a tertiary referral eye center during the five-year study period were prospectively included. GO was diagnosed in accordance with previously established criteria (21). All of the patients underwent a complete neuro-ophthalmic examination, including bestcorrected VA, applanation tonometry, pupillary reactions, extraocular motility evaluation, slit lamp examination, soft tissue and eyelid inflammation evaluation, lid fissure measurement, Hertel exophthalmometry, fundoscopy, and VF evaluation. VF testing was performed using manual perimetry and standard automated perimetry (SAP). Manual VF testing was performed using the Goldmann perimeter (GP) (HaagStreit AG, Bern, Switzerland). SAP was performed with a Humphrey Field Analyzer 750 (Zeiss-Humphrey, Dublin, CA) using the 24-2 SITA-Standard strategy. The patients’

Volume measurements of the orbital apex from coronal scans One-millimeter coronal CT slabs were reformatted from contiguous axial slices of the orbit, which includes the space between the interzygomatic line and the orbital apex (Figure 2). The orbital volume was calculated from the

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Volumetric CT analyses in dysthyroid optic neuropathy Gonc¸alves ACP et al.

Figure 1 - A, B and C. Examples of the area outlined on 3 axial slices of computed tomography scans, from which measurements were obtained. Representation of the soft tissue (mostly muscles; D) and orbital fat (E) volumes based on measurements of the sequential axial slices.

eight months after the first measurements. On a different occasion, the same group of orbits was independently measured by a senior radiologist (E.M.M.S.G.). Both readers were blinded to the presence or absence of DON. GO patients’ eyes for which visual function data clearly indicated the presence or absence of active DON were considered eligible for the study. The criteria used to diagnose DON included the following: 1) The presence of a confirmed VF defect on SAP or GP associated or not with diminished best-corrected VA and not caused by changes in transparency and 2) in unilateral and asymmetric cases, the presence of a relative pupillary defect. When the bestcorrected VA was normal and VF abnormalities were present on SAP, a repeat examination using SAP or GP was performed to rule out false-positive responses. Therefore, only eyes with confirmed VF defects were included in the analysis. Eyes with normal VA and nonreproducible VF abnormalities on repeat examinations were excluded. Patients with ocular or optic nerve diseases that could interfere with the diagnosis of DON were also excluded. All patients with DON had active disease, visual disturbance within three months of the onset of the disease and clinical signs of orbital congestive disease. Patients from the group without DON had a history of orbital disease ranging from two months to four years. The findings for the two groups were compared.

posterior portion of the orbit, which encompasses the tissues within a point in the optic nerve halfway between the globe and the orbital apex and up to the entrance of the optic canal. The fat and soft tissues were color-coded with the same settings used for the axial measurements. For each of the preset coronal planes, a region of interest was traced around the orbital bone rim (Figure 2). The software automatically calculated the total volume of fat and soft tissue in that region (Figure 2). The ratio of the soft tissue volume to the fat volume (using volume measures taken at the apex) was calculated to determine the volumetric orbital apex crowding index (VACI). The same reader repeated the measurements for 26 randomly selected orbits (13 with DON) approximately

Statistical analysis Descriptive statistics included the mean values¡SD for normally distributed variables. Receiver operating characteristic (ROC) curves were used to describe the ability of the VCI and the VACI to discriminate between orbits with and without DON. For each parameter, sensitivities at the fixed specificities of 80% and 95% were calculated. Sensitivity, specificity, likelihood ratio, and accuracy were calculated for the best cutoff values of VCI and VACI. The intraclass

Figure 2 - Percentage difference from rest to ventilatory anaerobic threshold (rest/VAT), rest to respiratory compensation point (rest/RCP) and rest to peak of exercise (rest/Peak) in patients with coronary artery disease subjected to continuous exercise training (panel A) and interval exercise training (panel B). VAT = ventilatory anaerobic threshold; RCP = respiratory compensation point. * p,0.05 vs. post-intervention.

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one patient, 0.6 in two patients, 0.4 in one patient, 0.5 in two patients, 0.2 in one patient, and 0.1 in one patient. Exophthalmometry measurements in the affected eyes ranged from 19 to 32 mm (mean¡SD: 25.14¡3.19 mm). Restrictive myopathy was present in all orbits with DON. The fundoscopic examination revealed optic disc edema in eight eyes, optic disc pallor in one and no abnormalities in the remainder. In the group without DON, the bestcorrected VA was 1.0 in all eyes, and exophthalmometry findings ranged from 16.0 to 31.5 mm (mean¡SD: 23.4¡2.7). Table 1 shows the mean¡SD and the ranges for the VCI and VACI. The mean VCI and VACI values were significantly higher in the orbits with DON (p,0.001). Table 2 compares the two groups’ areas under ROC curves for the two volumetric indexes, with confidence intervals and sensitivity at the 95% and 80% specificity rates. The area under the ROC curve was 0.86 for the VCI and 0.92 for the VACI. Cutoff values were determined for the two indexes to calculate their sensitivity and specificity for differentiating orbits with and without DON. The best sensitivity/specificity ratio was achieved with a VACI cutoff of 4.14 (sensitivity: 90%, specificity: 87%, likelihood ratio: 6.9, accuracy for detecting DON: 88%; Table 2). The ICCs for the VCI and VACI as determined by the second observer were 0.90 and 0.91, respectively (p,0.001), indicating an excellent interobserver variability. Corresponding values for repeated measurements by the same observer were 0.90 and 0.89, indicating excellent intraobserver variability (p,0.001).

Table 1 - Means¡standard deviation of the axial volumetric orbital crowding index (VCI) and the coronal volumetric orbital apex crowding index (VACI) in the orbits of patients with Graves’ orbitopathy (GO) with or without dysthyroid optic neuropathy (DON).

Parameter Mean¡SD VCI 95% CI Range Mean¡SD VACI 95% CI Range *

Orbits with DON n = 41

Orbits without DON n = 61

1.84¡1.02 1.52–2.16 0.31–5.28 21.35¡21.09 14.69–28.01 1.03–86.52

0.83¡0.28 0.76–0.90 0.34–1.53 2.82¡2.28 2.24–3.40 0.59–10.97

p-value* ,0.001

,0.001

Student’s t-test. Significant values are in italics. CI = confidence interval.

correlation coefficient (ICC) was calculated to assess the interrater and intrarater variability for the VCI and VACI measurements. A p-value less than 0.05 was considered statistically significant.

RESULTS Thirty-two of the 93 patients were excluded based on theabove-described criteria. The remaining 61 patients (37 women and 24 men) had 102 orbits that were included in the study. The orbits were divided into two groups according to the presence or absence of DON. The first group included 41 orbits of 27 patients (12 women and 15 men; mean age¡SD: 53.8¡10.1 years) who met the diagnostic criteria for DON. Fourteen patients had bilateral DON; for those patients, both orbits were included in the study. Thirteen patients had only one orbit included in the DON group. Of these 13 patients, the contralateral eye of ten patients did not meet the DON criteria. Two patients had the contralateral eye excluded because the presence or absence of DON could not be clearly determined; in one patient, a history of central retinal artery occlusion unrelated to GO excluded the contralateral eye. The second group included 61 orbits in which DON was clearly absent from 34 patients (25 women and nine men; mean age¡SD: 42.3¡10.2 years). In this group, seven orbits were excluded from analysis because of the uncertainty of the DON diagnosis. Six of these patients had questionable VF abnormalities on SAP even after repeat examinations. One patient had an eye with severe deviations caused by extraocular muscle involvement, which precluded proper VF examination. For the 41 orbits with DON, the best-corrected VA was 1.0 in 20 patients, 0.9 in seven patients, 0.8 in six patients, 0.7 in

DISCUSSION GO can be an insidious disease, and DON is a significant complication that requires prompt diagnosis and treatment to prevent permanent visual damage. Although research has suggested that DON may be caused by inflammatory and vascular mechanisms, the most widely accepted theory for DON development is direct compression of the optic nerve by enlarged extra ocular muscles at the orbital apex (3,6,23,24). The diagnosis of DON depends mainly on clinical features based on visual function assessment, afferent pupillary defect and fundoscopic abnormalities. Nevertheless, DON is often subclinical and in many cases, it is difficult to diagnose because of confounding signs and symptoms. Furthermore, patients with severe orbitopathy tend to have more congestive and oculomotor symptoms, which may outweigh or mask subtle visual deterioration (6). Therefore, the development of imaging techniques that facilitate the diagnosis of DON is highly desirable.

Table 2 - Area under the receiver operating characteristics curves (AUC) and sensitivities at fixed specificities for the volumetric crowding index (VCI) and the volumetric apical crowding index (VACI). Sensitivity, specificity, likelihood ratio (LR) and accuracy are presented for the best cutoff values of VCI and VACI. Parameter

AUC (SE)

Sensitivity/specificity Specificity $ 95%

Specificity $ 80%

Cutoff value

Sensitivity/specificity

LR

Accuracy

at cutoff value

(95% CI)

(95% CI)

3.6 (2.1-6.2) 6.9 (3.9-10.8)

76% (67%-85%) 88% (80%-93%)

VCI

0.86 (0.04)

56/95

71/80

1.08

71%/80%

VACI

0.92 (0.31)

66/95

93/80

4.14

90%/87%

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Volumetric CT analyses in dysthyroid optic neuropathy Gonc¸alves ACP et al.

Figure 3 - A, B and C. Examples of the area outlined on 3 coronal slices of computed tomography scans, from which measurements were obtained. Representation of the soft tissue (mostly muscles) (D) and orbital fat (E) apical volumes based on sequential coronal orbital slices.

MDCT scanner, and the measurement of the orbital structures with the volumetric analysis software was straightforward. The technology has mainly been used in studies to estimate the increase in orbital fat, the effect of orbital radiotherapy or the effect of orbital decompression on orbital structures. However, to our knowledge, no previous study has evaluated the technology’s ability to predict DON (19,27-31). According to published guidelines for the radiologic measurement of tissue volumes in GO patients, there is currently no consensus on how to measure orbital fat and extraocular muscle volume (32). To obtain volume measurements, we used a computer-assisted algorithm based on the density of soft tissue and orbital fat. We then calculated the following two volumetric indexes: the VCI, which is the ratio of the extraocular muscle volume to the orbital fat volume and is based on axial scans of the entire orbit; and the VACI, which is the ratio of the extraocular muscle volume to the orbital fat volume and is based on coronal scans of the orbital apex. The indexes appeared to be reliable and reproducible, as suggested by the high ICC values found in this study. In our study, all patients were carefully and prospectively evaluated for the presence or absence of DON, and a large number of orbits with optic neuropathy were included. Both indexes were efficient at differentiating patients with DON from patients without DON. The VACI, which evaluates the orbital apex, was particularly effective; it achieved a sensitivity of 90% and a specificity of 87% at a cutoff of 4.14, whereas the VCI achieved only 71% sensitivity and 80% specificity at a cutoff of 1.08. These findings indicate that volumetric orbital crowding measurements are better at detecting DON in patients with GO when they are restricted to the orbital apex. Our analysis of the performance of the VACI indicates a significant improvement over previous indexes in identifying patients with DON. The VACI was more efficient than

Previous CT studies have demonstrated a direct correlation between orbital apex crowding by enlarged extraocular muscles and the development of DON (6,12,13,18,24,25). Recent studies also suggest that bony orbit anatomy may play an important role in the development of DON because narrow orbits are more susceptible to apex crowding (11). In addition, other CT features (such as intracranial fat prolapse, lacrimal gland displacement, exophthalmos severity and superior ophthalmic vein dilatation) have been proposed but have not been confirmed as useful for detecting DON (4,6,11,17,24,26). Because the presence of apical crowding on CT imaging is strongly correlated with DON in GO, several authors have proposed indexes for detecting DON. Barret et al. (13) calculated a linear muscle index from CT images and found it to be reproducible and reasonably efficient for detecting DON. In a previous study, we investigated the sensitivity and specificity of this muscle index calculation with MDCT and found the best combination (79% sensitivity and 72% specificity) at a muscle index of 60% (5). Nugent et al. (14) proposed a categorical scale for apical crowding, which was later reproduced by many other authors (4,11,16,17). Using this method, Nugent et al. (14) found severe apical orbital crowding in 12 out of 18 orbits with DON, but only in 16 out of 124 orbits without DON. While it seems clear that crowding scores are useful for detecting DON, the accurate use of the scores is difficult because the authors of these studies did not provide clear definitions of the positions along the orbit where the coronal plan was used to determine their scores. In our study, we attempted to improve on existing CT indexes with a volumetric quantification of orbital crowding. In previous studies, extraocular muscle volume estimates were useful for detecting DON, but the manual quantitative analysis of the extraocular measurements was time-consuming and complex (12,18,25). In our study, orbital structures could be precisely reformatted using an

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10. Dayan CM, Dayan MR. Dysthyroid optic neuropathy: a clinical diagnosis or a definable entity? Br J Ophthalmol. 2007;91(4):409-10, http:// dx.doi.org/10.1136/bjo.2006.110932. 11. Chan LL, Tan HE, Fook-Chong S, Teo TH, Lim LH, Seah LL. Graves ophthalmopathy: the bony orbit in optic neuropathy, its apical angular capacity, and impact on prediction of risk. AJNR Am J Neuroradiol. 2009;30(3):597-602, http://dx.doi.org/10.3174/ajnr.A1413. 12. Feldon SE, Muramatsu S, Weiner JM. Clinical classification of Graves’ ophthalmopathy. Identification of risk factors for optic neuropathy. Arch Ophthalmol. 1984;102(10):1469-72. 13. Barrett L, Glatt HJ, Burde RM, Gado MH. Optic nerve dysfunction in thyroid eye disease: CT. Radiology. 1988;167(2):503-7. 14. Nugent RA, Belkin RI, Neigel JM, Rootman J, Robertson WD, Spinelli J, et al. Graves orbitopathy: correlation of CT and clinical findings. Radiology. 1990;177(3):675-82. 15. Ozgen A, Alp MN, Ariyurek M, Tutuncu NB, Can I, Gunalp I. Quantitative CT of the orbit in Graves’ disease. Br J Radiol. 1999;72(860):757-62. 16. McKeag D, Lane C, Lazarus JH, Baldeschi L, Boboridis K, Dickinson AJ, et al. Clinical features of dysthyroid optic neuropathy: a European Group on Graves’ Orbitopathy (EUGOGO) survey. Br J Ophthalmol. 2007;91(4):455-8, http://dx.doi.org/10.1136/bjo.2006.094607. 17. Birchall D, Goodall KL, Noble JL, Jackson A. Graves ophthalmopathy: intracranial fat prolapse on CT images as an indicator of optic nerve compression. Radiology. 1996;200(1):123-7. 18. Feldon SE, Lee CP, Muramatsu SK, Weiner JM. Quantitative computed tomography of Graves’ ophthalmopathy. Extraocular muscle and orbital fat in development of optic neuropathy. Arch Ophthalmol. 1985;103(2): 213-5. 19. Forbes G, Gorman CA, Brennan MD, Gehring DG, Ilstrup DM, Earnest Ft. Ophthalmopathy of Graves’ disease: computerized volume measurements of the orbital fat and muscle. AJNR Am J Neuroradiol. 1986;7(4):651-6. 20. Chapman VM, Grottkau BE, Albright M, Salamipour H, Jaramillo D. Multidetector computed tomography of pediatric lateral condylar fractures. J Comput Assist Tomogr. 2005;29(6):842-6, http:// dx.doi.org/10.1097/01.rct.0000175504.64707.e3. 21. Bartley GB, Gorman CA. Diagnostic criteria for Graves’ ophthalmopathy. Am J Ophthalmol. 1995;119(6):792-5. 22. Wall M, Neahring RK, Woodward KR. Sensitivity and specificity of frequency doubling perimetry in neuro-ophthalmic disorders: a comparison with conventional automated perimetry. Invest Ophthalmol Vis Sci. 2002;43(4):1277-83. 23. Kazim M, Trokel SL, Acaroglu G, Elliott A. Reversal of dysthyroid optic neuropathy following orbital fat decompression. Br J Ophthalmol. 2000;84(6):600-5, http://dx.doi.org/10.1136/bjo.84.6.600. 24. Kennerdell JS, Rosenbaum AE, El-Hoshy MH. Apical optic nerve compression of dysthyroid optic neuropathy on computed tomography. Arch Ophthalmol. 1981;99(5):807-9, http://dx.doi.org/10.1001/ archopht.1981.03930010807002. 25. Hallin ES, Feldon SE. Graves’ ophthalmopathy: I. Simple CT estimates of extraocular muscle volume. Br J Ophthalmol. 1988;72(9):674-7, http:// dx.doi.org/10.1136/bjo.72.9.674. 26. Hudson HL, Levin L, Feldon SE. Graves exophthalmos unrelated to extraocular muscle enlargement. Superior rectus muscle inflammation may induce venous obstruction. Ophthalmology. 1991;98(10):1495-9. 27. Gorman CA. Radiotherapy for Graves’ ophthalmopathy: results at one year. Thyroid. 2002;12(3):251-5, http://dx.doi.org/10.1089/105072502 753600232. 28. Hu WD, Annunziata CC, Chokthaweesak W, Korn BS, Levi L, Granet DB, et al. Radiographic analysis of extraocular muscle volumetric changes in thyroid-related orbitopathy following orbital decompression. Ophthal Plast Reconstr Surg. 2010;26(1):1-6, http://dx.doi.org/10.1097/ IOP.0b013e3181b80fae. 29. Alsuhaibani AH, Carter KD, Policeni B, Nerad JA. Orbital volume and eye position changes after balanced orbital decompression. Ophthal Plast Reconstr Surg. 2011;27(3):158-63. 30. Liao SL, Huang SW. Correlation of retrobulbar volume change with resected orbital fat volume and proptosis reduction after fatty decompression for Graves ophthalmopathy. Am J Ophthalmol. 2011;151(3):4659 e1, http://dx.doi.org/10.1016/j.ajo.2010.08.042. 31. Regensburg NI, Wiersinga WM, Berendschot TT, Saeed P, Mourits MP. Effect of smoking on orbital fat and muscle volume in Graves’ orbitopathy. Thyroid. 2011;21(2):177-81, http://dx.doi.org/10.1089/ thy.2010.0218. 32. Bijlsma WR, Mourits MP. Radiologic measurement of extraocular muscle volumes in patients with Graves’ orbitopathy: a review and guideline. Orbit. 2006;25(2):83-91, http://dx.doi.org/10.1080/01676830600675319.

both the Nugent scale, which was evaluated in several previous studies (4,14,16,17), and the linear muscle index described by Barret et al. (13). The latter suggested that an index of 67% or greater would have a diagnostic sensitivity of 67% for detecting DON (13). In a previous study that investigated the sensitivity and specificity of this muscle index using MDCT, the best sensitivity/specificity combination (79%/72%) was observed at a muscle index of 60% (5). This is clearly inferior to the performance of the indexes tested in the current study. In conclusion, our study tested the value of two volumetric indexes for assessing the degree of apical crowding, one based on measurements of the entire orbit and one based on measurements of the orbital apex only. Both indexes were found to be efficient at predicting DON, although the latter index was more effective. Further studies are necessary to validate our findings.

ACKNOWLEDGMENTS This work was supported by grants from Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo FAPESP (N˚ 2012/50392) Sa˜o Paulo, Brazil, and from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) (N˚ 306487/2011-0) Brası´lia, Brazil.

AUTHOR CONTRIBUTIONS Gonc¸alves AC contributed to the project design, data collection, data management, and writing the manuscript. Silva LN designed the project and collected data. Gebrim EM designed the project, collected data and revised the manuscript. Matayoshi S designed the project and revised the manuscript. Monteiro ML designed the project, analyzed data, and wrote and revised the manuscript.

REFERENCES 1. Naik VM, Naik MN, Goldberg RA, Smith TJ, Douglas RS. Immunopathogenesis of thyroid eye disease: emerging paradigms. Surv Ophthalmol. 2010;55(3):215-26, http://dx.doi.org/10.1016/ j.survophthal.2009.06.009. 2. Hallin ES, Feldon SE. Graves’ ophthalmopathy: II. Correlation of clinical signs with measures derived from computed tomography. Br J Ophthalmol. 1988;72(9):678-82. 3. Monteiro MLR, Moritz RBS, Angotti-Neto H, Moritz R, Benabou J. Color Doppler imaging of the superior ophthalmic vein in patients with Graves’ orbitopathy before and after treatment of congestive disease. Clinics. 2011;66(6):1329-34. 4. Giaconi JA, Kazim M, Rho T, Pfaff C. CT scan evidence of dysthyroid optic neuropathy. Ophthal Plast Reconstr Surg. 2002;18(3):177-82. 5. Monteiro ML, Goncalves AC, Silva CT, Moura JP, Ribeiro CS, Gebrim EM. Diagnostic ability of Barrett’s index to detect dysthyroid optic neuropathy using multidetector computed tomography. Clinics. 2008;63(3):301-6, http://dx.doi.org/10.1590/S1807-59322008000300003. 6. Neigel JM, Rootman J, Belkin RI, Nugent RA, Drance SM, Beattie CW, et al. Dysthyroid optic neuropathy. The crowded orbital apex syndrome. Ophthalmology. 1988;95(11):1515-21. 7. Bartley GB. The epidemiologic characteristics and clinical course of ophthalmopathy associated with autoimmune thyroid disease in Olmsted County, Minnesota. Trans Am Ophthalmol Soc. 1994;92:477588. 8. Ben Simon GJ, Syed HM, Douglas R, Schwartz R, Goldberg RA, McCann JD. Clinical manifestations and treatment outcome of optic neuropathy in thyroid-related orbitopathy. Ophthalmic Surg Lasers Imaging. 2006;37(4):284-90. 9. Monteiro ML, Portes AL, Moura FC, Regensteiner DB. Using frequencydoubling perimetry to detect optic neuropathy in patients with Graves’ orbitopathy. Jpn J Ophthalmol. 2008;52(6):475-82, http://dx.doi.org/ 10.1007/s10384-008-0579-x.

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CLINICS 2012;67(8):897-900

DOI:10.6061/clinics/2012(08)07

CLINICAL SCIENCE

Effects of arterial oxygen tension and cardiac output on venous saturation: a mathematical modeling approach Fernando Godinho Zampieri,I,II Marcelo Park,I,III Luciano Ce´sar Pontes Azevedo,I,III Marcelo Britto Passos Amato,IV Eduardo Leite Vieira CostaIII,IV I

Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Emergency Medicine Discipline, Intensive Care Unit, Sa˜o Paulo/SP, Brazill. Hospital Alema˜o Oswaldo Cruz, Sa˜o Paulo/SP, Brazil. III Hospital Sı´rio-Libaneˆs, Research and Education Institute, Sa˜o Paulo, Brazil. IV Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Cardio-Pulmonary Department, Pulmonary Divison, Respiratory Intensive Care Unit, Sa˜o Paulo/ SP, Brazil. II

OBJECTIVES: Hemodynamic support is aimed at providing adequate O2 delivery to the tissues; most interventions target O2 delivery increase. Mixed venous O2 saturation is a frequently used parameter to evaluate the adequacy of O2 delivery. METHODS: We describe a mathematical model to compare the effects of increasing O2 delivery on venous oxygen saturation through increases in the inspired O2 fraction versus increases in cardiac output. The model was created based on the lungs, which were divided into shunted and non-shunted areas, and on seven peripheral compartments, each with normal values of perfusion, optimal oxygen consumption, and critical O2 extraction rate. O2 delivery was increased by changing the inspired fraction of oxygen from 0.21 to 1.0 in steps of 0.1 under conditions of low (2.0 L.min-1) or normal (6.5 L.min-1) cardiac output. The same O2 delivery values were also obtained by maintaining a fixed O2 inspired fraction value of 0.21 while changing cardiac output. RESULTS: Venous oxygen saturation was higher when produced through increases in inspired O2 fraction versus increases in cardiac output, even at the same O2 delivery and consumption values. Specifically, at high inspired O2 fractions, the measured O2 saturation values failed to detect conditions of low oxygen supply. CONCLUSIONS: The mode of O2 delivery optimization, specifically increases in the fraction of inspired oxygen versus increases in cardiac output, can compromise the capability of the ‘‘venous O2 saturation’’ parameter to measure the adequacy of oxygen supply. Consequently, venous saturation at high inspired O2 fractions should be interpreted with caution. KEYWORDS: Mathematical Modeling; Central Venous Saturation, Cardiac Output. Zampieri FG, Park M, Azevedo LC, Amato MB, Costa EL. Effects of arterial oxygen tension and cardiac output on venous saturation: a mathematical modeling approach. Clinics. 2012;67(8):897-900. Received for publication on February 27, 2012; First review completed on March 22, 2012; Accepted for publication on April 8, 2012 E-mail: fgzampieri@gmail.com Tel.: 55 11 2661-6457

hemoglobin oxygen affinity, arterial partial pressure of oxygen (PaO2), and cardiac output (CO). It is possible to achieve comparable changes in DO2 by manipulating these variables within physiological limits, although the impact on VO2 might depend on what parameter is modified (6). Measuring oxygen consumption at the bedside is complex. Therefore, surrogates of the adequacy of oxygen delivery, such as lactate concentration, venous partial pressure of oxygen (PvO2), and mixed (SvO2) or central venous oxygen saturation, are commonly used during routine patient care (1,5,7). Venous oxygen saturation is part of the current guidelines for early resuscitation during sepsis (7). Nevertheless, SvO2 can respond differently to changes in DO2 if the changes are obtained through changes in different component of the DO2 equation (8), even when these changes lead to similar VO2 values. In this paper, we developed a mathematical model to investigate whether the

INTRODUCTION The ultimate goal of hemodynamic support is to provide enough oxygen delivery (DO2) to guarantee tissue oxygen needs and avoid organ dysfunction (1,2). Under normal conditions, DO2 exceeds oxygen consumption (VO2) to an extent that makes consumption independent of delivery (3,4). However, during regional low delivery, oxygen consumption becomes supply dependent (3-5). Oxygen delivery depends on the hemoglobin concentration (Hb),

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.

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perfusion fraction, its critical oxygen extraction rate and optimal VO2. In the first run of the model, mixed venous oxygen saturation and mixed venous content were calculated according to equation 1 using an arbitrary initial mixed venous oxygen partial pressure of 40 mmHg. Pulmonary capillary oxygen content was obtained through equations 2 and 3, and arterial oxygen content was estimated according to the pulmonary shunt fraction (equation 4). Oxygen delivery to each compartment was subsequently calculated by multiplying the compartment perfusion fraction by the global oxygen delivery. A new venous oxygen content value for each compartment was then calculated using equation 6, and the global mixed venous oxygen content was obtained from a perfusion-weighted average of the local venous contents (equation 7).

type of DO2 optimization, specifically increases in the arterial partial pressure of oxygen versus increases in cardiac output, would have different effects on SvO2.

METHODS The mathematical model was created based on the lungs and on seven peripheral compartments. Lung blood flow was divided into shunted and non-shunted areas. The peripheral compartments, each with its own perfusion, optimal VO2, and critical oxygen extraction rate, were created to simulate the behavior of the flow to organs. Having multiple peripheral compartments in the model was important to simulate the heterogeneity of the oxygen supply/demand in different organs and tissues to reproduce the complex relationship between mixed venous oxygen saturation and oxygen delivery. In all of the following notations, ‘‘x’’ represents venous (‘‘v’’), lung capillary (‘‘c’’) or arterial blood (‘‘a’’). Hemoglobin oxygen saturation (SxO2) was calculated according to equation 1 (9): nh i o{1 {1 PxO32 z150 : PxO2 : 23,400 z1 SxO2 ~

  DO2(n) {VO2 (n) CvO2(n) ~ Q(n) :CO

ð1Þ CvO2 ~

Blood oxygen content (CxO2) was calculated according to a standard formula (equation 2) (9): CxO2 ~1:36|Hb|SxO2 z0:0031|PxO2

ð2Þ

ð3Þ

ð4Þ

RESULTS

DO2 and VO2 in the peripheral compartments: We used seven compartments to simulate blood flow to the brain, heart, kidneys, muscles, splanchnic (liver), skin, and ‘others’, each with normal values of perfusion fraction, extraction rate, and optimal oxygen consumption (VO2optimal) (11). Because the arterial oxygen content was the same for all compartments, differences in oxygen supply from one compartment to the other occurred through differences in regional perfusion. For each compartment, the oxygen consumption was calculated according to equation 5:

nVO

2 ~DO2 :ER if DO2 :ERvVO2optimal VO2 ~VO2optimal if DO2 : ER§VO2optimal

ð7Þ

The mixed venous partial pressure of oxygen was determined from the mixed venous oxygen content by solving equations 1 and 2 to an acceptable error of 1:1,000 using the Newton-Raphson method. The new calculated value of the mixed venous partial pressure of oxygen was then reentered into the model, replacing the initial guess. We repeated these calculations until the difference between successive approximations of the mixed venous partial pressure of oxygen values was less than 1:1,000 of the previous value. The model outputted compartment and global final values of both the arterial and venous partial pressures of oxygen and saturation values. The model was designed using The R Project for Statistical Computing (www.r-project.org) with the rootsolve package.

As mentioned, the lungs were composed of shunted and non-shunted areas. Solving Berggren’s shunt equation for CaO2 gives the following equation (where Fshunt is the pulmonary shunt fraction): CaO2 ~CcO2 :ð1{Fshunt ÞzCvO2 :Fshunt

CvO2(n) :Q(n)

n~1

Lungs: The alveolar partial pressure of oxygen was calculated using the alveolar gas equation (equation 3) and used as an approximation of the capillary partial pressure of oxygen (10): PcO2 ~ð760{47Þ:FiO2 {PaCO2

7 X

ð6Þ

In all simulations, we maintained the following constant global values: Hb 14 g/dL, PaCO2 40 mmHg, pH 7.40, and a pulmonary shunt fraction of 0.1. Normal values of the perfusion fraction, which were critical for the oxygen extraction rate and optimal oxygen consumption, were also provided for each peripheral compartment (11). We modeled two different states: normal-high and low oxygen delivery. For each of these states, we varied the oxygen delivery by changing the FiO2 with a fixed CO and changing the CO with a fixed FiO2. The first step was to obtain increasing DO2 values by changing the FiO2 from 0.21 to 1.00 in steps of 0.10 (with the exception of the first step = 0.09) with two values of cardiac output: 1) CO = 2.0 L.min-1 (low oxygen delivery) and 2) CO = 6.5 L.min-1 (normal-high oxygen delivery). In the next step, with a constant fraction of inspired oxygen (0.21), we chose cardiac output values to match the DO2 obtained in the previous step. In the normal-high delivery state, oxygen demands were always met (VO2$197 mL.min-1), whereas in the low

ð5Þ

The mathematical model: The input variables for the model included global values of cardiac output, pulmonary shunt fraction, pH, arterial carbon dioxide tension, Hb, and fraction of inspired oxygen. Additionally, we supplied hemodynamic variables for each compartment, including its

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Effects of arterial tension on SvO2 Zampieri FG et al.

Table 1 - Effect of increasing the oxygen delivery on oxygen venous saturation by manipulating either the inspired fraction of oxygen or the cardiac output. Changes in FiO2 with fixed CO DO2

VO2 -1

Normal-high supply set

Low supply set

CO

FiO2 -1

(mL.min )

(mL.min )

1,208 1,238 1,256 1,271 1,286 1,300 1,315 1,329 1,344 368 377 383 387 392 396 401 405 410

197 197 197 197 197 197 197 197 197 125 128 129 130 131 133 134 135 136

SvO2 -1

0.21 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0.21 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

Changes in CO with fixed FiO2

(L.min )

(%)

6.50 6.50 6.50 6.50 6.50 6.50 6.50 6.50 6.50 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00

81 83 85 86 87 88 89 90 92 63 65 66 67 68 69 70 70 71

FiO2

CO

SvO2 -1

0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21

(L.min )

(%)

6.50 6.66 6.75 6.84 6.91 6.99 7.07 7.15 7.22 2.00 2.05 2.08 2.10 2.13 2.15 2.18 2.20 2.22

81 81 82 82 82 82 82 82 83 63 63 64 64 64 64 64 64 64

CO: cardiac output; DO2: oxygen delivery; FiO2: inspired fraction of oxygen; SvO2: venous oxygen saturation; VO2: oxygen consumption.

delivery state, the oxygen supplies were insufficient to meet the oxygen demands (VO2,197 mL.min-1). Normal-high supply set under this set of conditions, we simulated increases in DO2 under optimal oxygen consumption to all compartments (Table 1). The increase in CO caused a proportional increase in DO2, as expected. Conversely, DO2 could only be increased by 11% by nearly quintupling FiO2 from 0.21 to 1.0. This increase led to a

change in SvO2 from 81 to 92%; in contrast, when the same increase in DO2 was accomplished by increasing CO, the SvO2 only increased from 81 to 83%. In fact, even a 50% increase in DO2 (Figure 1A) accomplished through augmenting CO alone only increased SvO2 to 86%. Of note, for this set of conditions, the peripheral compartments could have been easily lumped together with no loss of information because VO2 was constant among the different conditions simulated.

Figure 1 - Panel A: Effect of increasing DO2 on the SvO2 through changes in the inspired fraction of oxygen (FiO2, solid line) or cardiac output (dashed line) under normal and high oxygen supply conditions sufficient to attain optimal oxygen consumption in all peripheral compartments. Panel B: Effect of increasing DO2 on the SvO2 through changes in the inspired fraction of oxygen (FiO2, solid line) or cardiac output (dashed line) under low oxygen supply conditions that caused the oxygen consumption to be supply limited.

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same amount (from 12 to 16 mL/dL), causing venous saturation to increase from 62 to 83%. The same 25% increase in DO2 caused by increasing CO will only decrease the arteriovenous difference in oxygen content from 4.0 to 3.2 mL/dL, thus increasing the venous oxygen content from 12 to 12.8 mL/dL. The result would be a modest increase in the central venous oxygen saturation from 62 to 66%. Our study has several limitations. We kept the values of pH and the arterial partial pressure of carbon dioxide, variables known to be affected by the hemodynamic condition, constant during all simulations. Although the model could accommodate changes in these variables, we decided to keep them constant to isolate the effects of the fraction of inspired oxygen and cardiac output. Additionally, for similar reasons, we ignored potential changes in the perfusion fraction that could be caused by the local control of blood flow during regional hypoxemia. Blood flow to tissues is usually driven by local metabolic demands to match supply and demand. Therefore, this model does not take into account changes in the local oxygen consumption that may occur after changes in the local partial pressures of oxygen and carbon dioxide. In conclusion, we showed that the type of DO2 optimization, specifically increases in FiO2 versus increases in CO, could affect the capability of venous oxygen saturation to measure the adequacy of oxygen supply. Interpreting venous saturation at high arterial partial pressures of oxygen values should be performed with caution.

Low supply set When VO2 was supply limited, the relation between DO2 and SvO2 became more complex as a consequence of the critical extraction rates being reached at different DO2 values (Table 1 and Figure 1). Interestingly, the modest 11% increase in DO2 produced by augmenting FiO2 markedly increased SvO2 to values above 70%, masking the 31% deficit of VO2 present at an FiO2 of 1.0 (Table 1). In contrast, increasing DO2 by the same amount by improving CO led to an SvO2 of 64%, indicating insufficient oxygen supply.

DISCUSSION In this paper, we showed that when oxygen supply was increased by the same amount by increasing either FiO2 or CO, the resulting SvO2 was higher when DO2 was increased by manipulating FiO2. Of note, we found that under conditions of low oxygen supply, high values of the fraction of inspired oxygen could increase the mixed venous saturation to normal levels, masking states of insufficient oxygen supply. Mixed venous saturation has long been used as a surrogate for adequate perfusion to tissues during resuscitation (5). Low levels of SvO2 are associated with poor prognosis for several clinical scenarios, and currently, guidelines suggest targeting SvO2 at 70% during the early phase of sepsis resuscitation, which is associated with a reduction in mortality (7). Nowhere in those guidelines, however, do the authors comment on the FiO2 at which the central venous blood gas should be collected. One strength of the theoretical modeling approach is that it is possible to compare the effects of FiO2 and CO on the mixed venous saturation at precisely the same oxygen supply/demand conditions, i.e., equal VO2 and DO2. Ho et al. have previously shown that in humans with shock, high levels of PaO2 can increase SvO2 levels (8). From their data, however, it was not possible to determine whether the increase in SvO2 was indeed reflecting a better oxygen supply/demand relationship or whether the SvO2 represented a false marker of the adequacy of such a relationship. Our findings of increases in SvO2 to levels considered normal caused by increasing the FiO2 in the setting of insufficient oxygen supply support the latter hypothesis. The mechanism by which increased values of FiO2 cause a steeper increase in SvO2 than that caused by increases in CO can be explained by the conservation of mass. When manipulating the FiO2 at fixed values of CO and oxygen consumption, the arteriovenous difference in oxygen content (CaO2 – CvO2) must remain constant because oxygen consumption is equal to the product of cardiac output and the arteriovenous difference in oxygen content. Thus, in this scenario, increases in arterial oxygen content will be followed by equal increases in venous oxygen content, explaining the roughly linear relationship between SvO2 and DO2 shown in Figure 1. Conversely, when manipulating CO while keeping arterial oxygen content and oxygen consumption constant, the increase in CO will be compensated for by an increase in the venous oxygen content proportional to the arteriovenous difference in oxygen content such that at high CO values, the venous oxygen content approaches the arterial oxygen content. For example, using the Fick principle, if one assumes a hypothetical initial arterial oxygen content of 16 mL/dL and a venous oxygen content of 12 mL/dL, a 25% increase in the DO2 by increasing the arterial oxygen content to 20 mL/ dL will cause an increase in venous oxygen content by the

AUTHOR CONTRIBUTIONS Zampieri FG wrote the manuscript, performed simulations with the model and wrote the mathematical model. Park M revised the manuscript and helped with model construction. Azevedo LCP performed simulations with the model and revised the manuscript. Amato MBP revised the manuscript and helped with model construction. Costa EDL designed and wrote the mathematical model and helped with manuscript preparation.

REFERENCES 1. Rivers E, Nguyen B, Havstad S, Ressler J, Muzzin A, Knoblich B, et al. Early goal-directed therapy in the treatment of severe sepsis and septic shock. N Engl J Med. 2001 8;345(19):1368-77, http://dx.doi.org/10.1056/ NEJMoa010307. 2. da Silva Ramos FJ, Azevedo LC. Hemodynamic and perfusion end points for volemic resuscitation in sepsis. Shock. 2010;34 Suppl 1:34-9, http://dx.doi.org/10.1097/SHK.0b013e3181e7e642. 3. Schumacker PT, Cain SM. The concept of a critical oxygen delivery. Intensive Care Med. 1987;13(4):223-9, http://dx.doi.org/10.1007/ BF00265110. 4. Ronco JJ, Fenwick JC, Tweeddale MG, Wiggs BR, Phang PT, Cooper DJ, et al. Identification of the critical oxygen delivery for anaerobic metabolism in critically ill septic and nonseptic humans. JAMA. 1993;270(14):1724-30, http://dx.doi.org/10.1001/jama.1993.03510140084034. 5. Marx G, Reinhart K. Venous oximetry. Curr Opin Crit Care. 2006;12(3):263-8, http://dx.doi.org/10.1097/01.ccx.0000224872.09077.dc. 6. Lorente JA, Landin L, De PR, Renes E, Rodriguez-Diaz R, Liste D. Effects of blood transfusion on oxygen transport variables in severe sepsis. Crit Care Med. 1993;21(9):1312-8, http://dx.doi.org/10.1097/00003246199309000-00013. 7. Dellinger RP, Levy MM, Carlet JM, Bion J, Parker MM, Jaeschke R, et al. Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008. Crit Care Med. 2008;36(1):296-327, http://dx.doi.org/10.1097/01.CCM.0000298158.12101.41. 8. Ho KM, Harding R, Chamberlain J. The impact of arterial oxygen tension on venous oxygen saturation in circulatory failure. Shock. 2008;29(1):3-6. 9. Severinghaus JW. Simple, accurate equations for human blood O2 dissociation computations. J Appl Physiol. 1979;46(3):599-602. 10. RILEY RL, Cournand A. Ideal alveolar air and the analysis of ventilationperfusion relationships in the lungs. J Appl Physiol. 1949;1(12):825-47. 11. Finch CA, Lenfant C. Oxygen transport in man. N Engl J Med. 1972;286(8):407-15, http://dx.doi.org/10.1056/NEJM197202242860806.

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DOI:10.6061/clinics/2012(08)08

CLINICAL SCIENCE

M2-polarized macrophages promote metastatic behavior of Lewis lung carcinoma cells by inducing vascular endothelial growth factor-C expression Bicheng Zhang,I Yafei Zhang,I,II Guoqing Yao,I Juan Gao,III Bo Yang,I Yong Zhao,I Zhiguo Rao,I Jianfei GaoI I

Wuhan General Hospital of Guangzhou Command, Department of Oncology, People’s Liberation Army, Wuhan, Hubei Province, China. II Third Military Medical University Southwest Hospital, Department of Gastroenterology, Chongqing, China. III Wuhan General Hospital of Guangzhou Command, Department of Gastroenterology, People’s Liberation Army, Wuhan, Hubei Province, China.

OBJECTIVES: Tumor-associated macrophages that generally exhibit an alternatively activated (M2) phenotype have been linked to tumor progression and metastasis. However, the role of M2-polarized macrophages in the growth and metastasis of lung adenocarcinoma remains enigmatic. The aim of this study was to explore the effect of M2 macrophages on the proliferation and migration of mouse Lewis lung carcinoma cells and tumor-induced lymphangiogenesis. METHODS: Trypan blue staining and the Transwell migration assay were performed to evaluate the effects of activated (M1 or M2) macrophages on the proliferation and migration of Lewis cells. Furthermore, vascular endothelial growth factor-C expression in Lewis cells and nitric oxide secretion from activated macrophages were detected during the co-culture assay. Following treatment with activated macrophages, lymphatic endothelial cells differentiated into capillary-like structures, and the induction of Lewis cell migration was assessed using a twodimensional Matrigel-based assay. RESULTS: In the co-culture Transwell system, the proliferation and migration of Lewis cells were promoted by M2 macrophages. Moreover, the co-culture significantly increased the expression of vascular endothelial growth factorC by Lewis cells and reduced the secretion of nitric oxide from M2 macrophages, which subsequently led to the capillary morphogenesis of lymphatic endothelial cells. Interestingly, following co-culture with Lewis cells, the function of RAW264.7 cells was polarized toward that of the M2 macrophage phenotype. CONCLUSION: M2-polarized macrophages promoted the metastatic behavior of Lewis cells by inducing vascular endothelial growth factor-C expression. Thus, the interruption of signaling between M2 macrophages and Lewis cells may be considered to be a new therapeutic strategy. KEYWORDS: M2-polarized macrophages; Lewis lung carcinoma; Proliferation; Migration; Lymphangiogenesis. Zhang B, Zhang Y, Yao G, Gao J, Yang B, Zhao Y, et al. M2-polarized macrophages promote metastatic behavior of Lewis lung carcinoma cells by inducing vascular endothelial growth factor-C expression. Clinics. 2012;67(8):901-906. Received for publication on February 2, 2012; First review completed on March 2, 2012; Accepted for publication on April 10, 2012 E-mail: jianfeigao1957@hotmail.com Tel.: 86 27 68878461

tumor growth, angiogenesis, and lymphangiogenesis and are the key regulators of the metastatic phenotype of cancer cells (1-3). Moreover, in lung adenocarcinoma, this pro-tumor role of TAMs is further supported by clinical studies demonstrating a correlation between high macrophages numbers in tumor tissue and poor patient prognosis (4-7). However, the activated phenotype and polarization status of TAMs in solid tumors are not well-understood, and the results of previous reports have often been contradictory. There appear to be at least two different subpopulations of activated macrophages coexisting in the tumor microenvironment (8,9). The first subpopulation is referred to as classically activated macrophages (M1 macrophages), which are characterized by the IL-12high, IL-23high, and IL-10low phenotype, and these macrophages can produce tumor necrosis factor (TNF)-a and nitric oxide (NO). Traditionally, M1 macrophages are regarded as potent effector cells that

INTRODUCTION Metastasis is the main cause of cancer-related mortality secondary to malignancy. It has been clearly demonstrated that inflammatory cells in the tumor microenvironment play an important role in tumor growth, progression, and metastasis. Within the tumor stroma, tumor-associated macrophages (TAMs) constitute a pivotal class of inflammatory cells, and compelling evidence has emerged to suggest that TAMs play a promoting role in processes such as carcinogenesis,

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.

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are able to kill microorganisms and tumor cells. The other subpopulation is termed alternatively activated macrophages (M2 macrophages). Exposure to IL-4, IL-13, vitamin D3, glucocorticoids, or transforming growth factor-b (TGF-b) decreases the antigen-presenting capability of macrophages and up-regulates the expression of macrophage mannose receptors (MMR, also known as CD206), scavenger receptors (SR-A, also known as CD204), CD163, dectin-1 and DC-SIGN (10). M2-polarized macrophages have the IL-12low, IL-23low, and IL-10high phenotype and are involved in stromal formation, tissue repair, tumor growth, angiogenesis, lymphangiogenesis, and immunosuppression. Although these two subpopulations have been extensively characterized, the role of M1- or M2-polarized macrophages in the progression and metastasis of lung cancer has not been clearly elucidated. Here, we investigated the effect of M2-polarized macrophages on the metastatic behavior of Lewis lung carcinoma (LLC) cells in a co-culture Transwell system and found that M2 macrophages could promote the proliferation and migration of LLC cells and stimulate tumor-induced lymphangiogenesis by inducing VEGF-C (vascular endothelial growth factor-C) expression.

iii) M2 group, and iv) RAW264.7 group. The proliferation of the LLC cells was determined by counting triplicate plates at the indicated times during a 6-day culture period using a Coulter counter. Cell viability for each experiment was determined by trypan blue staining. The results presented represent the mean values from three separate experiments.

Cell migration assay The migration of LLC cells was assayed in Transwell cell culture chambers with a porous (8.0 mm pore size) polycarbonate membrane filter (Millipore Corp., Bedford, MA, USA), as described previously (13). Briefly, 16104 LLC cells were seeded in the upper chamber. Following attachment, 1 ml of culture supernatant was added to the lower well. After 24 h of incubation, the membranes were removed and stained with hematoxylin and eosin (H&E), and the number of cells that had invaded the lower chamber was counted in three randomly selected fields under light microscopy.

Immunocytochemistry for VEGF-C expression Cell smears were prepared from the co-cultures described above and were then immunostained with a mouse monoclonal VEGF-C antibody (1:100; Boster, Wuhan, CHINA) at 4 ˚C overnight. The cells were subsequently exposed to a biotinylated secondary antibody for 20 min, which was followed by treatment with streptavidin peroxidase. For color development, the slides were stained with 3,3’-diaminobenzidine (DAB) and then counterstained with H&E. A redbrown precipitate in the cytoplasm of the LLC cells indicated a positive reaction. The true color multi-function CMIAS pathological image-analyzing system was used for spectrodensitometry, and the integral optical density (IOD) represented the relative intensity of positive VEGF-C expression. Five magnified fields (2006) were selected randomly, and the mean values were calculated for statistical comparison.

MATHERIAL AND METHODS Cell lines Mouse RAW264.7 macrophages and LLC cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and were maintained in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS; both from Invitrogen, Carlsbad, CA, USA). The isolation and culture of lymphatic endothelial cells (LECs) were performed as described in our previous report (11). Briefly, female Balb/c mice were intraperitoneally injected with emulsified incomplete Freund’s adjuvant (Sigma-Aldrich, St Louis, MO, USA) to induce lymphangioma formation. After 30 days of induction, tumors in the peritoneal cavity were removed and mechanically disrupted, and LECs were isolated and resuspended in endothelial cell basal medium-2 (EBM-2; Cambrex BioScience, Wokingham, UK) supplemented with 20% FBS and 50 ng/ml endothelial cell growth supplement (Cambrex BioScience, Wokingham, UK), and these cells were grown at 37 ˚C in a humidified atmosphere of 5% CO2. LECs were used in appropriate experiments or cultivated until the fourth passage.

NO production As described previously, NO production was estimated according to the accumulation of NO2- in the medium after 24 h of macrophage activation using the Greiss reagent (9). Briefly, equal volumes of culture supernatant and Greiss reagent (100 ml) were mixed for 10 min at room temperature. The absorbance at 540 nm was measured using a Labsystems Multiscan Ascent assay plate reader. A graded solution of NO2 was used to construct a standard curve, and the results presented represent the mean values from three separate samples.

Activation of RAW264.7 macrophages As described in our previous study (12), M2- or M1polaried macrophages were prepared by stimulating RAW264.7 macrophages with 10 IU/ml mouse recombinant IL-4 (Cytolab Ltd., Rehovot, Israel) or 100 IU/ml IFN-c (Cytolab Ltd., Rehovot, Israel) plus l0 ng/ml LPS (SigmaAldrich, St Louis, MO, USA) overnight, respectively.

In vitro capillary morphogenesis assay LEC differentiation into capillary-like structures was determined using a two-dimensional Matrigel-based assay. Initially, ice-cold growth factor-reduced Matrigel was placed into each well of a 24-well tissue culture plate. The plates were maintained at 37˚C until the Matrigel had fully solidified. LECs were harvested in complete media, washed once, and then resuspended in serum-free EBM media. LECs were mixed with media collected from LLC cells, and they were subsequently treated with activated macrophage-conditioned medium for 24 h. After 24 h of incubation, the formation of tube-like structures was monitored by microscopic observation at 1006 magnification, and more than 12 different fields in each well were photographed to measure the length of the tube-like structures, as described previously (14).

Cell proliferation assay A Transwell system with a porous (0.4 mm pore size) polycarbonate membrane filter (Millipore Corp., Bedford, MA, USA) and 24-well plastic tissue culture plates were used for the macrophage-LLC co-cultures. LLC cells were first seeded into 24-well culture plates at a subconfluent density of 16104 cells/well. Four hours later, different types of activated macrophages (16104/well) were washed three times in PBS and then added to the upper chambers. The resultant four groups were as follows: i) blank control group, ii) M1 group,

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Figure 1, the addition of both M2 macrophages and RAW264.7 cells significantly increased the proliferation of LLC cells compared to the control (p,0.05). Furthermore, the proliferation velocity of LLC cells in co-culture with M2 macrophages was greater than that observed for the RAW264.7 co-culture group. However, the proliferation of LLC cells was inhibited following co-culture with M1 macrophages.

M2 macrophages induce migration of LLC cells To determine the effect of activated macrophages on the migratory behavior of LLC cells, we utilized a Transwell system to evaluate cell migration. As shown in Figure 2, M2 macrophages and RAW264.7 cells significantly increased the migratory potential of LLC cells compared to the control condition (p,0.01), whereas M1 macrophages did not alter the migration of LLC cells. Furthermore, the migratory rate of LLC cells cultured with M2 macrophages was faster than that observed for the culture with RAW264.7 cells (p,0.05).

Figure 1 - Dynamic changes in LLC cell growth. Following culture of a blank control sample or co-culture with M1, M2, and RAW264.7 macrophages, the LLC cell number and viability were evaluated by trypan blue staining at the indicated times during a 6-day period. The data shown represent three experiments. * p,0.05 and ** p,0.01 compared to the blank control.

M2 macrophages increase VEGF-C expression in LLC cells

Statistical analysis The measurement data are expressed as the means¥SD. Differences were compared using a one-way ANOVA analysis followed by Student’s t-test. A p-value ,0.05 was considered to be statistically significant. All statistical analyses were performed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA).

We next explored whether activated macrophages could induce the production of pro-lymphangiogenic stimuli in LLC cells, which would lead to the capillary morphogenesis of LECs. Following co-culture, the numbers of LLC cells cocultured M2 macrophages or RAW264.7 cells were increased in comparison to the non-co-cultured group and the M1 macrophage co-culture group. In addition, changes in the morphology of LLC cells cultured with M2 macrophages or RAW264.7 cells were noted. Next, we sought to evaluate the expression of VEGF-C, a primary lymphatic vessel growth factor, using immunocytochemistry. With this technique, IOD values were used to represent the level of VEGF-C expression in LLC cells. As shown in Figure 3, the IODs for VEGF-C in the blank control, M1, M2 and RAW264.7

RESULTS M2 macrophages induce the proliferation of LLC cells To evaluate the effect of activated macrophages on the proliferation of LLC cells, cell viability was determined with trypan blue staining to create a growth curve. As shown in

Figure 2 - Effect of activated macrophages on the migration of LLC cells. (A) Following the culture of a blank control or the addition of M1 macrophages, M2 macrophages or RAW264.7 macrophages for 24 h, invasive LLC cells on the outer surface of the upper chambers were stained with H&E (2006). (B) Comparison to the penetrated cells. The data shown represent three experiments. ** p,0.01 compared to the blank control.

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Figure 3 - VEGF-C expression by LLC cells induced following exposure to activated macrophages. (A) VEGF-C could be detected in the blank control and in the M1, M2, and RAW264.7 macrophage co-cultured groups. (B) Comparison of the IODs for VEGF-C. The data shown represent three experiments. * p,0.05 and ** p,0.01 compared to the blank control.

co-cultured groups were 44.53¡8.76, 42.19¡3.52, 264.19¡ 13.36, and 118.51¡10.82, respectively. In comparison to the control condition, co-culture with M2 and RAW264.7 macrophages increased the expression of VEGF-C by LLC cells (p,0.05); however, M2 macrophages had a more pronounced effect on the expression of VEGF-C than the RAW264.7 macrophages. No significant change in VEGF-C expression was observed in the control or M1 macrophage group.

previous study demonstrated that M2-polarized TAMs in lung adenocarcinomas were associated with peritumoral lymphangiogenesis and a poor prognosis (22). Alternatively, in mouse Lewis lung adenocarcinoma, activated TAMs were shown to induce peritumoral lymphangiogenesis via the up-regulation of VEGF-C expression and the promotion of lymphangiogenesis-related behaviors in LECs (12). Moreover, recent studies have revealed that macrophages could enhance the invasiveness of cancer cells both in vitro and in vivo (23-26). It has also been shown that the presence of macrophages within tumors can be associated with a histologically more malignant phenotype that is characterized by an extensive stromal reaction, disorganized matrix deposition and neovascularization (27). Craig et al. (28) also found that the presence of macrophages increased tumor microvascular density in vivo and enhanced the rate of tumor growth. In support of these findings, the current study demonstrated that M2-polarized macrophages, but not M1 macrophages, could increase the proliferation and invasion of LLC cells. Moreover, following co-culture, nonactivated RAW264.7 macrophages were polarized towards the M2 phenotype and LLC cells exhibited higher proliferative rates and a greater invasive potential. In a previous report, Hagemann et al. (18) found that ovarian cancer cells were able to polarize co-cultured macrophages towards a tumorassociated phenotype, which resulted in a TAM activation pattern similar to that found in ovarian tumors in vivo. Hence, our results suggested that RAW264.7 macrophages and LLC cells could mutually alter each other’s behaviors. Furthermore, our data indicated that M2-polarized macrophages could induce the expression of VEGF-C in LLC cells, although they lost the ability to produce NO. In a previous report, Chen et al. (29) confirmed that the expression of specific genes, such as IL-6, IL-8, VEGF, VEGF-C, matrix metalloproteinase (MMP)-9 and MMP-1, was upregulated in non-small cell lung cancer following co-culture with macrophages. In another study, Jedinak et al. (24) demonstrated that activated

M2 macrophages do not produce NO We measured the accumulation of NO2– in culture supernatants using the Greiss assay. M1 macrophages produced relatively high levels of NO (234.12¡14.83 mmol/L), whereas M2 macrophages, RAW264.7 cells and the blank control culture failed to generate significant levels of NO.

M2 macrophages stimulate tumor-induced lymphangiogenesis Using a two-dimensional Matrigel-based assay, we next evaluated the differentiation of LECs into capillary-like structures. As shown in Figure 4 after 24 h of culture, the network of tube-like structures in the M2 macrophage and the RAW264.7 co-culture groups was more extensive than that observed in the M1 macrophage or control group (p,0.05), and there were no significant differences between the two former and latter culture conditions.

DISCUSSION Our data demonstrate that M2-polarized macrophages induced metastatic behavior in lung cancer cells by stimulating their proliferation and migration. In the past decade, various studies have suggested that TAMs adopt the M2polarized phenotype (15-17); however, more recent reports have described the polarization of activated TAMs (i.e., M1 and M2) within the tumor microenvironment (18-21). Our

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Figure 4 - Tumor-induced lymphangiogenesis stimulated by activated macrophages. (A) LECs were seeded in a 24-well plate pre-coated with Matrigel and maintained with media collected from LLC cells treated with activated macrophage-conditioned medium for 24 h. Capillary-like structures in the absence or presence of M1, M2 or RAW264.7 macrophages were determined 24 h later (2006 magnification). (B) Comparison of the tube-like structures formed by LECs following exposure to macrophages with different activated phenotypes. The data shown represent three experiments. * p,0.05 compared to the blank control.

TAMs increase tumor lymphangiogenesis and lymphovascular invasion in cervical cancer and invasive breast cancer. In the current study, we demonstrated that following interaction with M2 macrophages or RAW 264.7 cells, LLC cells expressed higher levels of VEGF-C, and this may have led to increased tumor-induced lymphangiogenesis. In conclusion, the interaction between M2 macrophages and lung adenocarcinoma cells induced the metastatic behavior of Lewis lung adenocarcinoma. Activated M2 macrophages have been shown to be involved in tumor progression and metastasis, which implies that they have the potential to become new targets for tumor therapy (38). Thus, the modulation of macrophage phenotype and function via the administration of immunoregulatory stimuli may provide therapeutic anti-cancer strategies. However, further study is necessary to investigate the detailed mechanisms of these interactions and to evaluate whether similar mechanisms are applicable in vivo.

macrophage-conditioned medium markedly induced the proliferation and migration of human HCT116 colon cancer cells and increased the activation of NF-kB and the secretion of VEGF from colon cancer cells, which subsequently induced the capillary morphogenesis of human aortic endothelial cells. In this study, we demonstrated that both M2 macrophages and RAW 264.7 cells could increase the expression of VEGF-C by LLC cells, which may contribute to the changes observed related to their metastatic behavior. Moreover, in addition to VEGF-C, other factors secreted by LLC cells or M2 macrophages were also likely responsible for the observed behavioral changes of LLC cells, although these factors were not specifically examined in the present study. Furthermore, the high NO levels detected in the M1 macrophage and LLC cell co-culture supernatant were likely responsible for the inhibition of the metastatic behavior of LLC cells. In addition, our data showed that M2-polarized macrophages stimulated tumor-induced lymphangiogenesis in part via the induction of VEGF-C expression. Lymphangiogenesis is considered the initial step and a necessary event in lymphatic and regional lymph node metastasis (30,31). Similar to angiogenesis, lymphangiogenesis consists of a complex multistep process involving endothelial proliferation, migration, and tube-like formation. In addition, these processes can be triggered by the binding of lymphangiogenic growth factors, such as VEGF-C and VEGF-D, to their receptors (including VEGFR-2 and VEGFR-3), which leads to lymphatic spread (32,33). Studies have also shown that macrophages support lymphangiogenesis in two ways: by transdifferentiation and direct incorporation into the endothelial layer or by stimulating the division of preexisting local LECs (34,35). Schoppmann et al. (36,37) reported that the peritumoral inflammatory reaction and VEGF-C-expressing

ETHICS STATEMENT: This study was approved by the Ethics Committee of Wuhan General Hospital, Guangzhou Command of the People’s Liberation Army.

ACKNOWLEDGMENTS We thank Manli Qi (Department of Pathology, Wuhan General Hospital of Guangzhou Command, People’s Liberation Army, Wuhan, China) for her excellent technical assistance. This study was supported by the Natural Science Foundation of Hubei Province, China (No. 2010CDB09204) and the Youth Dawn Plan of Science and Technology in Wuhan, China (No. 201150431137).

AUTHOR CONTRIBUTIONS Zhang B drafted the first manuscript and performed the experiments. Zhang Y performed the experiments, analyzed the data, prepared the

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19. Kawamura K, Komohara Y, Takaishi K, Katabuchi H, Takeya M. Detection of M2 macrophages and colony-stimulating factor 1 expression in serous and mucinous ovarian epithelial tumors. Pathol Int. 2009;59(5):300-5, http://dx.doi.org/10.1111/j.1440-1827.2009.02369.x. 20. Komohara Y, Ohnishi K, Kuratsu J, Takeya M. Possible involvement of the M2 anti-inflammatory macrophage phenotype in growth of human gliomas. J Pathol. 2008;216(1):15-24, http://dx.doi.org/10.1002/ path.2370. 21. Niino D, Komohara Y, Murayama T, Aoki R, Kimura Y, Hashikawa K, et al. Ratio of M2 macrophage expression is closely associated with poor prognosis for Angioimmunoblastic T-cell lymphoma (AITL). Pathol Int. 2010;60(4):278-83, http://dx.doi.org/10.1111/j.1440-1827.2010.02514.x. 22. Zhang B, Yao G, Zhang Y, Gao J, Yang B, Rao Z, et al. M2-polarized tumorassociated macrophages are associated with poor prognosis due to accelerated lymphangiogenesis in lung adenocarcinoma. Clinics. 2011;66(11):1879-86, http://dx.doi.org/10.1590/S1807-59322011001100006. 23. Hagemann T, Wilson J, Kulbe H, Li NF, Leinster DA, Charles K, et al. Macrophages induce invasiveness of epithelial cancer cells via NF-kB and JNK. J Immunol. 2005;175(2):1197-205. 24. Jedinak A, Dudhgaonkar S, Sliva D. Activated macrophages induce metastatic behavior of colon cancer cells. Immunobiology. 2010;215(3): 242-9, http://dx.doi.org/10.1016/j.imbio.2009.03.004. 25. Wyckoff J, Wang W, Lin EY, Wang Y, Pixley F, Stanley ER, et al. A paracrine loop between tumor cells and macrophages is required for tumor cell migration in mammary tumors. Cancer Res. 2004;64(19):70229, http://dx.doi.org/10.1158/0008-5472.CAN-04-1449. 26. Condeelis J, Pollard JW. Macrophages: obligate partners for tumor cell migration, invasion, and metastasis. Cell. 2006;124(2):263-6, http:// dx.doi.org/10.1016/j.cell.2006.01.007. 27. Oosterling SJ, van der Bij GJ, Meijer GA, Tuk CW, van Garderen E, van Rooijen N, et al. Macrophages direct tumour histology and clinical outcome in a colon cancer model. J Pathol. 2005;207(2):147-55, http:// dx.doi.org/10.1002/path.1830. 28. Craig M, Ying C, Loberg RD. Co-inoculation of prostate cancer cells with U937 enhances tumor growth and angiogenesis in vivo. J Cell Biochem. 2008;103(1):1-8, http://dx.doi.org/10.1002/jcb.21379. 29. Chen JJ, Lin YC, Yao PL, Yuan A, Chen HY, Shun CT, et al. Tumorassociated macrophages: the double-edged sword in cancer progression. J Clin Oncol. 2005;23(5):953-64. 30. Alitalo K, Tammela T, Petrova TV. Lymphangiogenesis in development and human disease. Nature. 2005;438(7070):946-53, http://dx.doi.org/ 10.1038/nature04480. 31. Stacker SA, Farnsworth RH, Karnezis T, Shayan R, Smith DP, Paavonen K, et al. Molecular pathways for lymphangiogenesis and their role in human disease. Novartis Found Symp. 2007;281:38-43; discussion 44-53, 208-9. 32. Mandriota SJ, Jussila L, Jeltsch M, Compagni A, Baetens D, Prevo R, et al. Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis. EMBO J. 2001;20(4):672-82, http:// dx.doi.org/10.1093/emboj/20.4.672. 33. Stacker SA, Caesar C, Baldwin ME, Thornton GE, Williams RA, Prevo R, et al. Vascular endothelial growth factor-D promotes the metastatic spread of cancer via the lymphatics. Nature Med. 2001;7(2):186-91. 34. Maruyama K, Ii M, Cursiefen C, Jackson DG, Keino H, Tomita M, et al. Inflammation-induced lymphangiogenesis in the cornea arises from CD11b-positive macrophages. J Clin Invest. 2005;115(9):2363-72, http:// dx.doi.org/10.1172/JCI23874. 35. Kerjaschki D. The crucial role of macrophages in lymphangiogenesis. J Clin Invest. 2005;115(9):2316-9, http://dx.doi.org/10.1172/JCI26354. 36. Schoppmann SF, Birner P, Sto¨ckl J, Kalt R, Ullrich R, Caucig C, et al. Tumor-associated macrophages express lymphatic endothelial growth factors and are related to peritumoral lymphangiogenesis. Am J Pathol. 2002;161(3):947-56, http://dx.doi.org/10.1016/S0002-9440(10)64255-1. 37. Schoppmann SF, Fenzl A, Nagy K, Unger S, Bayer G, Geleff S, et al. VEGFC expressing tumor-associated macrophages in lymph node positive breast cancer: Impact on lymphangiogenesis and survival. Surgery. 2006;139(6):839-46, http://dx.doi.org/10.1016/j.surg.2005.12.008. 38. Colombo MP, Mantovani A. Targeting myelomonocytic cells to revert inflammation-dependent cancer promotion. Cancer Res. 2005;65(20): 9113-6, http://dx.doi.org/10.1158/0008-5472.CAN-05-2714.

figures, and performed the statistical analysis. Yao G, Gao J, Yang B, Zhao Y, Rao Z and Gao J were responsible for the experiments. Zhang B and Zhang Y contributed equally to this work.

REFERENCES 1. Lewis CE, Pollard JW. Distinct role of macrophages in different tumor microenvironments. Cancer Res. 2006;66(2):605-12, http://dx.doi.org/ 10.1158/0008-5472.CAN-05-4005. 2. Porta C, Subhra Kumar B, Larghi P, Rubino L, Mancino A, et al. Tumor promotion by tumor-associated macrophages. Adv Exp Med Biol. 2007;604:67-86, http://dx.doi.org/10.1007/978-0-387-69116-9_5. 3. Mantovani A. La mala educacio´n of tumor-associated macrophages: diverse pathways and new players. Cancer Cell. 2010;17(2):111-2, http:// dx.doi.org/10.1016/j.ccr.2010.01.019. 4. Ryder M, Ghossein RA, Ricarte-Filho JC, Knauf JA, Fagin JA. Increased density of tumor-associated macrophages is associated with decreased survival in advanced thyroid cancer. Endocr Relat Cancer. 2008;15(4): 1069-74, http://dx.doi.org/10.1677/ERC-08-0036. 5. Lee CH, Espinosa I, Vrijaldenhoven S, Subramanian S, Montgomery KD, Zhu S, et al. Prognostic significance of macrophage infiltration in leiomyosarcomas. Clin Cancer Res. 2008;14(5):1423-30, http:// dx.doi.org/10.1158/1078-0432.CCR-07-1712. 6. Shieh YS, Hung YJ, Hsieh CB, Chen JS, Chou KC, Liu SY. Tumorassociated macrophage correlated with angiogenesis and progression of mucoepidermoid carcinoma of salivary glands. Ann Surg Oncol. 2009;16(3):751-60, http://dx.doi.org/10.1245/s10434-008-0259-6. 7. Zhang BC, Gao J, Wang J, Rao ZG, Wang BC, Gao JF. Tumor-associated macrophages infiltration is associated with peritumoral lymphangiogenesis and poor prognosis in lung adneocarcinoma. Med Oncol. 2011;28(4):1447-52, http://dx.doi.org/10.1007/s12032-010-9638-5. 8. Mosser DM. The many faces of macrophage activation. J Leukoc Biol. 2003;73(2):209-12, http://dx.doi.org/10.1189/jlb.0602325. 9. Edwards JP, Zhang X, Frauwirth KA, Mosser DM. Biochemical and functional characterization of three activated macrophage populations. J Leukoc Biol. 2006;80(6):1298-307, http://dx.doi.org/10.1189/jlb.0406249. 10. Raes G, Brys L, Dahal BK, Brandt J, Grooten J, Brombacher F, et al. Macrophage galactose-type C-type lectins as novel markers for alternatively activated macrophages elicited by parasitic infections and allergic airway inflammation. J Leukoc Biol. 2005;77(3):321-7. 11. Wang J, Zhang B, Guo Y, Li G, Xie Q, Zhu B, et al. Artemisinin inhibits tumor lymphangiogenesis by suppression of vascular endothelial growth factor C. Pharmacology. 2008;82(2):148-55, http://dx.doi.org/ 10.1159/000148261. 12. Zhang B, Wang J, Gao J, Guo Y, Chen X, Wang B, et al. Alternatively activated RAW264.7 macrophages enhance tumor lymphangiogenesis in mouse lung adenocarcinoma. J Cell Biochem. 2009;107(1):134-43, http:// dx.doi.org/10.1002/jcb.22110. 13. Jiang J, Grieb B, Thyagarajan A, Sliva D. Ganoderic acids suppress growth and invasive behavior of breast cancer cells by modulating AP-1 and NF-kappaB signaling. Int J Mol Med. 2008;21(5):577-84. 14. Wang J, Guo Y, Zhang BC, Chen ZT, Gao JF. Induction of apoptosis and inhibition of cell migration and tube-like formation by dihydroartemisinin in murine lymphatic endothelial cells. Pharmacology. 2007;80(4):207-18, http://dx.doi.org/10.1159/000104418. 15. Mantovani A, Allavena P, Sica A. Tumour-associated macrophages as a prototypic type II polarised phagocyte population: role in tumour progression. Eur J Cancer. 2004;40(11):1660-7, http://dx.doi.org/ 10.1016/j.ejca.2004.03.016. 16. Sica A, Schioppa T, Mantovani A, Allavena P. Tumour-associated macrophages are a distinct M2 polarised population promoting tumour progression: potential targets of anti-cancer therapy. Eur J Cancer. 2006;42(6):717-27, http://dx.doi.org/10.1016/j.ejca.2006.01.003. 17. Martinez FO, Sica A, Mantovani A, Locati M. Macrophage activation and polarization. Front Biosci. 2008;13:453-61, http://dx.doi.org/10.2741/ 2692. 18. Hagemann T, Wilson J, Burke F, Kulbe H, Li NF, Plu¨ddemann A, et al. Ovarian cancer cells polarize macrophages toward a tumor-associated phenotype. J Immunol. 2006;176(8):5023-32.

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DOI:10.6061/clinics/2012(08)09

CLINICAL SCIENCE

Laparoscopic nephrectomy for xanthogranulomatous pyelonephritis – Are there predictive factors for success? Marcelo Lima, Ricardo Miyaoka, Juliano Moro, Carlos D’Ancona Universidade Estadual de Campinas (UNICAMP), Division of Urology, Campinas/SP, Brazil.

OBJECTIVES: Laparoscopic nephrectomy for xanthogranulomatous pyelonephritis is currently associated with great operative difficulty and surgical complications. Herein, we report on our single-center experience and describe predictive factors for successfully accomplishing this procedure. METHOD: Between March 1998 and April 2010, 66 patients (27 men and 39 women) underwent laparoscopic nephrectomy for the treatment of a unilateral nonfunctioning kidney. These patients had previous diagnoses of renal chronic inflammation associated with calculi and previous pyonephrosis. All of the nephrectomies were performed using the transperitoneal approach, and a similar technique was used for radical nephrectomy. RESULTS: Laparoscopic nephrectomy for the treatment of renal chronic inflammation was successful in 58/66 cases (87.9%). Eight cases were converted to the open technique because of difficulty in progression, which was related to the discovery of dense adhesions in the hilar or perirenal region. One major (colonic lesion) and two minor (wound infection) complications occurred in the conversion group. A diagnosis of xanthogranulomatous pyelonephritis was confirmed pathologically for all of the specimens. Of the factors examined, a longitudinal renal length greater than 12 cm (laparoscopy group - 7.2¡1.8 cm, versus open group - 13.6¡1.5 cm; p,0.05) and time to access the renal vessels (laparoscopy group - 32¡18 min, versus open group - 91¡11 min; p,0.05) were associated with a higher conversion rate. Although the number of patients in the conversion group was small, the majority of these patients received right-sided nephrectomy. CONCLUSIONS: Laparoscopic nephrectomy for the treatment of xanthogranulomatous pyelonephritis is feasible and associated with low levels of morbidity. Factors including the time required to control the renal vessels, renal length and right-sided nephrectomy were associated with higher chances of conversion into an open procedure. KEYWORDS: Xanthogranulomatous Pyelonephritis; Laparoscopic Nephrectomy; Complications; Treatment. Lima M, Miyaoka R, Moro J, D’Ancona C. Laparoscopic nephrectomy for xanthogranulomatous pyelonephritis – Are there predictive factors for success? Clinics. 2012;67(8):907-909. Received for publication on February 14, 2012; First review completed on March 16, 2012; Accepted for publication on April 10, 2012 E-mail: ml.unicamp@gmail.com Tel.: 55 19 3521-7481

remains no unanimous agreement as to the best surgical approach. The surgical options for such a procedure include open or laparoscopic techniques, which can be performed either with or without robotic assistance (1,4-10). Initial reports suggested that the benefits of laparoscopic nephrectomy did not extend to patients with XGP (3). However, the majority of recent series have agreed that laparoscopy may enable decreased blood loss, shorter convalescence time and superior cosmesis (1,5,8) and that this technique can be successfully completed in more than 70% of cases (5,7,8). The conversion rates to either the open approach or the hand-assisted technique vary from 20% to 36% and are generally associated with difficulty in progressing due to severe perirenal adhesions and fibrosis (5,8). In this study, we report on our single-center experience with nephrectomy for the treatment of XGP and the factors found to be associated with a higher conversion rate of laparoscopic to open surgery.

INTRODUCTION Xanthogranulomatous pyelonephritis (XGP) was first described in 1916 by Schlagenhaufer (1) and is assumed to be an uncommon chronic renal infection in which the renal parenchyma is destroyed and replaced by lipid-laden macrophages (2). XGP typically occurs in the presence of chronic obstruction and suppuration and has been shown to account for up to 19.2% of all cases of pyelonephritis in a recent case series (1). Although there is a consensus that the treatment of choice for this condition should be total nephrectomy (3,4), there

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.

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300.2¡142.4 mL, p,0.05) and total hospital stay (2.1¡1.4 versus 4.3¡2.3 days, p,0.05) between the conversion group and the pure laparoscopy group, respectively (Table 2). One major (colonic lesion) and two minor (wound infection) complications occurred in the conversion group. The colonic lesion was promptly identified and repaired with primary suturing without further complications (grade II, according to the Clavien Classification of Surgical Complications) (11). The wound infection was treated with oral first-generation cephalosporin antibiotics (grade I, according to Clavien Classification of Surgical Complications) (11). Of the factors assessed, a longitudinal renal length greater than 12 cm (7.2¡1.8 versus 13.6¡1.5 cm, p,0.05) and the time required to securely isolate the renal vessels (32¡18 versus 91¡11, p,0.05) were associated with a higher conversion rate. Although the number of patients in the conversion group was small, the majority of these patients underwent right-sided nephrectomy (6/8, 75% - Table 1).

MATERIALS AND METHODS We reviewed the records for all of the laparoscopic nephrectomies performed between March 1998 and April 2010 at our institution. In total, we examined 262 nephrectomy cases. This study was performed in accordance with the ethical standards of the institutional committee on human experimentation and of the Helsinki Declaration of 1975 (revised in 1983). Patients with histopathological diagnoses of XGP were included, which resulted in a total of 66 nephrectomy cases. All of these patients had a history of fever, lithiasis treatment and urinary infection and had been administered parenteral antibiotics until one year before the procedure. 99m Tc-DMSA scintigraphy was performed for all of the patients, and the results indicated an average relative kidney function of 8¡3%. All of the nephrectomies were performed using an extrafascial technique in which the dissection was maintained outside of Gerota’s fascia, which is similar to that performed for renal malignant conditions. Organ tissue was removed en bloc for pathological examination through a mini Pfannenstiel incision. The subjects were divided into two groups based on the full accomplishment of the procedure by laparoscopy (Group I) or the need for open conversion (Group II). The decision to perform a conversion procedure was made by the surgeon. All of the specimens had a confirmed histological diagnosis of XGP without concurrent malignancy or multicystic dysplasia. The XGP specimens were characterized by the presence of a focal or diffuse process with foam-laden macrophages within a background of chronic inflammation. Data concerning patient demographics, preoperative computed tomography (CT) scan features, time to securely clamp the hilum vessels, total operative time, conversion rate, total blood loss and hospital stay were accessed. These data were analyzed using the non-paired Student’s t test. A level of significance below 0.05 was adopted.

DISCUSSION

Pure laparoscopic nephrectomy for renal chronic inflammation was successfully accomplished in 58 of 66 cases (87.9%). Eight cases (12.1%) were converted to the open technique because of difficulty in progressing, which was related to the discovery of dense adhesions in the hilar or perirenal region. No differences were observed regarding age, body mass index (BMI) or gender distribution between the conversion and no conversion groups (p.0.05) (Table 1). However, significant differences were detected concerning the mean operative time (122.5¡15.8 versus 215.6¡38.3 minutes, p,0.05); total blood loss (191¡96.5 versus

For XGP kidneys, nephrectomy is recommended as the definitive treatment due to the extensive destruction of the parenchyma in these cases and because the differential diagnosis with renal malignancy is often difficult (3,4). The benefits of laparoscopy, compared with traditional open surgery for the treatment of XGP, have been well established in recent case seriest (1,5,7,8). However, even among experienced surgeons, close to 30% of all cases require conversion to open surgery due to technical difficulties and failure to progress (5,8). In the last decade, significant advances have been made in the field of minimally invasive surgery, especially regarding laparoscopic, endourologic and robotic technologies. However, it is important to note that these technologies involve high-cost materials, and the cost effectiveness of these techniques is directly related to their ability to successfully accomplish their surgical purpose without perioperative complications or the need for retreatment. These benefits are translated into shorter hospital stays, fewer hospital readmittance and a more rapid return to work, which ultimately represent financial savings for the medical system. For these reasons, it is important to judiciously select the cases that are more likely to be successfully resolved with minimally invasive techniques, especially for conditions such as XGP, for which there is controversy regarding the most suitable approach. The current study represents the largest XGP case series to date, and the results identified three aspects that may

Table 1 - Demographic data.

Table 2 - Intraoperative data.

RESULTS

N Age BMI Gender Side

Group I

Group II

58 41.5¡8.2 26.2¡4.1 34 F (58.6%) 24 M (41.4%) 26 Right (44.8%) 32 Left (55.2%)

8 42.5¡9.6 25.6¡4.2 05 F (62.5%) 03 M (37.5%) 06 Right (75%) 02 Left (25%)

Group I

p-value

N 58 Surgical time (min) 122.5¡15.8 Blood loss (cc) 191¡96.5 Hospital stay (days) 2.1¡1.4 Renal length (cm) 7.2¡1.8 Time to secure renal 32¡18 vessels (min)

p.0.05 p.0.05 p.0.05

Group II

p-value

8 215.6¡38.3 300.2¡142.4 4.3¡2.3 13.6¡1.5 91¡11

p,0.05 p,0.05 p,0.05 p,0.05 p,0.05

Abbreviations: min = minutes; cc = cubic centimeters; cm = centimeters.

Abbreviations: BMI = body mass index; F = female; M = male.

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have a causal correlation with higher conversion rates to open surgery when treating XGP by nephrectomy. One of the factors identified to be associated with a higher conversion rate was a longitudinal renal length greater than 12 cm, which is compatible with an enlarged kidney. Furthermore, the following computed tomography findings can be used to diagnose XGP: nephromegaly, obstructive uropathy, densities varying from 215 to 110 UH, rimenhancing multiple fluid-filled cavities, the presence of a staghorn calculus or other large calculus in the collecting system, thickening of Gerota’s fascia and infiltration of the perinephric space, such as the psoas muscle (3,4,7). Our findings corroborate the hypothesis that nephromegaly is associated with a higher conversion rate. Moreover, this condition is associated with enlarged kidneys, demonstrating more severe renal inflammation and, therefore, more severe perirenal adhesions. These factors may justify the difficulty of progressing in these cases, as the kidney becomes bulky, and the working space diminishes regardless of the use of the transperitoneal approach. The difficulty associated with dissecting in narrow spaces becomes especially challenging on the right side, where the proximity to the vena cava requires extra care to avoid vascular injury. Regarding the conversion group examined in the current study, the majority underwent right-sided nephrectomy. When considering the XGP scenario, both the previously mentioned fibrotic adhesions and the existence of lymphadenomegaly as a result of ganglionic inflammation and subsequent enlargement impose additional difficulty. Under these circumstances, the dissection and ligation of the renal pedicle may not be possible laparoscopically. Moreover, the history of pyonephrosis or renal abscesses in the conversion group further supports the hypothesis of extensive local inflammation in this region. In addition, the time required to securely isolate the renal vessels was shown to be associated with a higher rate of conversion to open surgery. Thus, considering the obstacles previously mentioned, these results are the first to suggest a realistic time limit related to a greater likelihood to fail in the full laparoscopic approach. Although we should note the limitations of our study, especially its retrospective nature, it should be kept in mind that XGP is an unusual presentation and as such, more expressive data and additional evidence would only be achievable if a multicenter study was performed. Furthermore, we believe that our complication rate was low due to the experience and careful technique of the authors who performed the surgeries.

Laparoscopic nephrectomy is the definitive treatment for XGP and is feasible and associated with low levels of morbidity. Factors including the time required to control the renal vessels, renal length and right-sided nephrectomies were associated with higher rates of conversion to the open procedure.

AUTHOR CONTRIBUTIONS Lima M designed and conceived the study, collected, analyzed and interpreted the data and drafted the manuscript. Miyaoka R and Moro J collected, analyzed and interpreted the data and revised the manuscript. D’Ancona C revised the manuscript.

REFERENCES 1. Korkes F, Favoretto RL, Bro´glio M, Silva CA, Castro MG, Perez MD. Xanthogranulomatous pyelonephritis: clinical experience with 41 cases. Urology. 2008;71(2):178-80, http://dx.doi.org/10.1016/j.urology.2007.09.026. 2. Petronic V, Buturovic J, Isvaneski M. Xanthogranulomatous pyelonephritis. Br J Urol. 1989;64(4):336-8, http://dx.doi.org/10.1111/j.1464410X.1989.tb06036.x. 3. Bercowsky E, Shalhav AL, Portis A, Elbahnasy AM, McDougall EM, Clayman RV. Is the laparoscopic approach justified in patients with xanthogranulomatous pyelonephritis? Urology. 1999;54(3):437-42, http:// dx.doi.org/10.1016/S0090-4295(99)00261-7. 4. Rosoff JS, Raman JD, Del Pizzo JJ. Feasibility of laparoscopic approach in management of xanthogranulomatous pyelonephritis. Urology. 2006; 68(4):711-4, http://dx.doi.org/10.1016/j.urology.2006.04.031. 5. Kapoor R, Vijjan V, Singh K, Goyal R, Mandhani A, Dubey D, et al. Is laparoscopic nephrectomy the preferred approach in xanthogranulomatous pyelonephritis? Urology. 2006;68(5):952-5, http://dx.doi.org/ 10.1016/j.urology.2006.07.009. 6. Manohar T, Desai M, Desai M. Laparoscopic nephrectomy for benign and inflammatory conditions. J Endourol. 2007;21(11):1323-8, http:// dx.doi.org/10.1089/end.2007.9883. 7. Tunc L, Biri H, Onaran M, Krac M, Yesil S, Bozkirli I. Laparoscopic nephrectomy for xanthogranulomatous pyelonephritis in the absence of kidney stones or clinical urinary infection. Surg Laparosc Endosc Percutan Tech. 2007;17(6):570-2, http://dx.doi.org/10.1097/SLE.0b013e31812e5360. 8. Duarte RJ, Mitre AI, Chamboˆ JL, Arap MA, Srougi M. Laparoscopic nephrectomy outside gerota fascia for management of inflammatory kidney. J Endourol. 2008;22(4):681-6, http://dx.doi.org/10.1089/ end.2007.0291. 9. Rogers C, Laungani R, Krane LS, Bhandari A, Bhandari M, Menon M. Robotic nephrectomy for the treatment of benign and malignant disease. BJU Int. 2008;102(11):1660-5, http://dx.doi.org/10.1111/j.1464-410X. 2008.07895.x. 10. Guzzo TJ, Bivalacqua TJ, Pierorazio PM, Varkarakis J, Schaeffer EM, Allaf ME. Xanthogranulomatous pyelonephritis: presentation and management in the era of laparoscopy. BJU Int. 2009;104(9):1265-8, http://dx.doi.org/10.1111/j.1464-410X.2009.08547.x. 11. Dindo D, Demartines N, Clavien PA. Classification of surgical complications: a new proposal with evaluation in a cohort of 6336 patients and results of a survey. Ann Surg. 2004;240(2):205-13, http://dx.doi.org/ 10.1097/01.sla.0000133083.54934.ae.

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DOI:10.6061/clinics/2012(08)10

CLINICAL SCIENCE

Serum testosterone, sex hormone-binding globulin and total calcium levels predict the calcaneal speed of sound in men Kok-Yong Chin,I Ima-Nirwana Soelaiman,I Isa Naina Mohamed,I Wan Zurinah Wan NgahII I

Universiti Kebangsaan Malaysia, Faculty of Medicine, Pharmacology Department, Kuala Lumpur, Malaysia. II Universiti Kebangsaan Malaysia, Faculty of Medicine, Biochemistry Department, Kuala Lumpur, Malaysia.

OBJECTIVES: Variations in sex hormones and the calcium balance can influence bone health in men. The present study aimed to examine the relationship between the calcaneal speed of sound and biochemical determinants of bone mass, such as sex hormones, parathyroid hormones and serum calcium. METHODS: Data from 549 subjects from the Malaysian Aging Male Study, which included Malay and Chinese men aged 20 years and older residing in the Klang Valley, were used for analysis. The subjects’ calcaneal speed of sound was measured, and their blood was collected for biochemical analysis. Two sets of multiple regression models were generated for the total/bioavailable testosterone and estradiol to avoid multicollinearity. RESULTS: The multiple regression results revealed that bioavailable testosterone and serum total calcium were significant predictors of the calcaneal speed of sound in the adjusted model. After adjustment for ethnicity and body mass index, only bioavailable testosterone remained significant; the total serum calcium was marginally insignificant. In a separate model, the total testosterone and sex hormone-binding globulin were significant predictors, whereas the total serum calcium was marginally insignificant. After adjustment for ethnicity and body mass index (BMI), the significance persisted for total testosterone and SHBG. After further adjustment for age, none of the serum biochemical determinants was a significant predictor of the calcaneal speed of sound. CONCLUSION: There is a significant age-dependent relationship between the calcaneal speed of sound and total testosterone, bioavailable testosterone and sex hormone-binding globulin in Chinese and Malay men in Malaysia. The relationship between total serum calcium and calcaneal speed of sound is ethnicity-dependent. KEYWORDS: Calcaneal Speed of Sound; Quantitative Ultrasound; Testosterone; Estradiol; Calcium; Parathyroid; Age; Men. Chin KY, Soelaiman IN, Mohamed IN, Ngah WZ. Serum testosterone, sex hormone-binding globulin and total calcium levels predict the calcaneal speed of sound in men. Clinics. 2012;67(8):911-916. Received for publication on March 22, 2012; First review completed on April 7, 2012; Accepted for publication on April 11, 2012 E-mail: imasoel@medic.ukm.my Tel.: 03-40405514

system will continue to grow, especially in developing countries (4). Bone mineral density (BMD) measurement using dualenergy X-ray absorptiometry (DXA) is the current gold standard for diagnosing osteoporosis (5). However, the cost and availability of DXA prevent its wide usage for osteoporosis screening in developing countries. Quantitative ultrasound (QUS) technology is an emerging technology that provides an alternative to DXA. It is relatively less costly, easier to handle, free of ionizing emission, portable and thus more accessible than DXA (6). Calcaneal speed of sound (SOS), which is a QUS index, has been shown to correlate strongly with bone density (7,8). The role of testosterone in bone health has been confirmed by the observation that hypogonadal males have a lower BMD (9,10). The relationship between estradiol and bone health has also been shown in several ‘‘natural experiments’’. Males who express mutated estradiol receptors (11) or malfunctioning aromatase enzymes (12,13) have been

INTRODUCTION Osteoporosis is a systemic disease that is characterized by low bone density and deterioration of the bone microarchitecture, which lead to bone fragility and subsequent fractures (1). Men and women both suffer from osteoporosis, but the prevalence of fracture is lower in men than in women because men have a comparatively higher peak bone density and do not undergo a phase of accelerated bone loss (2). However, men suffer from greater morbidity and mortality after fracture than women (3). With the continual increase in lifespan, the burden of male osteoporosis on the healthcare

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.

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conducted by qualified physicians. Subjects with the following conditions were excluded: 1) mobility impairment (requiring walking aids); 2) bone fracture six months prior to screening; 3) major systemic diseases affecting bone metabolism, such as osteoporosis, osteomalacia, osteogenesis imperfecta, rickets, Paget’s disease, hyper/hypocalcemia and hyper/hypoparathyroidism; and 4) taking medications known to affect bone metabolism, such as testosterone, thyroid hormones, thiazide, diuretics, glucocorticoids, bisphosphonate, anticonvulsants and lithium. All of the subjects received detailed information regarded the study, and written consent was obtained.

reported to experience abnormal bone growth and low bone density. This finding is further confirmed by experimental studies in which male subjects with suppressed expression of testosterone, estradiol, or both sex hormones exhibited higher bone turnover (14,15). Fewer studies have established a relationship between sex hormones and QUS indices. Changes in calcium absorption can also contribute to osteoporosis in men. Studies have shown that there is a concurrent decline in BMD when calcium absorption is reduced in men (16). The decline in calcium absorption may trigger a feedback mechanism via the parathyroid (PTH) hormone, whereby bone resorption is increased to maintain the calcium balance in the blood (17). The relationship between PTH levels and BMD has been established (18). However, no study to date has attempted to correlate the variations in serum calcium and PTH levels with calcaneal QUS indices. We previously indicated that the calcaneal SOS measured using a CM-200 sonometer (Furuno, Nishinomiya City, Japan) demonstrates an age-related decline in a healthy Malaysian male population (19). The present study aimed to explore whether the decline in calcaneal SOS is related to variations in biochemical determinants such as sex hormones, serum calcium and PTH levels in a population of healthy Malaysian men composed of men aged 20 years and older from two major ethnic groups, Chinese and Malays. The information from this study will provide a better understanding of the biochemical variables that influence QUS indices in men, thus enabling a wider application of the technology for osteoporosis screening. It will also help to identify potential areas of intervention that can prevent the progress of osteopenia and osteoporosis in aging men.

Body anthropometric measurements The subjects’ weight in light clothing and without shoes was determined using a standardized balance beam scale and was recorded to the nearest 0.1 kg. The subjects’ standing height without shoes was determined using a portable stadiometer and was recorded to the nearest 0.1 cm. The subjects’ body mass index (BMI) was calculated using the formula BMI (kg/m2) = body weight (kg)/height squared (m2).

Calcaneal speed of sound measurement The subjects’ calcaneal SOS was determined using a CM200 sonometer (Furuno, Noshinomiya City, Japan), which measured the speed of sound (SOS) passing through the subject’s calcaneus as a determinant of bone health status. The CM-200 is a gel-coupled (dry) system that consists of two transducers. The subjects were required to place their right foot on the foot patch, which was adjusted to their foot size. The sound waves emitted from one transducer were transmitted through the calcaneus and received by another transducer. The signal was then analyzed and sent to the computer for storage and display. Three readings with repositioning were obtained for each subject. All measurements were performed by a trained technician. The instruments were calibrated prior to each screening session, and quality control was conducted using a phantom. The short-term in-vivo coefficient of variation for the device was approximately 0.1%.

MATERIALS AND METHODS Study design The present study was conducted as part of the Malaysian Aging Male Study, which aimed to determine the nutritional, oxidative and bone health of healthy Malaysian men aged 20 years and older. It was a cross-sectional study, and subjects were recruited from September 2009 to September 2011. Purposive sampling was used, and subjects were recruited via advertisements in major newspapers, radio broadcasts, flyers and public announcements through community centers and religious facilities. The details of the study, including the specific inclusion and exclusion criteria, were clearly stated in the advertisement. The original sample size derived from the Malaysian Aging Male study was 840 subjects; 570 of these subjects consented to blood draws for biochemical testing, and their data were used for analysis. This study was approved by the Ethics Committee of Universiti Kebangsaan Malaysia Medical Center (UKMMC), Research Project Code: UKM-AP-TKP09-2009 and FF-376-2010.

Laboratory assays All of the subjects were required to fast for at least eight hours before attending the screening sessions. During the fasting period, they were not allowed to consume any food or beverages except plain water. Venipuncture was performed between 08:30 and 10:30. The blood was collected in plain tubes, and the serum was extracted. Part of the serum was sent immediately for total testosterone, total estradiol, total calcium, inorganic phosphate and albumin assays. The remaining serum was stored at -70 ˚C for sex hormonebinding globulin (SHBG) and intact parathyroid (PTH) level measurement. The storage period for the serum was one to six months. Total testosterone and total estradiol levels were measured using an ADVIA Centaur immunoassay system (Siemens Healthcare Diagnostics, Illinois, USA) based on competitive immunoassay with direct chemiluminescent technology. The free and bioavailable fractions of testosterone and estradiol were calculated using methods previously described by So¨derga˚rd et al. (1982) (20). Total calcium, inorganic phosphate and albumin were measured with the ADVIA 2400 (Siemens Healthcare Diagnostics, Illinois, USA) using colorimetric methods. SHBG and PTH were

Subjects The subjects who volunteered for this study were males aged 20 years and older of Malay and Chinese ethnicity who resided in the Klang Valley of Peninsular Malaysia (Kuala Lumpur, Shah Alam, Klang, Petaling Jaya, Gombak). All of the subjects were screened using a detailed demographic questionnaire and their previous medical records. Physical examinations and medical history interviews were

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measured using solid phase enzyme-linked immunosorbent assay (ELISA) kits based on the sandwich principle (IBL International, Hamburg, Germany). The manufacturers’ test principles and procedures were followed.

Table 1 - Characteristics of the study population, stratified by ethnicity. Ethnicity Variable

Data analysis

Age Height Weight BMI Calcaneal SOS Total T Bioavailable T Total calcium Inorganic phosphate

The normality of the data was determined using the Shapiro-Wilk test. A log10 transformation was attempted for the SHBG and PTH levels, which reverted to normal; thus, the log10 values were used for analysis. However, the estradiol levels remained skewed after conventional transformation methods were attempted. Therefore, the estradiol levels were recoded into tertiles as ‘low’, ‘moderate’, and ‘high’ levels for analysis. Normally distributed data were presented as the mean (standard deviation [SD]) and skewed data were presented as the median (interquartile range [IQR]). Age, body anthropometry, calcaneal SOS and serum biochemical determinants were compared between the Chinese and Malay subjects using independent t-tests for normal data and Mann-Whitney U-tests for skewed data. A univariate analysis with adjustment for confounding variables, such as age and BMI, was performed when necessary. A multiple linear regression was performed to evaluate the relationship between the calcaneal SOS value and biochemical determinants. Two separate models were generated for the total/bioavailable testosterone and total/ bioavailable estradiol to prevent multicollinearity. Estradiol levels were entered into the regression models as dummy variables, using the ‘low’ level as the reference group to which the ‘moderate’ and ‘high’ estradiol level groups were compared. For continuous data, the standardized coefficient beta (b) explained the extent of variation in calcaneal SOS when predictors of interest changed by 1 SD, whereas for dichotomous data (dummy variables), b explained the standardized difference of the group in comparison to the reference group. Significance was set at p,0.05. All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) Version 16.0 (SPSS Inc., Chicago, USA).

SHBG Total E2 Bioavailable E2 Intact PTH

Malays Mean (SD) 45.29 (17.38)* 165.98 (6.37)* 71.54 (14.36)* 25.94 (4.78)* 1521.30 (27.94) 19.18 (6.94)9 11.09 (3.66) 2.29 (0.12)* 1.12 (0.15)* Median (IQR) 38.63 (28.04) 87.50 (86.00) 60.65 (57.15) 44.58 (21.55)

Chinese Mean (SD)

Total Mean (SD)

46.80 (13.08) 46.14 (15.11) 168.42 (6.31) 167.36 (6.45) 69.06 (12.54) 70.14 (13.40) 24.31 (3.89) 25.02 (4.37) 1515.80 (26.82) 1518.24 (27.43) 18.76 (6.39) 18.94 (6.63) 10.62 (3.46) 10.82 (3.56) 2.23 (0.10) 2.26 (0.11) 1.08 (0.15) 1.10 (0.15) Median (IQR) Median (IQR) 41.12 (28.31) 39.69 (28.00) 82.00 (112.50) 86.00 (101.00) 55.13 (77.64) 58.03 (68.91) 43.43 (22.34) 43.96 (22.00)

Abbr: SD, standard deviation; IQR, interquartile range; BMI, body mass index; E2, estradiol; PTH, parathyroid hormone; SOS, calcaneal SOS; T, testosterone.*Indicates significant differences between Malay and Chinese subjects (p,0.05). Normally distributed data are presented as the mean (standard deviation), whereas skewed variables are presented as the median (interquartile range).

(b = 0.091, p,0.05) were significant predictors of the SOS in men. However, after adjustment for BMI and ethnicity, only bioavailable testosterone (b = 0.162, p,0.05) was a significant predictor of the SOS in the study population, and the serum total calcium was marginally not significant (b = 0.081, p = 0.07). After further adjustment for age, the relationship between the calcaneal SOS value and all biochemical determinants was not significant (Table 2). Multiple regression was repeated using total testosterone, total estradiol and SHBG as predictors. Total testosterone (b = 0.120, p,0.05) and SHBG (b = -0.149, p,0.05) were significant predictors of SOS in the study population. The relationship between total serum calcium and calcaneal SOS was marginally not significant (b = 0.081, p = 0.068). The significance for total testosterone (b = 0.156, p,0.05) and SHBG (b = 0.132, p,0.05) persisted after adjustment for BMI and ethnicity. However, after further adjustment for age, none of the biochemical determinants was a significant predictor of calcaneal SOS (p.0.05).

RESULTS A total of 570 subjects completed the required screening procedures, calcaneal SOS measurement, body anthropometric measurements and blood collection. After adjustment for missing values and outliers, data from 549 subjects (96.32%) were available for analysis. Two hundred forty subjects (43.7%) were Malay, and 309 subjects (56.3%) were Chinese. The subjects’ ages ranged from 20 to 83 years, with a mean of 46.1 years (SD = 15.1 years). The Malay subjects were significantly younger than the Chinese subjects were (p,0.05). They also had a significantly higher weight and shorter stature, thus presenting a higher BMI than the Chinese subjects (p,0.05). The difference in calcaneal SOS values between the two ethnic groups was not significant after adjustment for age and BMI (p.0.05). The Malay subjects also had significantly higher total serum calcium and inorganic phosphate levels than the Chinese subjects (p,0.05), and the significance persisted after adjustment for age. The differences in all sex hormones (total and bioavailable fractions), intact PTH and SHBG levels between the two ethnic groups were not significant (p,0.05; Table 1). Multiple regression analyses revealed that bioavailable testosterone (b = 0.132, p,0.05) and serum total calcium

DISCUSSION Bone mineral density (BMD) has been associated with variations in sex hormones. A cross-sectional study conducted by Khosla et al. (1998) revealed that in males, the BMD at various sites correlated significantly with the bioavailable testosterone, total estradiol and bioavailable estradiol, but not with total testosterone. A multiple regression model in the same study also indicated that bioavailable estradiol and non-bioavailable estradiol were significant determinants of BMD (21). This finding was confirmed by Araujo et al. (2008), who showed that after various adjustments, total estradiol and free estradiol but not testosterone levels were significantly correlated with BMD in males. However, the bioavailable fraction of sex hormones was not considered in their study (22). The calcaneal SOS has been shown to reflect bone mineral density, but its association with sex hormones remains

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Table 2 - Results of a stepwise multiple regression between calcaneal speed of sound values and biochemical determinants. Standardized regression coefficient (b)

Predictors

1 Bioavailable T Serum total calcium Intact PTH Moderate vs. low bioavailable E2 High vs. low bioavailable E2 2 Total T SHBG Serum total calcium Intact PTH Moderate vs. total E2 High vs. low total E2

Unadjusted

Adjusted for ethnicity and BMI

Adjusted for ethnicity, BMI and age

0.132* 0.091* -0.056 0.079 0.061 0.120* -0.149* 0.081 -0.066 0.082 0.063

0.162* 0.081 -0.069 0.068 0.058 0.156* 0.132* 0.074 -0.077 0.078 0.066

0.044 -0.006 -0.038 0.062 0.018 0.046 0.045 -0.011 -0.042 0.067 0.031

Abbr: E2, estradiol; T, testosterone; SHBG, sex hormone-binding globulin. * Indicates p.0.05. For continuous data, the standardized coefficient beta (b) explained the extent of the variation in calcaneal SOS when the predictor of interest changed by 1 SD, whereas for dichotomous data (dummy variables), b explained the standardized difference of the group compared with the reference group.

uncertain. A study conducted by Gennari et al. (2003) revealed that after adjustment for BMI and age, the calcaneal SOS correlated significantly with estradiol levels (total, bioavailable and free fractions), but not with testosterone levels (total, bioavailable and free fractions) (23). In comparison, Kuchuk et al. (2007) found significant differences in the calcaneal SOS value for subjects in the highest quartile of bioavailable testosterone compared with lower quartiles; however, similar results were not found for the bioavailable estradiol level, even though the men in the lowest quartile for bioavailable estradiol had a lower BMD and higher bone turnover (24). Vanderschueren et al. (2010) found that the associations between calcaneal SOS and free and bioavailable testosterone, sex-hormone binding globulin levels and estradiol levels (total, bioavailable and free fractions) in 3,141 European males were significant, but no significant association was found between calcaneal SOS and total testosterone (25). In this study, calcaneal SOS measured with the CM-200 was moderately correlated with BMD (r = 0.68) (26). The calcaneal SOS was significantly associated with bioavailable testosterone in the unadjusted model; however, it was not independent of age (when adjusted for age, the relationship became insignificant). This finding is different from that of Vanderschueren et al. (2010), who found that the relationship between calcaneal SOS and bioavailable testosterone was significant when the analysis was adjusted for age (25). Most studies on the association between calcaneal SOS and total testosterone indicate a nonsignificant relationship (23,25); however, in the present study, the relationship between total testosterone and calcaneal SOS was significant. The association of total and free estradiol levels with calcaneal SOS was generally positive but not significant. In general, the subjects in the lowest tertile for estradiol levels had insignificantly lower calcaneal SOS values than subjects in the higher tertiles. This finding is similar to that of Kuchuk et al., who reported an insignificant relationship between SOS and bioavailable estradiol. Kuchuk et al. also reported a significant relationship between the broadband attenuation of sound and bioavailable testosterone (24), which suggests that bioavailable estradiol may exert effects on bone components that are undetectable using SOS. The level of sex hormone-binding globulin was significantly and

inversely related to the calcaneal SOS in this study. Sex hormone-binding globulin reduces the bioavailability of sex hormones; therefore, it has a negative impact on bone health (27) that has been shown in previous studies in which lower SHBG levels in men appeared to offer protection against osteopenia (28), and men with higher SHBG levels had greater fracture risk (29). Variations in calcium absorption play an important role in bone loss in men. Previous studies indicated a concurrent decline of calcium absorption and BMD in men (16) that may result from a decrease in renal function, which in turn reduces the renal secretion of calcitriol and leads to the malabsorption of calcium. Consequently, PTH secretion is induced, and bone resorption occurs (30). Other causes of calcium imbalance include decreased absorption of vitamin D (31) and a decreased intestinal response to calcitriol (32). Most studies have established a significant relationship between BMD and PTH levels (18,33). Studies examining the association between calcaneal SOS and PTH or serum calcium levels are scarce. In the present study, calcaneal SOS values correlated with total serum levels but not with PTH level. The transient increase in PTH levels in our subjects was mild and may not have caused bone mass variations that were detectable with the QUS technique. It should be noted that previous findings related to the association between BMD and PTH levels were established at sites other than the calcaneus (for example, the hip (34) and the femoral neck (33)). Hence, it is uncertain how much PTH variations affect bone mass at the calcaneus. The positive and significant association between serum total calcium level and calcaneal SOS suggests that other underlying causes mediate the relationship between calcium balance and calcaneal SOS value. A high-protein diet has been hypothesized to increase calcium excretion (35), but this possibility was not examined in the present study. Calcium excretion has also been shown to increase with age (36), and its effects may not be compensated for, thus creating a continuous negative calcium balance in the body that stimulates bone resorption. Several limitations must be considered in the interpretation of this study’s results. The sampling method used in the present study was a purposive, nonrandomized sampling method; consequently, substantial selec-

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tion bias may have been introduced during recruitment, and the results should be generalized with caution. The QUS device used, the CM-200, generates only one QUS index: calcaneal SOS. Other indices, such as broadband attenuation of sound and stiffness index, were not evaluated. The true calcium absorption and excretion were not measured in the present study; consequently, the calcium balance observed was based on the serum total calcium level alone and may not be adequate. Calcitriol levels and dietary calcium consumption among the subjects were not investigated in the present study; consequently, the variations in calcium level could not be fully explained. We experienced substantial difficulty in comparing the regression model generated in the present study with the literature because previous studies did not report their linear regression procedures in detail. Multicollinearity issues created by entering the total and bioavailable or free sex hormones into the same regression model were also apparent in previous studies. In the present study, the total and bioavailable sex hormones were entered into two separate regression models so that the results presented are valid and unaffected by multicollinearity. In conclusion, SHBG and total and bioavailable testosterone are significantly associated with calcaneal SOS values in men, and the relationships are age-dependent. The relationship between total serum calcium and calcaneal SOS is ethnicity-dependent. Pharmacologic interventions for the age-dependent deterioration of bone health in men should aim to maintain optimal bioavailable testosterone and calcium levels.

8. 9. 10. 11. 12. 13.

14.

15.

16.

17. 18.

19.

ACKNOWLEDGMENTS This project received financial support from an Arus Perdana Grant (UKMAP-TKP-09-2009) and a postgraduate research grant (FF-376-2010) from Universiti Kebangsaan Malaysia Medical Centre.

20.

AUTHOR CONTRIBUTIONS

21.

Chin KY contributed to subject recruitment, calcaneal speed of sound measurement, research implementation and the writing of the manuscript. Soelaiman IN planned the research, provided critical review and gave final approval of the manuscript. Mohamed IN provided the critical statistical review of the manuscript. Ngah WZ contributed to the research construction and obtained ethics approval, financial support and supervised the project.

22.

23.

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DOI:10.6061/clinics/2012(08)11

CLINICAL SCIENCE

A clinical follow-up of 35 Brazilian patients with PraderWilli Syndrome Caio Robledo D’Angioli Costa Quaio,I Tatiana Ferreira de Almeida,I Lilian Maria Jose´ Albano,I Israel Gomy,I Debora Romeo Bertola,I Monica Castro Varela,II Celia P. Koiffmann,II Chong Ae KimI I

Instituto da Crianc¸a da Faculdade de Medicina da Universidade de Sa˜o Paulo, Genetics Unit, Sa˜o Paulo/SP, Brazil. Evolutionary Biology, Instituto de Bioscieˆncias da Universidade de Sa˜o Paulo, Sa˜o Paulo/SP, Brazil.

II

Department of Genetics and

OBJECTIVE: Prader-Willi Syndrome is a common etiology of syndromic obesity that is typically caused by either a paternal microdeletion of a region in chromosome 15 (microdeletions) or a maternal uniparental disomy of this chromosome. The purpose of this study was to describe the most significant clinical features of 35 Brazilian patients with molecularly confirmed Prader-Willi syndrome and to determine the effects of growth hormone treatment on clinical outcomes. METHODS: A retrospective study was performed based on the medical records of a cohort of 35 patients diagnosed with Prader-Willi syndrome. The main clinical characteristics were compared between the group of patients presenting with microdeletions and the group presenting with maternal uniparental disomy of chromosome 15. Curves for height/length, weight and body mass index were constructed and compared between Prader-Willi syndrome patients treated with and without growth hormone to determine how growth hormone treatment affected body composition. The curves for these patient groups were also compared with curves for the normal population. RESULTS: No significant differences were identified between patients with microdeletions and patients with maternal uniparental disomy for any of the clinical parameters measured. Growth hormone treatment considerably improved the control of weight gain and body mass index for female patients but had no effect on either parameter in male patients. Growth hormone treatment did not affect height/length in either gender. CONCLUSION: The prevalence rates of several clinical features in this study are in agreement with the rates reported in the literature. Additionally, we found modest benefits of growth hormone treatment but failed to demonstrate differences between patients with microdeletions and those with maternal uniparental disomy. The control of weight gain in patients with Prader-Willi syndrome is complex and does not depend exclusively on growth hormone treatment. KEYWORDS: Obesity; Body composition; Genetics; Prader-Willi syndrome. Quaio CR, Almeida TF, Albano LM, Gomy I, Bertola DR, Varela MC, et al. A clinical follow-up of 35 Brazilian patients with Prader-Willi Syndrome. Clinics. 2012;67(8):917-921. Received for publication on May 19, 2012; First review completed on June 6, 2012; Accepted for publication on June 8, 2012 E-mail: chong.kim@icr.usp.br Tel.: 55 11 2661-8671

poor suck reflex and a failure to thrive beginning at birth that typically improves within the first year of life (1-10). The underlying genetic cause of this pleiotropic disorder is the lack of expression of paternal genes in the critical chromosome region 15q11-13. This lack of expression is due to a de novo paternal microdeletion of this region in 75% of cases (microdeletions), a maternal uniparental disomy (mUPD) of chromosome 15 in 20% of cases and either structural chromosomal aberrations or imprinting center defects in roughly 5% of cases (1-13). The most important complications of PWS are related to the cardiovascular and respiratory involvement caused by obesity. These complications are directly responsible for the high incidence of death among children and adults with PWS, close to 3% per year (2-4). However, the literature lacks comprehensive information on the long-term survival of PWS patients.

INTRODUCTION One of the most common causes of syndromic obesity is Prader-Willi syndrome (PWS), which has a peculiar evolution that is characterized by childhood-onset obesity, facial dysmorphisms, hypogonadism, short stature, intellectual handicap and an insatiable appetite, leading to significant clinical complications later in life. Interestingly, these symptoms are preceded by marked neonatal hypotonia, 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.

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To determine how GHt affected body composition, curves for height/length, weight and body mass index (BMI) were visually compared between PWS patients treated with and without GHt. These growth curves were also compared with the reference growth charts for normal populations from the World Health Organization and the National Center for Health Statistics (WHO/NCHS; referred to as the normal population curve). The means and standard deviations (SDs) of all ages (from 0 to 19 y; calculated using the mean of each individual for each age) were adjusted using the distance-weighted least squares method to calculate the growth curves (Lam, 1983; McLain, 1974) for our patents and for the normal population. Each patient contributed between 1 and 22 measurements at different ages, ranging from birth to 23 y 5 mo. Graphical and statistical analyses were performed using STATISTICA 7.0 and Microsoft Excel.

There is no specific treatment for PWS. The clinical followup is based on constant surveillance and the treatment of common complications. In addition, growth hormone replacement therapy (GHt) is often used to improve final stature and to control weight gain (1,9,10). The purpose of this study was to describe the most significant clinical features during a long-term follow-up of 35 Brazilian patients with molecularly confirmed PWS and to determine the effects of GHt on the clinical outcomes.

METHODS A retrospective study was performed based on the medical records of a cohort of 35 patients (18 males and 17 females) diagnosed with PWS and followed in our service. Twenty-three patients had undergone regular follow-up visits in our service, six patients had incomplete clinical information, and six patients died from complications related to PWS. All subjects included in this study had a typical DNA methylation pattern of the PWS critical region. The DNA was modified by bisulfite treatment, and SNURF-SNRPN exon 1 was amplified by PCR (14). The characteristic PWS pattern is defined by the presence of only the 313 bp maternal band. Three microsatellite markers within the critical region of 15q11-q13 (D15S11, D15S113 and GABRB3) and at least one marker outside this region (D15S984, D15S131, D15S117, D15S115 and CYP19) were studied in 24 patients and their parents to distinguish between deletions and maternal uniparental disomy; 16 patients had microdeletions (eleven males and five females), and eight had mUPD (two males and six females). All patients presented with normal peripheral blood karyotypes. In 11 cases (five males and six females), molecular tests to differentiate between mUPD and microdeletions were not performed. The clinical features for which the data were not normally distributed were compared between the mUPD and microdeletion groups using Fisher’s exact test. Variables with normal distributions, including weight, BMI and height, were compared with Student’s t test.

RESULTS The most relevant clinical features of our cohort of PWS patients and the comparisons between patients with microdeletions and mUPD are summarized in Table 1. Some of the main clinical findings in our cohort were as follows: preterm birth (3%), birth by Cesarean section (90%), hypotonia (100%), poor suck reflex and feeding problems (92%), developmental delay (97%), cryptorchidism in males (43%) and hypogonadism (57%). The birth weight ranged from 1,020 g to 3,650 g (mean: 2,717 g), and the birth length ranged from 34.5 cm to 54.0 cm (mean: 47.7 cm). The mean age at walking was 2.4 years (range: 1.2 y-4.5 y). Hyperphagia presented between 1 y and 5 y (mean: 2.8 y) of age, and obesity developed between 1 y and 6 y (mean: 2.6 y) of age. GHt was started in 11 patients (eight male and three female patients) between 1.2 y and 11.5 y (mean: 6.5 y) of age, with the length of therapy varying from 0.5 y to 6.6 y (mean: 3.8 y). No significant differences were found when comparing the clinical parameters between patients with microdeletions and those with mUPD.

Table 1 - The distribution of relevant clinical features in individuals with Prader-Willi syndrome. The columns ‘‘mUPD’’ and ‘‘microdeletion’’ show the numbers of patients with each feature in the maternal uniparental disomy of chromosome 15 and microdeletion groups, respectively. The numerator shows the absolute value, and the denominator represents the number of individuals in whom the feature was evaluated. The column ‘‘Total’’ shows the data for all 35 patients with Prader-Willi syndrome. Note that the row ‘‘Age’’ presented the mean age and the corresponding standard deviation for each group. Clinical Features

mUPD (n = 8)

Microdeletion (n = 16)

Total (n = 35)

Gender: female male Age: mean (SD) Assisted delivery Hypotonia Feeding problems Developmental delay Facial dysmorphisms Hypogonadism Cryptorchidism Sleep disturbances Death Patients diagnosed before 4 y.o. Use of growth hormone therapy

6/8 2/8 12.2 y (5.9 y) 8/8 8/8 6/7 8/8 8/8 3/7 2/7 4/5 1/8 4/8 4/8

5/16 11/16 12.9 y (4.6 y) 13/13 14/14 10/11 14/14 10/10 9/13 7/13 3/4 3/14 8/15 5/14

17/35 18/35 13.7 y (6.9 y) 25/26 32/32 24/26 30/31 24/24 12/27 15/30 12/14 4/35 17/31 13/32

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with the results published in the literature, such as the frequencies of microdeletion/mUPD, hypotonia, facial dysmorphisms, developmental milestone delays, feeding difficulties, hypogonadism and cryptorchidism (1,11,12).

Six patients died between 1.5 y and 19 y (mean: 11.6 y) of age. The cause of death was determined in four cases; three patients died due to cardiovascular/respiratory complications from obesity, and one died from endocarditis at 1.5 y of age. In the two other cases, no autopsy was performed, and the cause of death was not determined. Of the six deceased patients, three had microdeletions, one had mUPD, and in the remaining two, the molecular test to determine the genetic mechanism of deletion was not performed. Comparing the death rate between the mUPD and microdeletion groups showed no significant difference (p,0.05). The weight, BMI and length/height curves for PWS patients were compared with the WHO/NCHS curves for the normal population (Figure 1).

Clinical findings Hypotonia was present in the first year of life for all patients, leading to feeding problems in the great majority of patients. In this study, only six patients were diagnosed with PWS before 12 months of age. This low number of early diagnoses reflects the difficulty in correctly diagnosing PWS and the lack of proper diagnostic methods available to the public health system in Brazil. The late diagnoses may have skewed our results. Interestingly, we observed that hyperphagia onset and the time of PWS diagnosis were positively correlated. The presence of severe hypotonia or significant feeding problems should indicate the possibility of PWS and warrants genetic testing to confirm the diagnosis. Obesity and hyperphagia began early in life. This observation suggests that the pathophysiology of obesity in PWS is complex and is not only related to high caloric intake but also to hypothalamic and hormonal dysregulation and low energy expenditure (5,6). Our study showed a tendency for children with PWS to have birth lengths and weights below the -2 SD curve for the normal population. Children with PWS then exhibit an increase in weight at approximately two years of age, when they exceed the +2 SD curve. The height curve also showed an increase, but this increase was modest and not sufficient to pull the patients above the -2 SD curve for height. These results are concordant to those in the literature (1,13). The mortality rate (17%) observed in our study was high for a young population. It is remarkable that all six patients died prematurely before adulthood (mean age: 11.6 y).

Weight/BMI curves Males: The use of GHt in males did not decrease weight gain. The mean weight for PWS patients treated with growth hormone exceeded +2 SDs of the mean weight of the normal population by age 4, and the mean weight of PWS patients not treated with growth hormone exceeded 2 SDs of the normal mean weight by the age of 7. Thereafter, both remained groups above +2 SDs compared with the WHO/NCHS curve for the normal population. When comparing the BMI curves of male patients with or without GHt, both groups started below -2 SDs and later exceeded +2 SDs of the WHO/NCHS curve for the normal population during infancy. This increase in BMI occurred earlier in patients previously treated with growth hormone, reflecting rapid weight gain and the inability of the length/height increase to counterbalance the weight gain. Females: Female patients treated with growth hormone showed improvement in weight gain. Female patients treated with growth hormone had a weight curve that remained close to the mean of the normal population, whereas female patients not treated with growth hormone had a curve that exceeded +2 SDs by the age of four, with a continuing upward trend. When comparing BMI curves, a similar trend was observed. Female patients previously treated with growth hormone remained between the mean and +2 SDs of the normal population, whereas female patients not treated with growth hormone exceeded +2 SDs at the age of two and continued a rapid upward trend.

Microdeletions and mUPD We did not find significant differences in any of the other clinical parameters measured between patients with microdeletions and mUPD. We believe that our sample, though considerable for a genetic disorder, was not large enough to reach statistical significance.

Growth hormone replacement therapy Length/Height

In our study, GHt considerably improved the control of weight gain and the BMI for female patients but not for male patients. GHt did not improve height/length in either gender. The control of weight gain in patients with PWS is complex and does not depend exclusively on GHt; other factors, such as food-intake control, exercise and active participation from the family, were not addressed and may have overcome the benefits of GHt in male patients (1,8). The benefits of GHt in individuals with PWS have been demonstrated in multiple studies. GHt improves linear growth velocity and ultimate height, body composition (i.e., increased lean body mass and decreased fat mass), muscle function and the level of activity (9,10). However, in the present study, there was a large heterogeneity among patients receiving GHt, and the variability in the length of treatment may have directly influenced the efficacy of GHt, especially for the male patients. Furthermore, there may have been a selection bias because fewer data were available for older patients. The treatment of PWS patients involves identifying and managing symptoms. A multidisciplinary approach, including controlling food access, hormone replacement therapies,

Males: When comparing the length/height curves for male patients, we observed that patients who received GHt started below -2 SDs in the WHO/NCHS curve of normal children and, by the age of six, were closer to the mean, whereas those without GHt fell below the -2 SD curve. Despite these early differences, when male patients with or without GHt reached eighteen years old, the curves crossed at a point close to -2 SDs. Females: When analyzing the length/height curves for female patients, female patients without GHt started below 2 SDs and, by the age of six, approached the mean of the WHO/NCHS curve of the normal population and stayed at the same level until the age of 15, when a negative slope was noted. The GHt group always remained below -2 SDs.

DISCUSSION We have presented the most relevant clinical features of a large case series of Prader-Willi syndrome patients in Brazil. Several findings in our retrospective analysis are consistent

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Figure 1 - Weight, BMI and length/height curves for PWS patients compared with the WHO/NCHS curve for the normal population. The ‘‘Normal’’ curve represents the mean of the WHO/NCHS curve for the normal population; ‘‘+2SD’’ and ‘‘-2SD’’ represent, respectively, two standard deviations above and two standard deviations below the mean of the normal population. The ‘‘GHt+’’ curve represents the mean of the patients who received at least six months of growth hormone treatment, and ‘‘GHt-’’ represents the mean of the patients who did not undergo growth hormone treatment.

the medical community about PWS as an important cause of syndromic obesity and to characterize the most serious clinical outcomes in Brazil. We observed a high mortality rate in a young population. Moreover, GHt did not improve the

special education, and psychological follow-up, is crucial for the successful management of the disease (1). We presented our observations from a clinical follow-up of a large cohort of Brazilian PWS patients to raise awareness in

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Follow-up of Prader-Willi syndrome patients Quaio CR et al. 5. Swaab DF, Purba JS, Hofman MA. Alterations in the hypothalamic paraventricular nucleus and its oxytocin neurons (putative satiety cells) in Prader-Willi syndrome: a study of five cases. J Clin Endocrinol Metab. 1995;80(2):573-9, http://dx.doi.org/10.1210/jc.80.2.573. 6. Purtell L, Sze L, Loughnan G, Smith E, Herzog H, Sainsbury A, et al. In adults with Prader-Willi syndrome, elevated ghrelin levels are more consistent with hyperphagia than high PYY and GLP-1 levels. Neuropeptides. 2011;45(4):301-7, http://dx.doi.org/10.1016/j.npep.2011.06.001. 7. Crino` A, Schiaffini R, Ciampalini P, Spera S, Beccaria L, Benzi F, et al. Hypogonadism and pubertal development in Prader-Willi syndrome. Eur J Pediatr. 2003;162(5):327-33. 8. Holland AJ, Treasure J, Coskeran P, Dallow J, Milton N, Hillhouse E. Measurement of excessive appetite and metabolic changes in PraderWilli syndrome. Int J Obes Relat Metab Disord. 1993;17(9):527-32. 9. Hoybye C. Five-years growth hormone (GH) treatment in adults with Prader-Willi syndrome. Acta Paediatr 2007;96(3):410-3, http:// dx.doi.org/10.1111/j.1651-2227.2006.00051.x. 10. Festen DA, de Lind van Wijngaarden R, van Eekelen M, Otten BJ, Wit JM, Duivenvoorden HJ, et al. Randomized controlled GH trial: effects on anthropometry, body composition and body proportions in a large group of children with Prader-Willi syndrome. Clin Endocrinol (Oxf). 2008;69(3):443-51, http://dx.doi.org/10.1111/j.1365-2265.2008. 03228.x. 11. Lin H-Y, Lin S-P, Chuang C-K, Chen M-R, Yen J-L, Lee Y-J, et al. Genotype and phenotype in patients with Prader-Willi Syndrome in Taiwan. Acta Paediatrica 2007;96(6):902-5, http://dx.doi.org/10.1111/ j.1651-2227.2007.00284.x. 12. Gunay-Aygun M, Schwartz S, Heeger S, O’Riordan MA, Cassidy SB. The changing purpose of Prader-Willi syndrome clinical diagnostic criteria and proposed revised criteria. Pediatrics. 2001;108(5):E92, http:// dx.doi.org/10.1542/peds.108.5.e92",-1,"xxx/5.e92. 13. Butler MG. Prader-Willi Syndrome: Obesity due to Genomic Imprinting. Curr Genomics. 2011;12(3):204-15. 14. Zeschnigk M, Lich C, Buiting K, Doerfler W, Horsthemke B. A single tube PCR test for the diagnosis of Angelman and Prader-Willi syndrome based on allelic methylation differences at the SNRPN locus. Eur J Hum Genet. 1997;5(2):94-8.

control of the majority of the clinical parameters that were analyzed, demonstrating that the control of weight gain in patients with PWS is complex and does not depend exclusively on GHt.

ACKNOWLEDGMENTS The publication was supported by FAPESP, Brazil (2012/50300-8).

AUTHOR CONTRIBUTIONS Quaio CR, Almeida TF, Albano LM, Bertola DR and Kim CA designed the study and were responsible for the patient data collection and manuscript writing. Almeida TF was also responsible for statistical analysis. Varela MC and Koiffmann CP performed the molecular analysis. Gomy I revised the manuscript.

REFERENCES 1. Cassidy SB, Driscoll DJ. Prader-Willi syndrome. Eur J Hum Genet. 2009;17(1):3-13, http://dx.doi.org/10.1038/ejhg.2008.165. 2. Schrander-Stumpel CT, Curfs LM, Sastrowijoto P, Cassidy SB, Schrander JJ, Fryns JP. Prader-Willi syndrome: causes of death in an international series of 27 cases. Am J Med Genet A. 2004;1;124A(4):333-8, http:// dx.doi.org/10.1002/ajmg.a.20371. 3. Butler JV, Whittington JE, Holland AJ, Boer H, Clarke D, Webb T. Prevalence of, and risk factors for, physical ill-health in people with PraderWilli syndrome: a population-based study. Dev Med Child Neurol. 2002;44(4):248-55, http://dx.doi.org/10.1017/S001216220100202X. 4. Whittington JE, Holland AJ, Webb T, Butler J, Clarke D, Boer H. Population prevalence and estimated birth incidence and mortality rate for people with Prader-Willi syndrome in one UK Health Region. J Med Genet. 2001;38(11):792-8, http://dx.doi.org/10.1136/jmg.38.11.792.

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DOI:10.6061/clinics/2012(08)12

BASIC RESEARCH

Preconditioning of the response to ischemia/ reperfusion-induced plasma leakage in hamster cheek pouch microcirculation Fabiana Gomes da Conceic¸a˜o,I Cristiane Maria Simonato Conde,I Erik Svensjo¨,II Daniel Alexandre Bottino,I Eliete BouskelaI I Laboratory for Clinical and Experimental Research on Vascular Biology, Biomedical Center, State University of Rio de Janeiro/RJ, Brazil. II Institute of Biophysics Carlos Chagas Filho, Health Sciences Center, Federal University of Rio de Janeiro/RJ, Brazil.

OBJECTIVE: Ischemic preconditioning and some drugs can protect tissues from injury by preserving microcirculation. This study evaluated vascular permeability in a hamster cheek pouch preparation using either short ischemic periods or bradykinin as preconditioning stimuli followed by 30 min of ischemia/reperfusion. METHOD: Sixty-six male hamsters were divided into 11 groups: five combinations of different ischemic frequencies and durations (one, three or five shorts periods of ischemia, separated by one or five minutes) with 10 min intervals between the ischemic periods, followed by 30 min ischemia/reperfusion; three or five 1 min ischemic periods with 10 min intervals between them followed by the topical application of histamine (2 mM); bradykinin (400 nM) followed by 30 min of ischemia/reperfusion; and three control groups (30 min of ischemia/reperfusion or histamine or bradykinin by themselves). Macromolecular permeability was assessed by injection of fluorescein-labeled dextran (FITC-dextran, MW = 150 kDa; 250 mg/Kg body weight), and the number of leaks/cm2 was counted using an intravital microscope and fluorescent light in the cheek pouch. RESULTS: Plasma leakage (number of leaks/cm2) was significantly reduced by preconditioning with three and five 1 min ischemic periods, one and three 5 min ischemic periods and by bradykinin. Histamine-induced macromolecular permeability was also reduced after three periods of 5 min of ischemia. CONCLUSION: Short ischemic periods and bradykinin can function as preconditioning stimuli of the ischemia/ reperfusion response in the hamster cheek pouch microcirculation. Short ischemic periods also reduced histamineinduced macromolecular permeability. KEYWORDS: Ischemic Preconditioning; Bradykinin Preconditioning; Vascular Permeability; Microcirculation; Hamster Cheek Pouch. Conceic¸a˜o FG, Conde CM, Svensjo¨ E, Bottino DA, Bouskela E. Preconditioning of the response to ischemia/reperfusion-induced plasma leakage in hamster cheek pouch microcirculation. Clinics. 2012;67(8):923-929. Received for publication on December 21, 2011; First review completed on January 10, 2012; Accepted for publication on March 2, 2012 E-mail: dbottino1@yahoo.com.br Tel.: 55 21 2334 0703

ischemia. However, after blood flow normalization, reperfusion injury also restricts cell survival. Consequently, larger myocardial infarct areas predict worse prognosis due to the loss of contractile mass and inadequate ventricle remodeling through hypertrophy, eventually causing heart failure (2). Preconditioning refers to a phenomenon wherein tissues are rendered resistant to the deleterious effects of prolonged ischemia and reperfusion by prior exposure to oxidants, brief periods of vascular occlusion, endotoxin derivatives, heat shock, a variety of pharmacological agents (chemical or pharmacological preconditioning), or chronic ethanol consumption (3). Of these perturbations, our best understanding of the mechanisms involved in conferring protection is related to ischemic preconditioning (IPC) (4). Myocardial preconditioning has been developed to minimize the effects of acute ischemia to the heart. In 1986, Murry et al. reported that four brief episodes of 5

INTRODUCTION Mortality by cardiovascular disease is still an important concern worldwide. According to World Health Organization (WHO), in 2004, coronary heart disease was the second most common cause of death in low- and middle-income countries and the most common cause of death in high-income ones (1). During an acute myocardial infarction, many attempts to reestablish blood flow are performed by physicians to save as many cardiomyocytes as possible before they are damaged by

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.

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minutes of ischemia in the circumflex coronary in dogs followed by 5 minutes of reperfusion reduced the infarct size by 75% compared to controls (5). Since the publication of that report, many studies have tried to explain the mechanistic steps involved in the preconditioning process, such as mitochondrial ATP-sensitive K+ channels, nitric oxide, adenosine and bradykinin (BK) (6-8). Several studies in the hamster cheek pouch (HCP) on experimental perturbations with drugs and the subsequent responses suggest that it could be a useful model for studies of IPC and other types of preconditioning (9-12). In a study of ischemia/reperfusion (I/R) in the HCP, a period of 30 minutes of ischemia resulted in a 30% reduction in plasma leakage in response to a second ischemic period, although the response to histamine appeared unchanged (12). The main element affected by ischemia that determines cell survival is the microcirculation, the smallest vessels of the vascular system; the endothelium plays a key role in the ischemic process. Endothelial barrier dysfunction is a wellrecognized response to ischemia, which involves inflammation (13). Many changes occur in the structure of the endothelial monolayer; one example is the widening of paracellular junctions caused by the dissociation of junctional proteins or cytoskeletal contractions (14,15). Increased microvascular permeability results in edema and organ failure. The magnitude of albumin leakage from postischemic venules during ischemia-reperfusion injury was reportedly proportional to the number of adherent and emigrated leucocytes (16). However, histamine, normally associated with allergic reactions and increases in vascular permeability through H2 action, has been shown to decrease inflammation by reducing chemotactic responsiveness of leukocytes (17,18). Adachi et al. demonstrated that diphenhydramine, a histamine H1 receptor antagonist, inhibits histamine-N-methyltransferase, which is a histamine-inactivating enzyme in the brain. Thus, brain histamine ameliorated reperfusion injury after cerebral ischemia in rats (19). The combination of biogenic amines such as histamine, bradykinin and ischemic preconditioning may minimize the deleterious effects of ischemia-reperfusion injury on the microcirculation. The main objectives of this work were to test the use of the hamster cheek pouch as a model of preconditioning and the effects of different frequencies and durations of brief ischemic preconditioning (IP) episodes followed by 30 minutes of total ischemia and reperfusion or histamine compared to the topical application of bradykinin followed by ischemia/reperfusion on microvascular permeability in hamster cheek pouch preparations.

of waiting time and 30 minutes of ischemia; 1ischem59 – 5 minutes of ischemia (IP) followed by 20 minutes of waiting time and 30 minutes of ischemia; 3ischem59 - three 5- minute periods of ischemia with 10-minute intervals between them (IP), followed by 20 minutes of waiting time and 30 minutes of ischemia; 3ischem19_Hista – three one minute periods of ischemia with 10-minute intervals between them (IP), followed by 20 minutes of waiting time and the topical application of histamine (2 mM); 3ischem59_Hista - three 5 minutes periods of ischemia with 10-minute intervals between them (IP), followed by 20 minutes of waiting time and topical application of histamine (2 mM); control_Hista - topical application of histamine (2 mM); BK - preconditioning with bradykinin (BK) was achieved by the topical application of 400 nM BK for 5 minutes, followed after 20 minutes by either 30 minutes of ischemia or no treatment (control) (Figure 1).

Hamster cheek pouch preparation The hamster cheek pouch was prepared according to studies by Duling (1973) (20), Svensjo¨ et al. (1978) (21), modified by Persson et al. (1985) (12) and by Bouskela and Grampp (1992) (22) (Figure 2A and B). Anesthesia was induced by an intraperitoneal injection of sodium pentobarbital (0.2 ml/100 g body weight, pentobarbital sodium, Sanofi, Paris, France, 60 mg/ml). After cannulation of the femoral vein, anesthesia was maintained with an injection of alpha-chloralose (2.5 ml/kg body weight, Merck, Darmstadt, Germany). Thirty minutes after the preparation was completed, fluorescein-labeled dextran (FITC-dextran, MW = 150 kDa; 250 mg/kg body weight) was injected intravenously as a macromolecular tracer. The cheek pouch (Figure 2B) was subjected to ischemic periods of different durations with an inflatable cuff made of thin latex tubing, mounted around the neck of the everted pouch where it leaves the mouth of the hamster. The cuff did not disturb the local blood flow, and an intracuff pressure of 200–220 mmHg created by air compression with a syringe resulted in complete arrest of the microvascular blood flow within few seconds (12). Increases in microvascular permeability for large molecules (plasma leakage) was quantified by counting sites with extravasation of fluorescent plasma (leaky sites = leaks) at post-capillary venules at 2–5 min intervals after the onset of reperfusion and/or topical application of bradykinin (400 nM) or histamine (2 mM) for 5 minutes. The results are reported as the number of leaks counted 10 minutes after the onset of reperfusion.

Statistical Analysis Results are reported as means¡standard deviations unless otherwise noted. For statistical analyses, ANOVA was used to compare groups to controls. p,0.05 was considered to indicate a statistically significant difference.

MATERIALS AND METHODS This study was approved by the Ethical Committee of the State University of Rio de Janeiro, RJ, Brazil (CEA/215/2007). Sixty-six male Syrian golden hamsters (Mesocricetus auratus, Botucatu, Sa˜o Paulo, SP, Brazil), weighing between 80 g to 100 g, were randomly divided into eleven experimental groups: control (C) - 30 minutes of ischemia; 1ischem19 - 1 minute of ischemia (IP) followed by 20 minutes of waiting time and 30 minutes of ischemia; 3ischem19 - three 1 minute periods of ischemia with 10-minute intervals between them (IP), followed by 20 minutes of waiting time and 30 minutes of ischemia; 5ischem19 - five 1 minute periods of ischemia with 10-minute intervals between them (IP), followed by 20 minutes

RESULTS Brief 1- to 5-minute periods of ischemia followed by reperfusion caused no visible increases in the number of leaks or plasma leakage. One period of 1 minute of ischemia had influence (p.0.05) on the subsequent 30-minute period of I/R on plasma leakage, but several brief ischemic periods reduced the response to the 30-minute period of ischemia as shown in Figure 3A. Three periods of 5 minutes of ischemia produced a significant reduction of the response to the

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Figure 1 - The experimental protocol of the present study. Groups with one, three, and five one-minute ischemic periods: 1ischem19, 3ischem19, and 5ischem19. Groups with one and three five-minute ischemic periods: 1ischem59 and 3ischem59. Groups with three oneminute and three five-minute ischemic periods followed by histamine: 3ischem19-Hista and 5ischem19_Hista. All ischemic periods had 10-minute rest intervals between them. Bradykinin group (BK) and three control groups: ischemia/reperfusion, histamine, and bradykinin (control, Control_Hista, Control_BK).

applications of 400 nM bradykinin given 30 minutes prior to I/R reduced the response to I/R-induced plasma leakage from 125 to 40 leaks/cm2 (32% of the I/R control group) and reduced the bradykinin-induced plasma leakage from 305 to 226 leaks/cm2 (74% of the control group) (Figure 5). Table 1 displays the results as means and standard deviations of leaks in the 3ischem19, 3ischem19_Hista, 3ischem59, and 3ischem59_Hista groups. Additionally, it is possible to visualize the percentage of leaks from each group with the associated control values. There was no statistical difference

subsequent 30 minutes of I/R. When logarithmic values of the maximal number of leaks induced by 30 minutes of ischemia were plotted versus the total duration of ischemia during IPC, a linear correlation could be observed (Figure 3B). Figure 4 shows two IPC groups with three one-minute and three five-minute ischemia periods followed by the topical application of histamine compared to control. Three occlusions of five minutes produced the fewest leaks compared to the control group. Bradykinin also contributed microcirculatory protection. Five-minute topical

Figure 2 - A. Hamster cheek pouch (HCP) model. B. The inflatable cuff mounted around the neck of the prepared cheek pouch (HCP).

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Figure 3 - Effect of brief periods of ischemia and reperfusion prior to a period of 30 minutes of ischemia. A. Plasma leakage (maximal number of leaks¡SD) induced at reperfusion after 30 minutes of ischemia following brief periods of ischemia/reperfusion in six groups of hamsters (each group, n = 6) prior to the final 30 minute-ischemic period. # = p, 0.05, * = p,0.01 as compared with I/R controls. B. Log-linear relationship between the number of leaks and the total ischemic period prior to the final 30 min I/R.

of IPC on I/R-, histamine and bradykinin induced plasma leakage has not been studied in the HCP. This study confirmed that repeated minor episodes of ischemia have a protective effect on the microcirculation. This is a natural phenomenon where several primary pathways converge to cardioprotective effect (24,25). Not all of the mechanisms involved in this cardioprotection are known, and our first question in this study was how long the cheek pouch vessels should be occluded to decrease the microvascular permeability during I/R. We found that a one-minute ischemic period was not enough to reduce the number of leaks during reperfusion. We tested three and five one-minute ischemic periods with ten-minute reflow periods, and the results revealed a decreasing number of leaks. The greatest reduction in plasma leakage induced by 30 min I/R were observed with one and three five-minute ischemic periods with the same reflow time. Murry et al. used 15-minute occlusions to precondition myocardial

between the 3ischem19 and 3ischem19_Hista groups concerning the percentage of leaks. However, the decreases in the percentage of leaks from the 3ischem59 and 3ischem59_Hista groups compared to 3ischem19 and 3ischem19_Hista were highly significant. Further, the decrease in the percentage of leaks was much more evident in the 3ischem59 group than in the 3ischem59_Hista group.

DISCUSSION The hamster cheek pouch as prepared for intravital microscopy has been used in several studies to elucidate the mechanisms behind I/R-induced increases in plasma leakage. A 30-minute period of ischemia was originally selected because it resulted in statistically significant and reproducible increases in plasma leakage during reperfusion, whereas 5-minute ischemic periods produce no visible changes in plasma leakage (12). However, the possible effect

Figure 4 - Effect of brief periods of I/R prior to histamine application. Plasma leakage (maximal number of leaks¡SD) induced by histamine (2 mM). Three periods of 1 or 5 minutes of ischemia reduced the subsequent response to histamine. * = p,0.05, # p,0.01 as compared with histamine controls (2 mM).

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Figure 5 - Plasma leakage (maximal number of leaksÂĄSD) induced by ischemia/reperfusion (I/R) and 400 nM bradykinin (BK) without (Triangles) or with (Diamonds) bradykinin prior to I/R. Bradykinin prior to I/R reduced the subsequent response to I/R (from 125 to 40 leaks - 32% of control) and to bradykinin (from 305 to 225.7 leaks - 74% of control), * p,0.05.

and I/R to 26%, 32% and 31%, respectively, of the control group without antioxidant pretreatment (23). Delbin et al. have shown that histamine did not influence pulmonary ischemia/reperfusion injury in sedentary or preconditioned exercised rats (five days a week for eight weeks) (29). However, increased nitric oxide (NO) release after regular physical exercise is cardioprotective (30). Perhaps ischemic preconditioning protection is different from the protection developed from exercise training. Indeed, IPC prevented the impairment of pulmonary endothelium-dependent vasodilation partially due to nitric oxide (NO) release in isolated rat lungs (31). In this study we observed a significant preconditioning effect of three one-minute and three five-minute ischemic periods (Table 1) when tested with 30 min I/R and with histamine (2mM). There was no difference in the preconditioning effect with one-minute ischemic periods (78 % and 79% of control). However, the three five-minute ischemic periods induced a greater preconditioning effect in the 30 min I/R group (39% of control) than in the histamine group (63% of control) suggesting a difference in the mechanism of plasma leakage induction due to ROS formation and the direct receptor stimulation with histamine. Indeed during ischemia in the HCP the presence of reactive oxygen species have been observed (32), and with the addition of histamine, calcium channels may exhibit more prominent action after three five-minute ischemic periods. Some studies have shown the contribution of histamine to ischemic preconditioning and microcirculation. Kandilci et al. prevented the decrease in pulmonary vasodilator response to histamine and acetylcholine following two hours of hypothermic ischemia in rats using two 5-minute cycles of ischemia and reperfusion (33). Moreover, Wang et al. demonstrated that mast cell granules are not affected by ischemic preconditioning. The theory behind this finding was that an initial brief ischemia would result in mast cell degranulation without significant cardiovascular damage, but when a prolonged ischemic episode would occur, no more degranulation could be possible (34).

tissue in dogs with high mortality (5). When they attempted to use more occlusions with smaller durations, including four five-minute occlusions with 5-minute reflow between them, they observed protective effects on the myocardium. At 10 minutes reflow period, the maximum number of leaks occured and for that reason we have chosen this time to report. We could also demonstrate that the log of IPCinduced reduction was linearly correlated to the total duration of ischemia for 15 minutes (Figure 3B) suggesting that the preconditioning effect was related to the total time of ischemic rather than the number of ischemic periods. Preconditioning can protect the heart and other tissues from a subsequent sustained period of ischemia. Early preconditioning is independent of protein synthesis, and the prolonged insult should be less than 2 hours (28). Our model used 30 minutes of total ischemia. Prolonged ischemia will produce impaired endothelial-dependent vasodilation, capillary plugging, increased adhered and transmigrated leukocytes and venous protein leakage (28). Ischemic preconditioning protects endothelial cells and reverses microcirculatory injury caused by ischemia (3). The effect of IPC (361 min) on histamine-induced plasma leakage did not differ from that observed with I/R; the respective effects were 79% and 78% of the non-IPC-control group response. When the IPC-period was prolonged (365 min), further 63% and 40% reductions were observed in the histamine and I/R groups, respectively, compared to the non-IPC-control group. A similar histamine-preconditioning effect was observed by Erlansson et al. (1985), who reported that in a series of four repeated histamine challenges, the second, third, and fourth challenges produced plasma leakages that were each 65% of the first challenge (12). Taken together, these results may suggest that the preconditioning effect is more related to the strength of the stimuli (the duration of ischemic periods or the dose of histamine) on endothelial cells, rather than the nature of preconditioning (IPC or histamine). In this context, it is interesting to note that in a study using HCP an antioxidant (flavonoid) reduced the plasma leakage responses induced by histamine, bradykinin

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Table 1 - Comparison of the effects of three ischemic periods (of one minute or three minutes duration) on subsequent I/ R (30min) and on histamine (2 mM) induced plasma leakage.

Number of leaks¡ SD % p-value

I/R Control

I/R after 3 ischemic periods of one minute

I/R after 3 ischemic periods of five minutes

Control Histamine after 3 ischemic histamine periods of one minute

Histamine after 3 ischemic periods of five minutes

137¡7

107¡4

54¡3

386¡27

306¡15

245¡11

100 -

78 ,0.05

39 ,0.01

100 -

79 ,0.05

63 ,0.01

10. Mabanta L, Valane P, Borne J, Frame MD. Initiation of remote microvascular preconditioning requires K(ATP) channel activity. Am J Physiol Heart Circ Physiol. 2006;290:H264-71, http://dx.doi.org/ 10.1152/ajpheart.00455.2005. 11. Frame MD, Mabanta L. Remote microvascular preconditioning alters specific vasoactive responses. Microcirculation. 2007;14(7):739-51, http://dx.doi.org/10.1080/10739680701410074. 12. Persson NH, Erlansson M, Svensjo E, Takolander R, Bergqvist D. The hamster cheek pouch–an experimental model to study post-ischemic macromolecular permeability. Int J Microcirc Clin Exp. 1985;4(3):257-63. 13. Rodrigues SF, Granger DN. Role of blood cells in ischaemia-reperfusion induced endothelial barrier failure. Cardiovasc Res. 2010;87(2):291-9, http://dx.doi.org/10.1093/cvr/cvq090. 14. Kumar P, Shen Q, Pivetti CD, Lee ES, Wu MH, Yuan SY. Molecular mechanisms of endothelial hyperpermeability: implications in inflammation. Expert Rev Mol Med. 2009;11:e19, http://dx.doi.org/10.1017/ S1462399409001112. 15. Aghajanian A, Wittchen ES, Allingham MJ, Garrett TA, Burridge K. Endothelial cell junctions and the regulation of vascular permeability and leukocyte transmigration. J Thromb Haemost. 2008;6(9):1453-60, http://dx.doi.org/10.1111/j.1538-7836.2008.03087.x. 16. Kurose I, Anderson DC, Miyasaka M, Tamatani T, Paulson JC, Todd RF, et al. Molecular determinants of reperfusion-induced leukocyte adhesion and vascular protein leakage. Circ Res. 1994;74(2):336-43, http:// dx.doi.org/10.1161/01.RES.74.2.336. 17. Hiraga N, Adachi N, Liu K, Nagaro T, Arai T. Suppression of inflammatory cell recruitment by histamine receptor stimulation in ischemic rat brains. Eur J Pharmacol. 2007;557(2-3):236-44, http:// dx.doi.org/10.1016/j.ejphar.2006.11.020. 18. Azuma Y, Shinohara M, Wang PL, Hidaka A, Ohura K. Histamine inhibits chemotaxis, phagocytosis, superoxide anion production, and the production of TNFalpha and IL-12 by macrophages via H2-receptors. Int Immunopharmacol. 2001;1(9-10):1867-75, http://dx.doi.org/10.1016/ S1567-5769(01)00112-6. 19. Adachi N, Liu K, Ninomiya K, Matsuoka E, Motoki A, Irisawa Y, et al. Reduction of the infarct size by simultaneous administration of lhistidine and diphenhydramine in ischaemic rat brains. Resuscitation. 2011;82(2):219-21, http://dx.doi.org/10.1016/j.resuscitation.2010.10.024. 20. Duling BR. The preparation and use of the hamster cheek pouch for studies of the microcirculation. Microvasc Res. 1973;5(3):423-9, http:// dx.doi.org/10.1016/0026-2862(73)90059-9. 21. Svensjo E, Arfors KE, Arturson G, Rutili G. The hamster cheek pouch preparation as a model for studies of macromolecular permeability of the microvasculature. Ups J Med Sci. 1978;83(1)71-9, http://dx.doi.org/ 10.3109/03009737809179115. 22. Bouskela E, Grampp W. Spontaneous vasomotion in hamster cheek pouch arterioles in varying experimental conditions. Am J Physiol. 1992;262(2 Pt 2):H478-H85. 23. Bouskela E, Donyo KA. Effects of oral administration of purified micronized flavonoid fraction on increased microvascular permeability induced by various agents and on ischemia/reperfusion in diabetic hamsters. Int J Microcirc Clin Exp. 1995;15(6):293-300, http://dx.doi.org/ 10.1159/000179078. 24. Baines CP, Zhang J, Wang GW, Zheng YT, Xiu JX, Cardwell EM, et al. Mitochondrial PKCepsilon and MAPK form signaling modules in the murine heart: enhanced mitochondrial PKCepsilon-MAPK interactions and differential MAPK activation in PKCepsilon-induced cardioprotection. Circ Res. 2002;90(4):390-7, http://dx.doi.org/10.1161/01.RES.0000012702. 90501.8D. 25. Goto M, Liu Y, Yang XM, Ardell JL, Cohen MV, Downey JM. Role of bradykinin in protection of ischemic preconditioning in rabbit hearts. Circ Res. 1995;77(3):611-21, http://dx.doi.org/10.1161/01.RES.77.3.611. 26. Reimer KA, Murry CE, Yamasawa I, Hill ML, Jennings RB. Four brief periods of myocardial ischemia cause no cumulative ATP loss or necrosis. Am J Physiol. 1986;251(6 Pt 2):H1306-15. 27. Swain JL, Sabina RL, Hines JJ, Greenfield JC Jr, Holmes EW. Repetitive episodes of brief ischaemia (12 min) do not produce a cumulative depletion of high energy phosphate compounds. Cardiovasc Res. 1984;18(5):264-9, http://dx.doi.org/10.1093/cvr/18.5.264.

We have observed the preconditioning effect of bradykinin on ischemia/reperfusion by reducing plasma leakage compared to the control group. In clinical practice, exogenous administration of bradykinin prior to cardiopulmonary bypass in coronary artery bypass grafting reduces myocardial injury (35). Bradykinin can also be considered an endothelium protector. Indeed, in vitro experiments with pig cerebral endothelial cells exposed to necrotic and apoptotic cell inducers (H2O2) demonstrated enhanced expression of the cytoprotective proteins COX-2 and (Cu/Zn) SOD when the cells were pretreated with bradykinin (36). Nevertheless, nitrite production in response to bradykinin was enhanced in microvessels during second window protection (SWOP) in dogs subjected to myocardial preconditioning with 10 minutes of coronary artery occlusion (37). Further, endothelial nitric oxide synthase (eNOS) protein in the SWOP myocardium was twofold higher than in control groups. Our study has demonstrated a preconditioning effect of short periods of ischemia and reperfusion when measured as plasma leakage induced by a 30 min I/R-period. This preconditioning effect (reduction in the response to 30 min I/R-induced plasma leakage) that was log-linear related to the total time of brief ischemic periods. When the same preconditioning protocol was applied before histamine application a similar but smaller reduction in the plasma leakage was observed. A preconditioning effect was also observed after topical application of bradykinin 30 minutes prior to I/R-induced plasma leakage that was of the same magnitude as that induced by brief ischemic periods.

REFERENCES 1. World Health Organization. http://www.who.int/mediacentre/factsheets/fs310/en/index4.html.World Health Organization. 2010. Accessed 13-12-22010. 2. Yang X, Cohen MV, Downey JM. Mechanism of cardioprotection by early ischemic preconditioning. Cardiovasc Drugs Ther. 2010;24(3):22534, http://dx.doi.org/10.1007/s10557-010-6236-x. 3. Dayton C, Yamaguchi T, Warren A, Korthuis RJ. Ischemic preconditioning prevents post-ischemic arteriolar, capillary, and post-capillary venular dysfunction: signaling pathways mediating the adaptive metamorphosis to a protected phenotype in preconditioned endothelium. Microcirculation. 2002;9(2):73-89. 4. Das M, Das DK. Molecular mechanism of preconditioning. IUBMB Life. 2008;60(4):199-203, http://dx.doi.org/10.1002/iub.31. 5. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986;74(5):1124-36, http://dx.doi.org/10.1161/01.CIR.74.5.1124. 6. Ardehali H. Role of the mitochondrial ATP-sensitive K+ channels in cardioprotection. Acta Biochim Pol. 2004;51(2):379-90. 7. Bertuglia S, Giusti A. Role of nitric oxide in capillary perfusion and oxygen delivery regulation during systemic hypoxia. Am J Physiol Heart Circ Physiol. 2005;288(2):H525-31. 8. Noda K, Sasaguri M, Ideishi M, Ikeda M, Arakawa K. Role of locally formed angiotensin II and bradykinin in the reduction of myocardial infarct size in dogs. Cardiovasc Res. 1993;27(2):334-40, http:// dx.doi.org/10.1093/cvr/27.2.334. 9. Bertuglia S. Intermittent hypoxia modulates nitric oxide-dependent vasodilation and capillary perfusion during ischemia-reperfusion-induced damage. Am J Physiol Heart Circ Physiol. 2008;294(4):H1914-22, http:// dx.doi.org/10.1152/ajpheart.01371.2007.

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Preconditioning and microcirculation Conceic¸a˜o FG et al. 33. Kandilci HB, Gumusel B, Demiryurek AT, Lippton H. Preconditioning modulates pulmonary endothelial dysfunction following ischemiareperfusion injury in the rat lung: role of potassium channels. Life Sci. 2006;78(19):2172-8, http://dx.doi.org/10.1016/j.lfs.2006.07.011. 34. Wang P, Downey JM, Cohen MV. Mast cell degranulation does not contribute to ischemic preconditioning in isolated rabbit hearts. Basic Res Cardiol. 1996;91(6):458-67, http://dx.doi.org/10.1007/BF00788727. 35. Wang X, Wei M, Kuukasjarvi P, Laurikka J, Rinne T, Moilanen E, et al. The anti-inflammatory effect of bradykinin preconditioning in coronary artery bypass grafting (bradykinin and preconditioning). Scand Cardiovasc J. 2009;43(1):72-9, http://dx.doi.org/10.1080/1401743080218 0449. 36. Bovenzi V, Savard M, Morin J, Cuerrier CM, Grandbois M, Gobeil F Jr. Bradykinin protects against brain microvascular endothelial cell death induced by pathophysiological stimuli. J Cell Physiol. 2010;222(1):168-76, http://dx.doi.org/10.1002/jcp.21933. 37. Kim SJ, Zhang X, Xu X, Chen A, Gonzalez JB, Koul S, et al. Evidence for enhanced eNOS function in coronary microvessels during the second window of protection. Am J Physiol Heart Circ Physiol. 2007;292(5): H2152-8, http://dx.doi.org/10.1152/ajpheart.00326.2006.

28. Carden DL, Granger DN. Pathophysiology of ischaemia-reperfusion injury. J Pathol. 2000;190(3):255-66, http://dx.doi.org/10.1002/ (SICI)1096-9896(200002)190:3,255::AID-PATH526.3.0.CO;2-6. 29. Delbin MA, Moraes C, Camargo E, Mussi RK, Antunes E, de NG, et al. Influence of physical preconditioning on the responsiveness of rat pulmonary artery after pulmonary ischemia/reperfusion. Comp Biochem Physiol A Mol Integr Physiol. 2007;147(3):793-8, http:// dx.doi.org/10.1016/j.cbpa.2006.08.037. 30. Woodman CR, Thompson MA, Turk JR, Laughlin MH. Endurance exercise training improves endothelium-dependent relaxation in brachial arteries from hypercholesterolemic male pigs. J Appl Physiol. 2005;99(4): 1412-21, http://dx.doi.org/10.1152/japplphysiol.00293.2005. 31. Kandilci HB, Gumusel B, Topaloglu E, Ucar G, Korkusuz P, Ugur Y, et al. Effects of ischemic preconditioning on rat lung: role of nitric oxide. Exp Lung Res. 2006;32(7):287-303, http://dx.doi.org/10.1080/019021406008 17473. 32. Erlansson M, Bergqvist D, Marklund SL, Persson NH, Svensjo¨ E. Superoxide dismutase as an inhibitor of postischemic microvascular permeability increase in the hamster. Free Radical Biology & Medicine. 1990;9:59-65.

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DOI:10.6061/clinics/2012(08)13

BASIC RESEARCH

Vitreous pharmacokinetics and electroretinographic findings after intravitreal injection of acyclovir in rabbits Francisco Max Damico,I Mariana Ramos Scolari,I Gabriela Lourenc¸on Ioshimoto,II Beatriz Sayuri Takahashi,I Armando da Silva Cunha Jr.,III Sı´lvia Ligo´rio Fialho,IV Daniela Maria Bonci,II Fabio Gasparin,I Dora Fix VenturaII I

Faculdade de Medicina da Universidade de Sa˜o Paulo, Sa˜o Paulo/SP, Brazil. II Universidade de Sa˜o Paulo, Instituto de Psicologia, Sa˜o Paulo/SP, Brazil. Universidade Federal de Minas Gerais, Faculdade de Farma´cia, Belo Horizonte/MG, Brazil. IV Fundac¸a˜o Ezequiel Dias, Belo Horizonte/MG, Brazil.

III

OBJECTIVES: Acute retinal necrosis is a rapidly progressive and devastating viral retinitis caused by the herpesvirus family. Systemic acyclovir is the treatment of choice; however, the progression of retinal lesions ceases approximately 2 days after treatment initiation. An intravitreal injection of acyclovir may be used an adjuvant therapy during the first 2 days of treatment when systemically administered acyclovir has not reached therapeutic levels in the retina. The aims of this study were to determine the pharmacokinetic profile of acyclovir in the rabbit vitreous after intravitreal injection and the functional effects of acyclovir in the rabbit retina. METHODS: Acyclovir (Acyclovir; Bedford Laboratories, Bedford, OH, USA) 1 mg in 0.1 mL was injected into the right eye vitreous of 32 New Zealand white rabbits, and 0.1 mL sterile saline solution was injected into the left eye as a control. The animals were sacrificed after 2, 9, 14, or 28 days. The eyes were enucleated, and the vitreous was removed. The half-life of acyclovir was determined using high-performance liquid chromatography. Electroretinograms were recorded on days 2, 9, 14, and 28 in the eight animals that were sacrificed 28 days after injection according to a modified protocol of the International Society for Clinical Electrophysiology of Vision. RESULTS: Acyclovir rapidly decayed in the vitreous within the first two days after treatment and remained at low levels from day 9 onward. The eyes that were injected with acyclovir did not present any electroretinographic changes compared with the control eyes. CONCLUSIONS: The vitreous half-life of acyclovir is short, and the electrophysiological findings suggest that the intravitreal delivery of 1 mg acyclovir is safe and well tolerated by the rabbit retina. KEYWORDS: Acyclovir; Pharmacokinetics; Electroretinography; Drug Toxicity; Retina. Damico FM, Scolari MR, Ioshimoto GL, Takahashi BS, Cunha Jr AS, Fialho SL, et al. Vitreous pharmacokinetics and electroretinographic findings after intravitreal injection of acyclovir in rabbits. Clinics. 2012;67(8):931-937. Received for publication on February 23, 2012; First review completed on March 18, 2012; Accepted for publication on April 1, 2012 E-mail: fmdamico@usp.br Tel.: 55 11 3212-0200

of existing retinal lesions usually ceases approximately 2 days after the initiation of treatment with intravenous acyclovir, and visible regression may be observed after approximately 4 days (1). Complete regression occurs after an average of 32.5 days when the retina may have already been irreversibly damaged (1). To penetrate the eye, drugs must overcome the bloodocular barrier, which greatly decreases their bioavailability to the retina (6). Intravitreal injections bypass the bloodretinal barrier and thus provide immediate drug delivery at therapeutic levels directly to the target site. There is evidence that the time between diagnosis and the initiation of treatment may be a prognostic factor and that the reduction of this interval may improve the outcome of ARN (7). We postulate that the intravitreal injection of acyclovir as soon as the diagnosis of ARN has been established may be a potential adjuvant therapy because it may help to control this type of aggressive retinitis within the crucial first 48 hours, when systemic acyclovir has not reached therapeutic levels in the retina.

INTRODUCTION Acute retinal necrosis (ARN) is a rapidly progressive viral retinitis that is caused by members of the herpesvirus family and has an unfavorable visual prognosis (1,2). Prompt recognition of ARN is important, and patients who are treated early with high doses of intravenous acyclovir have a better visual outcome and a lower incidence of retinal detachment (3). Acyclovir is a potent systemic antiviral that has been the drug of choice for the treatment of ARN because of its selectivity against the herpes simplex virus and the varicella-zoster virus (4,5). The goal of treatment is to stop the progression of retinal lesions. Clinically, the progression

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.

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segment and lens were discarded, and the vitreous body was removed and frozen at -18˚C for a high-performance liquid chromatography (HPLC) assay. Peripheral blood was drawn at the time of sacrifice to determine the systemic concentration of acyclovir after intravitreal injection. The samples were frozen at -18˚C for HPLC. HPLC for the determination of vitreous and systemic acyclovir levels has been previously described (8-11). Briefly, the chromatographic system consisted of a MerckHitachi LaChrom Elite apparatus that was equipped with an autosampler with a sample loop of 100 mL (model L2200, Merck-Hitachi, Germany), a pump with a constant flow rate of 1.4 mL/min (model L-2130, Merck-Hitachi, Germany), and a diode array detector with a wavelength of 215 nm (model L-2450; Merck-Hitachi, Germany). Separation chromatography was performed using an Ace 5 C18 column (25064.6 mm id, Advanced Chromatography Technologies, Scotland) that was maintained at 50 ˚C (column oven model L-2300, Merck-Hitachi, Germany). A mixture of acetonitrile (Merck, Darmstadt, Germany) and 40 mM phosphoric acid buffer (Omega, Belo Horizonte, Brazil) at pH 3.0 (32:68 v/v) was used as the mobile phase. Under these experimental conditions, the retention time was 14.0 min. Sample treatment: Frozen vitreous samples were thawed at ambient temperature. After brief mixing, 500 mL of the mobile phase was added to 500-mL aliquots of the thawed vitreous and blood samples. After mixing for 1.0 min, the samples were filtered (Durapore, 0.2 mm, Millipore) and 100 mL was injected into the column. To reduce experimental variability, all the samples were measured together in the same assay. A standard stock solution was prepared in methanol that contained 1 mg/mL acyclovir. This solution was added to drug-free rabbit vitreous and blood to prepare six non-zero concentrations in the range of 0.25–15.0 mg/mL acyclovir (0.25, 0.5, 2.0, 5.0, 10.0, and 15.0 mg/mL). The ocular pharmacokinetic model was developed using previously described studies as a reference (12,13). All the data were fit with a single exponent according to Equation 1, and the estimated half-time (t1/2) of acyclovir elimination was calculated with Equation 2.

In this study, we investigated the pharmacokinetic profile and functional effects of acyclovir in the rabbit vitreous and the retina, respectively, at different time points after an intravitreal injection.

METHODS Animals In total, 32 New Zealand white rabbits (weight, 2.0-2.5 kg) were included in this study. The animals were treated according to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. The experiments were approved by the Research Ethics Committee of the Universidade de Sa˜o Paulo, Faculdade de Medicina and by the Committee for Ethics in Animal Research of the Instituto de Psicologia at the Universidade de Sa˜o Paulo (Sa˜o Paulo, Brazil). The animals were housed in individual cages under a 12/ 12-hour light-dark cycle with free access to water and food. Before every procedure (intravitreal injection, ERG, and sacrifice), the animals were anesthetized with an intramuscular injection of 50 mg/kg ketamine hydrochloride (Ketamina; Agener, Sa˜o Paulo, Brazil) and 6.7 mg/kg xylazine hydrochloride (Calmiun; Agener, Sa˜o Paulo, Brazil). The pupils were dilated with topical 0.5% tropicamide (Mydriacyl; Alcon, Sa˜o Paulo, Brazil), and the eyes were anesthetized with 0.5% proxymetacaine hydrochloride (Anestalcon; Alcon, Sa˜o Paulo, Brazil). A slit-lamp examination and indirect fundus ophthalmoscopy were performed in all of the eyes prior to the intravitreal injections and after 2, 9, 14, and 28 days to detect signs of inflammation or infection. All of the animals were sacrificed with an intravenous injection of 70 mg/kg sodium pentobarbital (Euthanyle; Brouwer, Buenos Ayres, Argentina) under deep anesthesia.

Acyclovir preparation The original solution of acyclovir (Acyclovir; Bedford Laboratories, Bedford, OH) was diluted with sterile saline solution (saline) to reach a concentration of 10 mg/mL for injection, and 0.1 mL (1 mg) was injected directly into the vitreous.

Intravitreal injection

CðtÞ~ Co exp({kt)

Before the injections, anterior chamber paracentesis (0.1 mL of the aqueous humor) was performed with a 27gauge needle to prevent an increase in intraocular pressure and minimize drug reflux. The intravitreal injections were performed using a 30-gauge needle that was attached to a 1mL tuberculin syringe, which was inserted approximately 3 mm posterior to the limbus, and 0.1 mL acyclovir was slowly injected directly into the vitreous. The right eye of each rabbit was injected with the acyclovir solution and the left eye was injected with saline as a control.

t1=2 ~0:693=k where C (mg/mL) and C0 (mg/mL) represent respectively the acyclovir concentration at any time and at t0, t (day) is the time after injection, and k (day-1) represents a rate constant.

Electroretinography

High-performance liquid chromatography for vitreous half-life determination

To evaluate the effect of acyclovir on retinal function, electroretinograms (ERGs) were recorded in the 8 animals that were sacrificed 28 days after injection. ERG recordings were obtained on days 2, 9, 14, and 28 after intravitreal injection. The ERG protocol was based on the international standard for electroretinography from the International Society for Clinical Electrophysiology of Vision (ISCEV) (14). The

To determine the half-life of acyclovir in the vitreous, the animals were divided into four groups of eight animals each. All the animals received an intravitreal injection of 1 mg/ 0.1 mL acyclovir in the right eye and 0.1 mL saline in the left eye. The animals were sacrificed on days 2, 9, 14, and 28 after intravitreal injection. The eyes were enucleated, the anterior

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animals were dark-adapted for 1 h and anesthetized with a mixture of ketamine hydrochloride (50 mg/kg) and xylazine hydrochloride (6.7 mg/kg), and the pupils were dilated 15 min before ERG using a topical application of 0.5% tropicamide (Mydriacyl; Alcon, Sa˜o Paulo, Brazil). Immediately before the ERG recordings, the eyes were topically anesthetized with 0.5% proxymetacaine hydrochloride (Anestalcon; Alcon, Sa˜o Paulo, Brazil). The ERG responses from both eyes were recorded using a corneal bipolar contact lens electrode (GoldLens; Doran Instruments Inc., Littleton, MA). Reference electrodes were placed in the skin near the lateral canthus of the eyes, and a ground electrode (model E5; Grass Technologies, West Warwick, RI) was placed on the ear. Light stimulation was provided by a Ganzfeld LED stimulator (Q450 SC Roland-Consult, Germany) and was controlled by a computerized system (RetiPort, Roland Consult, Germany). ERGs were recorded according to a modified ISCEV protocol (14,15): 1) Dark-adapted ERG: Five stimulus intensities were tested after dark adaptation: a) 10 flashes were presented at 0.0003 cd.s/m2 with 5-s interflash intervals; b) 6 flashes were presented at 0.003 cd.s/m2 with 5-s interflash intervals; c) after 20 s of dark adaptation, 6 flashes were presented at 0.03 cd.s/m2 with an interflash interval of 10 s; d) after 1 min of dark adaptation, 6 flashes were presented at 0.3 cd.s/m2 with an interflash interval of 10 s; and e) after 1 min of dark adaptation, 3 flashes were presented at 3.0 cd.s/m2 with an interflash interval of 15 s. 2) Light-adapted ERG: A 2-min light adaptation was conducted with a background of 25 cd.s/m2 (white light) using an average of 6 flashes at 3.0 cd.s/m2 with an interflash interval of 5 s. ERG data analysis: The a- and b-wave amplitudes and the implicit times were measured. The a-wave amplitude was measured from the baseline to the minimum amplitude after light stimulus onset. The a-wave time to peak or implicit time was measured from flash onset to the a-wave peak. The b-wave amplitude was measured from the awave through the b-wave peak amplitude. Similarly, the bwave implicit time corresponded to the time of occurrence of its peak amplitude (14). The correlation between the dark-adapted b-wave amplitude and the stimulus luminance was modeled using the Naka-Rushton function. The following three parameters were obtained: the b-wave saturating amplitude (Vmax); the dark-adapted sensitivity, which was defined as the intensity necessary for a response amplitude of 50% of the Vmax (k); and the exponential of the Naka-Rushton equation (n), which is related to the slope in the linear phase of the sigmoid function and represents the homogeneity of the retinal sensitivity.

RESULTS Clinical findings No cataracts, anterior chamber cells, vitreous cells, retinal lesions, or cases of endophthalmitis were detected in the eyes that were injected with acyclovir or saline at any time point during the study period.

Acyclovir pharmacokinetics The HPLC method was validated, and a calibration curve was obtained. The HPLC sensitivity was 0.1 mg/mL. The vitreous concentrations of acyclovir over 28 days are presented in Figure 1. The mean intravitreal concentrations of acyclovir (mg/mL) were 0.28, 0.09, 0.06, and 0.08 on days 2, 9, 14, and 28 after injection, respectively. The half-life of acyclovir in the rabbit vitreous could not be estimated because the drug was quickly eliminated and only a small amount was detected on the second day after injection. Acyclovir was not detected in the peripheral blood of any animal that was tested.

ERG The representative dark- and light-adapted ERG recordings of one animal at different time points are shown in Figure 2. The eyes that were injected with acyclovir were compared with the eyes that were injected with saline. In the dark- and light-adapted states, no significant differences were found between the means of the a- and b-wave amplitudes and the implicit times for any time point or light intensity. The data from the dark-adapted ERG recordings are shown in Table 1. The dark- and light-adapted a-wave and b-wave mean amplitudes were plotted against the log light intensity (VlogI) using the Naka-Rushton equation. The curves at each time point were compared to the mean baseline curve. No significant differences were found for the a-wave or b-wave amplitudes on days 9, 14, and 28 compared with the baseline. The data for the b-wave amplitudes are presented in Figure 3. The Vmax, k, and n values were calculated for the b-wave amplitudes, and no significant differences were found between the eyes that were injected with acyclovir and saline. The b-wave Vmax, k, and n values are presented in Table 2. The b-wave to a-wave correlations for the dark-adapted state (30 cd.s/m2) are shown in Figure 4. No significant differences were found among the different time points.

Analysis The statistical analysis was performed using Statistica software (StatSoft v6.0, Inc., Tulsa, OK, USA,). The assessment of the significant differences among the groups was performed using the repeated-measures ANOVA test, which takes into account the strength of the correlations and provides information regarding the variables. The Fisher’s Least Significant Differences Test was used to determine the differences between the group means in the repeated-measures ANOVA tests.

Figure 1 - Acyclovir concentrations in the vitreous at different time points after intravitreal injection.

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Figure 2 - Representative ERG recordings of the eyes of one animal at different time points [dark-adapted (30 cd.s/m2) and lightadapted (3 cd.s/m2) states].

Oscillatory potentials could not be detected in any of the eyes that were studied.

The injected dose was estimated based on the dose that was used for the intravenous injections and on a dose-escalating study (Damico FM et al., unpublished), which demonstrated that 1 mg acyclovir injected into the vitreous does not cause morphological changes in the rabbit retina, according to light microscopy. ERG is a useful tool that is commonly employed to monitor retinal toxicity (16). In addition to the determination of a- and b-wave amplitudes and implicit times in lightand dark-adapted states, many correlations and other parameters can be analyzed. The analysis of the correlation between b-wave amplitude and light intensity at different

DISCUSSION Our ERG results suggest that intravitreal injection of acyclovir 1 mg in 0.1 mL does not cause functional changes in the retina of New Zealand white rabbits according to binocular indirect ophthalmoscopy and ERG during a 28-day follow-up period. This is the first report of experimental data on the retinal effects of 1 mg acyclovir injected directly into the vitreous.

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Table 1 - a- and b-wave amplitudes and implicit time means at different light intensities and time points (dark-adapted state). Eight animals were included at each time point. SD = standard deviation; N.R. = not registered. Light energy

a-wave Amplitude (mV) 2

(cd.s/m )

Baseline 0.003 0.03 0.3 3 30 9 days 0.003 0.03 0.3 3 30 14 days 0.003 0.03 0.3 3 30 28 days 0.003 0.03 0.3 3 30

Mean (SD)

b-wave Implicit time (ms)

Amplitude (mV)

Mean (SD)

Implicit time (ms)

Mean (SD)

Mean (SD)

Acyclovir

Saline

Acyclovir

Saline

Acyclovir

N.R. N.R. N.R. -72 (43) -143 (24)

N.R. N.R. N.R. -75 (28) -132 (42)

N.R. N.R. N.R. 45 (1) 42 (0)

N.R. N.R. N.R. 45 (4) 36 (2)

23 108 130 210 259

(8) (58) (32) (56) (47)

21 (7) 89 (14) 110 (21) 189 (34) 232 (55)

82 83 68 67 66

(3) (6) (2) (1) (1)

82 (19) 85 (4) 74 (4) 70 (12) 72 (13)

N.R. N.R. N.R. -58 (34) -127 (24)

N.R. N.R. N.R. -72 (13) -137 (39)

N.R. N.R. N.R. 45 (1) 42 (0)

N.R. N.R. N.R. 47 (6) 45 (2)

16 119 134 196 260

(9) (42) (37) (42) (53)

11 119 147 209 281

(4) (18) (25) (37) (59)

73 80 68 67 72

(9) (4) (1) (3) (8)

74 (9) 83 (6) 78 (4) 59 (4) 54 (15)

N.R. N.R. N.R. -74 (34) -118 (51)

N.R. N.R. N.R. -65 (42) -127 (44)

N.R. N.R. N.R. 47 (1) 43 (1)

N.R. N.R. N.R. 46 (5) 42 (2)

11 (8) 117 (59) 195 (94) 231 (105) 264 (109)

11 157 254 261 303

(3) (39) (53) (47) (63)

89 (0) 84 (4) 81 (11) 66 (1) 66 (2)

84 (10) 72 (5) 72 (5) 67 (4) 63 (6)

N.R. N.R. N.R. -80 (28) -116 (30)

N.R. N.R. N.R. -77 (38) -131 (42)

N.R. N.R. N.R. 46 (2) 42 (1)

N.R. N.R. N.R. 44 (5) 40 (2)

23 (19) 173 (65) 220 (94) 305 (102) 347 (85)

17 (4) 190 (73) 178 (49) 243 (64) 275 (101)

77 (21) 80 (5) 75 (11) 79 (13) 77 (11)

87 (16) 90 (6) 78 (5) 73 (5) 75 (9)

time points offers an estimate of the functional integrity of the retina. Different ERG components are related to different retinal structures. The cornea negative a-wave reflects the function of the photoreceptors; the b-wave reflects the function of bipolar and Mueller cells; and the oscillatory potentials reflect the activity of the inner retina (17). Moreover, the b-wave to a-wave amplitude ratio is an

Saline

Acyclovir

Saline

index of post-receptoral function that represents the effect of a given stimulus in the inner and outer retina (18). In this study, ERG recordings were obtained at baseline and at 3 different time points after intravitreal injection of acyclovir into the study eye and saline into the contralateral eye. ERG parameters are influenced by many factors, such as pupil size, dark adaptation time, electrode, stimulus intensity, age, breed, anesthetic drugs, and body temperature (19,20); however, it is difficult to control for these factors. Our results presented low variability (Table 1), which reflects the high quality of the ERG technique. There were no significant differences between the a- and b-wave amplitude values and the implicit time values, which suggests that the inner and outer retina were not functionally impaired by the dose of acyclovir that was used in this study. In addition, the b-wave to a-wave ratios were not different between the eyes that were injected with acyclovir and saline, which suggests that the 1-mg dose of acyclovir does not cause the deterioration of retinal function. However, full-field ERG is a mass electrical response of the retina to light stimulation, and acyclovir may cause focal damage to the retina that may not be detected by testing. Intravitreal injections of bevacizumab do not induce signs of toxicity in the rabbit retina according to ERG (21,22). However, inflammatory cells and signs of apoptosis in the photoreceptors were observed using transmission electron microscopy in these studies. Similar findings were obtained for a high-dose intravitreal injection of moxifloxacin (23). Experimental studies in the 1980s evaluated the retinal toxicity of acyclovir after intravitreal injection (24-26) and demonstrated that doses of up to 450 mg/0.1 mL do not

Figure 3 - Response versus log light intensity (VlogI) curve of the eyes that were injected with acyclovir and saline solution. The gray area represents the mean ยก1 standard deviation of the bwave amplitude of the eyes that were injected with saline solution.

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Table 2 - b-wave amplitude parameters obtained from the response versus log light intensity (VlogI) curve using the Naka-Rushton equation (dark-adapted state). b-wave amplitude

Time point Vmax

Baseline 7 days 14 days 28 days

k

n

Acyclovir

Saline

p-value

Acyclovir

Saline

p-value

Acyclovir

Saline

p-value

2.492 2.335 2.341 2.455

2.340 2.333 2.443 2.352

0.299 0.971 0.342 0.138

0.0082 0.0030 0.0052 0.0045

0.0019 0.0040 0.0041 0.0030

0.200 0.081 0.532 0.338

0.7367 0.9052 0.9984 1.0167

0.4743 0.9639 1.1895 2.1552

0.121 0.750 0.325 0.258

after oral intake has been observed to vary from 2.5 to 3 h (27,28). In rabbits, the systemic half-life is even lower, varying from 1 to 2 h (29). These results suggest that acyclovir may present a short half-life in the vitreous, which may explain the rapid clearance from the vitreous that was observed in this study. However, acyclovir would not be detected if it were bound to serum proteins (30). This hypothesis was not tested in the present study. In this study, the determination of the vitreous half-life of acyclovir was not possible because of the intervals that were chosen for sample collection. However, our results suggest that the vitreous half-life of acyclovir may be very short, such as less than 48 h. This study has several limitations. The relatively low number of animals that underwent ERG assessment (eight animals) may mask differences between the eyes that were injected with acyclovir and saline. However, our results had low variability, which suggests that they are reliable. In addition, acyclovir is highly soluble in water and vitreous clearance is likely to be very short. ERG was recorded at baseline and on day 9, when a small amount of acyclovir

cause retinal toxicity; however, the same animals were not used as controls. In contrast, in the present study the contralateral eye of the same animal was used as control. The use of different animals as controls may not be adequate because of the high inter-animal variability in ERG parameters. In addition, the ERG methods and data were not well described in the previous studies, which makes comparisons with our results difficult. The pharmacokinetic study revealed that systemic acyclovir absorption after intravitreal injection may not be significant because the drug was not detected in the peripheral blood of any animal. This is the first report on the vitreous half-life of acyclovir. HPLC indicated a rapid decay in the concentration of acyclovir in the vitreous during the first two days after intravitreal injection, after which the acyclovir was maintained at low levels from day 9 on. Acyclovir is a Biopharmaceutics Classification System Class III compound and is highly water-soluble with low permeability and a low molecular mass. In addition, the systemic half-life of acyclovir

Figure 4 - b-wave to a-wave (dark-adapted state, 30 cd.s/m2) correlations between the eyes that were injected with acyclovir and saline at different time points. Each point represents the a- and b-wave amplitudes from one eye.

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was present in the vitreous. Thus, acyclovir may have caused functional retinal damage within the first hours or days after intravitreal injection that was not detectable 9 days after injection. Therefore, extensive functional retinal recovery may have occurred. Finally, this is an experimental study and the limitations of the rabbit model include differences in retinal vascularization and eye volume when compared with the human eye. For these reasons, the results may not fully represent the effects in human eyes. In conclusion, this pharmacokinetic study suggests that the vitreous half-life of acyclovir is very short, and the clinical and electrophysiological findings suggest that intravitreal delivery of acyclovir 1 mg in 0.1 mL is safe and well tolerated by rabbit retina. Before intravitreal acyclovir can be used as an adjuvant therapy during early ARN to compensate for the delay in systemic acyclovir reaching therapeutic levels in the retina, additional studies are necessary to determine the precise vitreous half-life of acyclovir, and dose-escalating studies are necessary to determine safe doses for intravitreal injection.

9.

10.

11.

12.

13.

14.

15.

16.

Financial support: Francisco Max Damico (CNPq Postdoctoral Fellowship - 150614/2009-8). Mariana Ramos Scolari (FAPESP Undergraduate Research Fellowship - 2010/08331-8). Dora Fix Ventura (FAPESP Research Grants - 2011/06924-4, 2008/58731-2, and CNPq Fellowship – 1A).

17.

18.

AUTHOR CONTRIBUTIONS 19.

Damico FM contributed to the study design, collection, analysis, and interpretation of the data, manuscript writing, acquisition of financial support and general supervision of the research group. Scolari MR, Ioshimoto GL, Takahashi BS, Fialho SL, Bonci DM, and Gasparin F were responsible for data collection. Cunha Jr. AS contributed to the study design, collection, analysis and interpretation of the data, and drafting of the manuscript. Ventura DF contributed to the conception and design of the study, analysis and interpretation of the data, writing of the manuscript, acquisition of financial support and general supervision of the research group.

20.

21.

22.

23.

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CLINICS 2012;67(8):939-944

DOI:10.6061/clinics/2012(08)14

BASIC RESEARCH

Upregulation of matrix synthesis in chondrocyteseeded agarose following sustained bi-axial cyclic loading Belinda Pingguan-Murphy, Illida Nawi University of Malaya, Faculty of Engineering, Department of Biomedical Engineering, Kuala Lumpur/Malaysia.

OBJECTIVES: The promotion of extracellular matrix synthesis by chondrocytes is a requisite part of an effective cartilage tissue engineering strategy. The aim of this in vitro study was to determine the effect of bi-axial cyclic mechanical loading on cell proliferation and the synthesis of glycosaminoglycans by chondrocytes in threedimensional cultures. METHOD: A strain comprising 10% direct compression and 1% compressive shear was applied to bovine chondrocytes seeded in an agarose gel during two 12-hour conditioning periods separated by a 12-hour resting period. RESULTS: The bi-axial-loaded chondrocytes demonstrated a significant increase in glycosaminoglycan synthesis compared with samples exposed to uni-axial or no loading over the same period (p,0.05). The use of a free-swelling recovery period prior to the loading regime resulted in additional glycosaminoglycan production and a significant increase in DNA content (p,0.05), indicating cell proliferation. CONCLUSIONS: These results demonstrate that the use of a bi-axial loading regime results in increased matrix production compared with uni-axial loading. KEYWORDS: Chondrocytes; Mechanotransduction; Cellular; Glycosaminoglycans; Bioreactors. Pingguan-Murphy B, Nawi I. Upregulation of matrix synthesis in chondrocyte-seeded agarose following sustained bi-axial cyclic loading. Clinics. 2012;67(8):939-944. Received for publication on December 21, 2011; First review completed on February 2, 2012; Accepted for publication on April 11, 2012 E-mail: bpingguan@um.edu.my Tel.: 603 7967-4491

variable mix of static-, compressive-, and shear- loading components (12). While previous studies have examined the effect of static compression, directly or indirectly applied pressure (including hydrostatic pressure, osmotic pressure, pH and ion concentration and interstitial fluid flow–induced pressure), compressive stresses, and sliding shear stress on chondrocyte metabolism, there have been no studies published to date that have examined the effect of a bi-axial loading regime comprised of the cyclic application of both compressive and shear loading on isolated chondrocytes, which would ensure the full recovery of dimension within each cycle. Given that existing research indicates that chondrocytes are able to differentiate different loading regimes in terms of a differential response and given the complex nature of cartilage tissue, which makes consistent loading of chondrocytes difficult, the present study makes use of the wellestablished chondrocyte-seeded agarose model and a bioreactor capable of highly flexible combinations of compressive and shear loading over a sustained period. This study aims to test the hypothesis that bi-axial loading increases sulfate glycosaminoglycan synthesis, a key component of the extracellular matrix compared with compressive loading alone. In addition, based on previous reports suggesting that a post-isolation recovery period is beneficial,

INTRODUCTION Mechanical loading is an essential factor in the maintenance of articular cartilage matrix homeostasis (1) and is linked to both matrix composition (2) and remodeling (3,4). This interaction has been determined in both in vitro and in vivo studies, which have both demonstrated that mechanical loading effectively modulates chondrocyte metabolism (510). Through a complex and poorly understood mechanotransduction pathway, chondrocytes respond to a variety of loading regimes in different ways: static compression or the lack of loading has been shown to cause matrix degradation, but some forms of dynamic compression have been shown to maintain matrix homeostasis or foster an increase in matrix density (5,6,11). Physiological loading patterns produced by gait and walking activities comprise a combination of loading forms, varying in terms approximately equivalent to frequency, force, waveform and duration and including 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.

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the effect of a period of free-swelling recovery prior to the mechanotransduction by the chondrocytes is evaluated and quantified (5,13).

MATERIALS AND METHODS Biochemical reagents

Figure 1 - Schematic diagram showing six agarose constructs seeded with chondrocytes attached to porous glass strips. The top strip moves vertically to give a shearing effect, while the other strip moves horizontally for the compressive strain. The movement of the porous glass strips is controlled by the bioreactor.

All reagents, buffers and assays were obtained from Sigma-Aldrich (Petaling Jaya, Selangor, Malaysia) unless otherwise stated. The culture medium was Dulbecco’s Modified Eagle’s Medium (DMEM) (D5921) supplemented with 20% fetal bovine serum (FBS), 2% (v/v) HEPES buffer solution, 1% (v/v) penicillin-streptomycin, 1% (v/v) Lglutamine and 0.01% (w/v) L-ascorbic acid. The culture medium was sterilized using a 0.22 mm cellulose acetate filter.

two opposite faces to strips of sintered glass that were used in the subsequent mechanical manipulations.

Bi-axial Loading System An incubator-housed bioreactor (14) was used to facilitate the direct compression and shear compression applied to the chondrocyte/agarose constructs. The bioreactor uses strips of sintered glass to which the cell/agarose constructs are affixed to permit the long-term application of cyclic displacement in both the principal compressive and shear axes (Figure 1). For uni-axial loading, a 10% compressive strain was delivered to the constructs; for bi-axial loading, the 10% compressive strain was combined with a 1% shear strain. The loading frequency was maintained at 1 Hz, and the waveform was sinusoidal throughout all experiments. The system was operated according to the method of Yusoff et al. (14).

Preparation of chondrocyte/agarose constructs Bovine articular cartilage was obtained from cow joints from the local abattoir. The cells were isolated on the same day the 18–24 month old Bos indicus calf was slaughtered. Articular chondrocytes were isolated from dissected bovine metacarpal-phalangeal joints. The full thickness of the cartilage from the entire proximal surface of the joint was removed under sterile conditions. The explanted tissue was then enzymatically digested with 20 U?ml-1 protease at 37 ˚C for 1 hour and immersed in 200 U?ml-1 collagenase type II for a further 16 hours. In both cases, the enzymes were resuspended in DMEM supplemented with 20% FBS. To ensure that all cartilage fragments were fully exposed during digestion, the Falcon tubes containing the cartilage fragments were placed on a roller-mixer (ProBlot L12-2, LabNet, Malaysia). The supernatant containing the released chondrocytes was passed through a 70 mm cell sieve (BD Bioscience, Malaysia) into sterile Falcon tubes and washed twice with DMEM+20% FBS. Cell suspensions from several joints were pooled, resuspended to a uniform density of live cells, and mixed with an equal volume of 8% agarose (type VII) prepared in Earl’s Balanced Salt Solution (EBSS) to yield a 4% w/v agarose gel containing 46106 cells?ml-1. The cell/ agarose suspension was transferred while still fluid into a mold to form six 5 mm cubes, which were then affixed on

Experimental Design The created constructs were divided into three equal groups: one group was subjected to uni-axial loading, another to bi-axial loading, and the third left unloaded for an equivalent period. The loading took place for two 12-hour periods, separated by a 12-hour recovery time. An additional comparative subdivision across all groups added a freeswelling recovery period of 24 hours post-isolation and seeding before the loading was initiated. Each group contained 24 constructs, and the data were obtained across four time-separated repeats. The groups, divisions, loading regimes and repeats per group are summarized in Table 1.

Table 1 - Outline of the experiments performed to assess the influence of bi-axial loading and uni-axial loading and the need for pre-culturing prior to the chondrocytes being subjected to the mechanical loads. Uni-axial samples were exposed to 10% direct compressive strain and 0% shear strain, whereas bi-axial samples were exposed to 10% direct compressive strain and 1% shear strain. In the experimental setups, (#) signifies chondrocytes that were subjected to loads immediately upon cell seeding, and (N) signifies chondrocytes that were left pre-cultured for 24 hours prior to being subjected to any load. Group and sub-group Loading Free-swelling Loading Pattern -12–0 h: free swelling 0–12 h 12–24 h 24–36 h 36–48 h Repeats and samples per batch n (per batch) n (total) Total constructs

Uni-axial loading

Bi-axial loading

No loading

#

N

#

N

#

N

n/a Uni-axial Rest Uni-axial Rest

Rest Uni-axial Rest Uni-axial Rest

n/a Bi-axial Rest Bi-axial Rest

Rest Bi-axial Rest Bi-axial Rest

n/a Rest Rest Rest Rest

Rest Rest Rest Rest Rest

6 24

6 24

6 24

6 24

6 24

6 24

144

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Matrix synthesis following bi-axial cyclic loading Pingguan-Murphy B and Nawi I

were separated by one week, and fresh cells were obtained from new calves for each batch. There was an approximately 50% increase in GAG production after the addition of the 1% shear loading cycle. The DNA content of the samples that were exposed to sinusoidal loading was reduced approximately 10% when compared with the unstrained samples (p,0.05) (Figure 2B). However, even after normalization to the DNA content, GAG production in the bi-axially loaded constructs was 1.8fold higher than the production in the uni-axial loaded constructs (p,0.05) (Figure 2A).

If a batch was discovered to be infected, then it was immediately destroyed, and no results from that batch were included in the study; this situation occurred in two cases. Furthermore, if detachment of the constructs occurred during the course of the experiment, the data were omitted from analysis as it was no longer possible to determine how much load the constructs had been subjected to. This occurred once, and the experiment was repeated.

Biochemical Analysis Following mechanical stimulation, all constructs were enzymatically digested in 10 U?ml-1 of agarase and 2.8 U?ml-1 of papain (15). The suspension was analyzed for DNA content using Hoechst 33258 dye with calf thymus DNA as a standard (16). A microplate-based fluorometer (FLUOstar Optima, BMG Labtech, Ofdenberg, Germany) was used to obtain the fluorescence levels (450 nm emission, 355 nm excitation) (16,17). The glycosaminoglycan (GAG) content in both the medium and agarase/papain digests was assessed using a DMB assay with a chondroitin-4-sulfate standard (18). As collagen is not expected to be synthesized in significant quantities during the time span of this experiment, it was not measured.

Effects of pre-culturing on GAG synthesis and DNA content Figure 3 summarizes the results obtained in cells that had been pre-cultured or cells that were not pre-cultured. The pre-cultured (PC) samples demonstrated higher cellularity and GAG production than the non-pre-cultured (NPC) samples. There was a 30% increase in the DNA content of the PC samples when the constructs were subjected to biaxial loading (Figure 3A). Additionally, a statistically significant 10% increase in GAG production was observed in the PC samples (Figure 3B) (p,0.05).

Statistical Analysis

DISCUSSION

All data are presented as the mean normalized to the values obtained from the unstrained samples with the standard error of the mean values across the 24 replicates from 4 separate experiments used in each group. ANOVA was used to determine the significance of the differences between the groups tested. All statistical analyses were performed using SPSS (Version 17.0; SPSS Inc., Chicago, Illinois, USA, 2006), with the significance level set at a = 0.05. A post hoc LSD test was used for multiple comparisons at a = 0.05.

Worldwide, one in ten people has osteoarthritis, which is a recurring progressive degenerative disease that is usually caused by trauma or overuse of the afflicted joint (19). Although some tissues might be expected to undergo repair when given appropriate support and rest, repair of the articular cartilage is rarely observed. Two important factors of a tissue engineering solution for articular cartilage are the extent to which a functional matrix can be produced and the ability to stimulate and sustain cell proliferation. The role of mechanical load in cartilage tissue remodeling and chondrocyte signaling has been clearly established (20-22). Previous studies have shown that low amplitude dynamic compression induces the stimulation of GAG synthesis in cartilage explants (23-26) and isolated chondrocytes cultured in three-dimensional structures (5-9,15). Physiologically, articular cartilage in the load-bearing joints experiences complex mechanical loading consisting of combinations of compressive, shearing and tensile forces (27). Most studies have used animal models as primary animal material is more easily acquired, although a few studies have demonstrated comparable trends with human cartilage. Table 2 summarizes

RESULTS Effects of bi-axial and uni-axial loading on GAG synthesis and DNA content The levels of GAG synthesis observed from cells exposed either to 10% uni-axial compression or the same compression combined with 1% shear compressive strain are shown in Figure 2A. The presented data have been normalized to the unstrained control samples to limit the effect of batch-tobatch variation and any changes in environmental conditions. A total of 24 samples were used for each condition, with sets of six samples tested within each batch; the batches

Figure 2 - After a free-swelling pre-culture for 24 hours, the chondrocyte/agarose constructs were subjected to either 10% direct compressive and 1% shear compressive strain (Bi-axial) or 10% direct compressive strain without shear strain (Uni-axial). At the end of the experiment, (a) proteoglycan synthesis and (b) DNA content were quantified. The values are depicted as the percentage change and standard error. The values are normalized to unstrained samples as the control group (100%). The DNA content of each sample is treated as the baseline in (a). As determined using ANOVA, all data from the samples loaded bi-axially or uni-axially were significantly different. (*) p,0.05.

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Figure 3 - The (a) DNA content of and (b) proteoglycan production by chondrocytes seeded in agarose subjected to bi-axial (10% compressive + 1% shear strains) or uni-axial (10% compressive + 0% shear strains) loads immediately after cell seeding or after 24 hours of pre-culture under free-swelling conditions was measured. The data were taken from a total of 48 samples for each pre-cultured (PC) and non-pre-cultured (NPC) condition. The data are presented as the percentage change normalized to the non-pre-cultured samples. The DNA content of the chondrocytes in each sample is the baseline for all glycosaminoglycan measurements for all conditions in (b). Unstrained non-pre-cultured samples are treated as the control group. The error bar depicts the standard error of the mean. (*) p,0.05.

the results of some studies using human and bovine cartilage that have examined the effect of physiological mechanical loading on matrix synthesis by chondrocytes. In the present study, the differential effect of uni- and biaxial loading on the chondrocytes demonstrates that bi-axial loading is advantageous for the synthesis of GAG, resulting in an approximately 50% increase. Our findings are consistent with the results reported following the application of 1– 3% shear strain alone (24,31). Some of the possible mechanisms involved in the transduction of dynamic compression include altered fluid pressure, enhanced fluid flow, induced streaming potentials, cell-matrix interactions and growth factor release. In addition to cellular and nuclear deformation, dynamic loading enhances the convective transport of mobile solutes, especially larger molecules such as ADP and growth factors. Fluid flow that involves the convection of mobile counterions past ionized charge groups on immobilized macromolecules generates streaming potentials (5). Cell proliferation was significantly greater in pre-cultured chondrocyte/agarose constructs. The samples were left to freely swell for 24 hours upon seeding. The newly formed pericellular matrix around chondrocytes within agarose has a higher elastic modulus than agarose/chondrocyte constructs (13), which should influence the mechanical response of the cell. Furthermore, in support of the current study, Buschmann et al. (5) demonstrated that the readiness of the cells occurs within one day, as determined by hyaluronan and integrin assays.

As suggested by Hunter et al. (11), the mechanical signals transmitted to the cells may vary substantially between different scaffolding environments. Chondrocytes in native tissue bind to the ECM via cell adhesion, which can transfer matrix strains through mechanosensitive ion channels directly to the cytoskeleton, whereas in agarose, the cells adhere to polysaccharide molecules, making preculture advantageous when polysaccharide matrices such as agarose or alginate are used. With pre-culturing, the cells are able to bind to the new pericellular matrix as it is deposited, thus providing a biomechanical interaction between the pericellular environment and the chondrocytes (11,32). Hunter et al. (11) suggested that stimulation interspersed with periods of rest might enhance tissue formation. This resting period is essential to permit the restoration of cell sensitivity because continued compression is thought to cause mechanosensory saturation that would, in turn, decrease the sensitivity of the cells to additional mechanical stimuli (11,33). As shown in Figure 2B, the chondrocytes subjected to sinusoidal bi-axial loading demonstrated a marked reduction in DNA content compared with the unstrained control samples. Considering the multiple phases of the cell cycle, the cells might take a relatively long time to complete one cycle. Freshly isolated chondrocytes are highly metabolically active until they have had time to deposit some of their own matrix (34). Previous studies by Waldman et al. (10,35) have shown that intermittent multi-axial loading on chondrocytes

Table 2 - Comparison of previous in vitro studies indicating the positive effect of mechanical loading on articular chondrocytes. Type of load Dynamic compression

Regimen

Model System

Major Effect

Reference

15% amplitude strains at a frequency of 0.3 Hz

Bovine articular chondrocytes

After 48 hours, an increase in proteoglycans and collagens was observed Increased proteoglycan and collagen levels Increased Type II collagen and SOX9 gene expression Upregulated Aggrecan and type II collagen mRNA Increased Aggrecan mRNA

(9)

Dynamic compressive and shear 2–5% strain at a frequency loading of 0.5 Hz Intermittent hydrostatic 10 MPa at a frequency of 1 Hz pressure Intermittent hydrostatic 1, 5 and 10 MPa pressure Cyclic pressure–induced strain 0.33 Hz

Bovine articular chondrocytes Human osteoarthritis chondrocytes Human articular chondrocytes Human articular chondrocytes

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Matrix synthesis following bi-axial cyclic loading Pingguan-Murphy B and Nawi I

resulted in a decrease in the total DNA content of stimulated samples, which may have been caused by either cell death or cell cycle inhibition. An early study revealed that cell viability and cell cycle progression were shown to be initially inhibited by mechanical stimulation. However, once a proper ECM is built, mechanical stimulation begins to have positive effects. In fact, in a study by Huselstein et al. (36), the positive effect was only seen after 21 days. In summary, chondrocytes seeded in three-dimensional scaffolds and subjected to bi-axial loading (i.e., superimposed compressive and shear strain) synthesize increased levels of matrix when compared with chondrocytes subjected to only compression. In agreement with previous studies, this study also shows that the pre-culturing of chondrocytes is an important prerequisite for effective three-dimensional culture, specifically in terms of cell proliferation. However, there is still a need to investigate the effect of a longer period of preculture. Furthermore, the effects of variations in the loading parameters, including changes in the frequency, duration, amplitude and waveform applied during the loading period, as well as the effects of shear loading in isolation, remain unknown.

12.

13.

14.

15.

16.

17.

18.

ACKNOWLEDGMENTS

19.

This study was funded by the Ministry of Higher Education of Malaysia (UMRG: RG033/09AET and PPP: PS109/2010A). The authors would like to thank Mr. Adhli Iskandar for logistical support in the acquisition of the source tissue.

20.

21.

AUTHOR CONTRIBUTIONS

22.

Nawi I contributed substantially to the conception and design of the study, data acquisition, analysis and interpretation, and manuscript draft. Pingguan-Murphy B revised the manuscript and approved the final version of the manuscript.

23.

REFERENCES

24.

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DOI:10.6061/clinics/2012(08)15

REVIEW

Thyroid nodule management: clinical, ultrasound and cytopathological parameters for predicting malignancy Frederico F. R. Maia, Denise Engelbrecht Zantut-Wittmann University of Campinas, Department of Internal Medicine, Endocrinology Division, SaËœo Paulo/SP, Brazil.

Although fine-needle aspiration cytology is considered to be the reference method for evaluating thyroid nodules, the results are inaccurate in approximately 10-30% of cases. Several studies have attempted to predict the risk of malignancy in thyroid nodules based on age, nodularity, thyrotropin values, thyroid autoimmune disease, hot/cold nodule status, and ultrasound parameters. However, no consensus has been found, and none of these parameters has significantly affected patient management. The management of indeterminate thyroid nodules and re-biopsies of nodules with initially benign cytological results remain important and controversial topics of discussion. The Bethesda cytological system and several studies on the use of molecular markers to predict malignancy from cytological samples of thyroid nodules need further clarification. More in-depth discussions among and continuous education of the specialists involved in treating thyroid disease are necessary to improve the management of these patients. This review aims to examine the clinical, laboratory, ultrasound, and scintigraphic parameters that can be used for thyroid nodule management. KEYWORDS: Thyroid Nodule; Ultrasound; Fine-Needle Aspiration Cytology; Malignancy. Maia FF, Zantut-Wittmann DE. Thyroid nodule management: clinical, ultrasound and cytopathological parameters for predicting malignancy. Clinics. 2012;67(8):945-954. Received for publication on December 20, 2011; First review completed on January 26, 2012; Accepted for publication on March 19, 2012 E-mail: zantutw@fcm.unicamp.br Tel.: 55 19 35217775

Nodular hyperplasic lesions are characteristically present in multinodular goiter (MNG) and are caused by follicular cell hyperplasia. In some cases, hyperplasic nodules can grow and become autonomous even in the absence of external stimuli (6). Differentiated thyroid carcinomas (DTCs), which encompass papillary and follicular carcinomas, are relatively uncommon tumors. They are generally associated with a good prognosis, with an estimated incidence of 1 to 10 cases/ 100,000 people per year. They are the most common endocrine neoplasm in the world, but they represent only 1% of all malignancies (1,7,9). Undifferentiated or anaplastic carcinomas represent approximately 5% of all thyroid carcinomas, and medullary thyroid carcinoma (MTC), which is derived from parafollicular cells, may occur sporadically or familially (1,3,5,8). Due to the increased use of ultrasonography (US) and the increased access to cytology analysis through fine-needle aspiration biopsy (FNAB) guided by US (FNAB-US), the number of small-sized thyroid gland carcinoma diagnoses has increased in Brazil and in many other countries (5-8). Thus, carcinomas smaller than 1 cm in diameter are being detected more frequently. They are usually diagnosed in an unexpected manner ("incidentalomas") by US or histopathological examinations of surgically excised glands in cases with benign presentations, such as airway obstruction, large goiter, and uncontrolled hyperthyroidism (8,9). Epidemiological studies conducted in iodine-sufficient regions demonstrate a 5 to 10% prevalence of palpable

THYROID NODULES Thyroid nodules are one of the most common endocrine diseases in the world. They affect approximately 4 to 7% of the population in iodine-sufficient areas, with a markedly increased incidence in iodine-deficient regions (1). Thyroid nodules are classified as adenomas, carcinomas, or hyperplastic lesions based on their macroscopic and microscopic histological features (1,2). Adenomas consist of encapsulated lesions derived from the follicular epithelium, and they may be present in isolated, macrofollicular (colloid), microfollicular (fetal), and trabecular/solid (embryonic) forms (2,3). Adenomas may be functioning (autonomous), in which case they are proportionally larger than the rest of the parenchyma and produce excessive thyroid hormones, or non-functioning, in which case hormone levels are unchanged. Autonomous adenomas can occur at any age, but they are rarely toxic in individuals under 60 years of age (4). These nodules are generally considered benign, with rare cases of malignancy (5).

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.

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thyroid nodules in women and a 1 to 2% prevalence in men (1,2,8-10). US studies have revealed the existence of thyroid nodules in 19 to 67% of normal-risk female and elderly individuals (3,10-14). These findings have been corroborated by autopsy studies (15). The increased nodularity and diameter of the thyroid seems to be inversely proportional to thyroid-stimulating hormone (TSH) levels (16-18). An evolution to hyperthyroidism due to the development of functioning autonomous nodules occurs in approximately 10% of cases over a ten-year period (19-21). FNAB-US assessment is currently recommended for nodules with a diameter larger than 1 cm and for nodules smaller than 1 cm with suspicious features (hypoechogenicity, microcalcifications, border irregularity, and Doppler central flow) (8,9,13,22,23) detected by ultrasound. An ultrasound examination is indicated in all cases of suspected nodules, and an initial TSH serum assessment should be performed in addition to scintigraphy when a functional nodule is confirmed (9). The majority of patients with thyroid nodules can be treated conservatively because 90 to 95% of nodules are nonfunctional, benign nodules with associated mortality rates of less than 1% (1,8,9,13). In particular, female gender, age between 20 and 45 years, nodules smaller than 2 cm, lack of multicentricity on US, absence of a glandular capsule, and locoregional lymph node outbreaks are considered to be factors related to low malignancy risk (8,9,13,16). Thus, it is important to properly select candidates for surgery based on the suspicion of malignancy (16,21,22). Various clinical, ultrasound, and cytological parameters, such as age, gender, nodularity, TSH level, thyroid autoimmunity, and ultrasound findings (hypoechogenicity, microcalcifications, irregular borders, and increased central nodular flow), have been studied to improve diagnostic accuracy and differentiate between benign and malignant nodules (9,13-16,22). The literature indicates higher malignancy rates in individuals below 16 or above 45 years of age (8,13). There is no male or female predominance, even though the incidence of nodules is higher in women (8,9). Some studies have found a higher malignancy rate in patients with a solitary nodule than in patients with multiple lesions (21), although more recent reports have not confirmed this association (8,22). Therefore, the results are conflicting, and the samples studied are often not representative, due to either short follow-up times or low correlations between the factors studied (1,2,8,10,13-16). Cytopathologically characterizing and differentiating between benign and malignant follicular lesions is practically impossible (24). The identification of genetic or immunohistochemical markers that can distinguish both follicular adenoma from follicular carcinoma and papillary hyperplasia from papillary carcinoma remains a long-standing goal. However, these markers remain inadequate for use in clinical practice (24-27). This review aims to discuss current thyroid nodule management, diagnosis, and malignancy prediction using ultrasound imaging and clinical and cytopathological data. The following terms were used in a Medline/Pubmed search: thyroid nodule, management of thyroid nodules, thyroid ultrasound, thyroid cytology, and FNAB of thyroid nodules. Approximately 220 articles published in the period from 2000 to June 2011 were analyzed. The consensual results pertinent to the topic were described to both examine

the major diagnostic concerns and to explore more accurate therapeutic approaches for patients with thyroid nodules.

TSH LEVELS AND AUTOIMMUNE DISEASE IN THYROID NODULES Previous studies have shown that increased serum TSH levels may be associated with an increased risk of thyroid cancer in patients with nodular goiter (28-37). The risk of thyroid malignancy increases with higher TSH concentrations, even those within the normal range (28-30). Boelaert et al. (30) studied 1500 consecutive patients without thyroid dysfunction and found a higher risk of malignancy (an adjusted odds ratio (AOR) of 2.72) in subjects whose TSH levels ranged from 1.0 to 1.7 mU/L than in those with TSH levels ,0.4 mU/L (an AOR of 1.00), with a particularly high incidence found in those with TSH levels .1.8 mU/L (AOR 3.88). Males, younger patients, and patients with solitary nodules were also found to have a higher risk of malignancy (30). Fiore et al. (37) studied the relationship between TSH serum levels and papillary thyroid carcinoma (PTC) in patients with uni- or multi-nodular goiter who were or were not treated with levothyroxine. The treated patients had lower TSH serum levels and decreased PTC prevalence. The PTC prevalence was lower in the patients with TSH levels ,0.4 mU/ml and was greater in those with TSH levels .3.4 mU/ml (1.9% vs. 16.5%), with no influence of age or multiple nodularity. In contrast, Gerschpacher et al. (35) compared the TSH concentrations of 33 patients with papillary microcarcinoma who underwent total thyroidectomy with those of a control group with carcinomas larger than 1 cm (n = 54), and no significant TSH concentration differences were observed (1.40¡0.92 mU/L vs. 1.43¡1.44 mU/L). Moreover, these results were not observed in the patients with indeterminate nodules based on cytology (29,31-36). Retrospective studies have reported a correlation between thyroid malignancy and autoimmune thyroid disease (ATD) (33-39), as well as a higher rate of malignancy in Hashimoto’s thyroiditis (HT) nodules (22). In contrast, Anil et al. (38) observed a 1.0% malignancy rate in patients with HT vs. a 2.7% rate in a control group, a difference that was not statistically significant. Mukasa et al. (40) observed a higher malignancy rate in HT patients with nodules larger than 1 cm or with smaller nodules exhibiting suspicious US findings than in patients with Graves’ disease (1.77% vs. 0.97%). Adenomatous lesions were also more frequent in the HT group and in patients younger than 40 years of age. Thus, the most recent reports recommend measuring antiTPO and anti-thyroglobulin (anti-Tg) antibodies during initial thyroid nodule investigations in which an elevated TSH level (over the normal range) is found (8,9,22).

SCINTIGRAPHY AND THYROID NODULES Scintigraphy and thyroid uptake have been utilized for over 60 years. They are valuable procedures for investigating a number of thyroid dysfunctions, such as destructive thyroiditis, ectopic thyroid, and hyperfunctioning nodules. However, they have limited diagnostic value for iso- or hypo-functioning thyroid nodules (8-10,41,42). Scintigraphy (and thyroid uptake with radioactive iodine or perthecnetathe-Tc99m when TSH is subnormal) is recommended for

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814 were benign and 269 were malignant. Intranodular vascularity was frequently observed in the benign nodules, and vascularity was more typically absent in the malignant nodules. These findings corroborate the results of Cantisani et al. (34), who used Doppler to analyze vascularity in US readings from 1090 patients and concluded that flow pattern should not be used as the sole predictor of malignancy or other thyroid nodule characteristics; therefore, FNAB remains mandatory. Baier et al. (52) evaluated the US data and the clinical and laboratory characteristics of 944 patients with thyroid nodules and noted an association between malignant solid nodules and patient age younger than 45 years. In contrast, Choi et al. (53) found no association between age and malignancy in nodules with indeterminate cytology. The authors studied the cases of 165 patients who had been diagnosed with "follicular tumors’’ and found no significant associations between malignancy and gender, age ($45 years), diameter, or US characteristics, although there was a significant association with central flow by Doppler study. According to the literature, the malignancy rate in thyroid nodules that are 4 cm or larger, with indeterminate cytology, varies from 10 to 30% (22,40,42,49,50). Rosa´rio et al. (45) found malignancies in 23.5% of the cases with indeterminate cytology. They found suspicious characteristics in the ultrasounds of 76% of these nodules, compared with 6.5% of the nodules with no suspicious aspects. In a recent analysis, Kihara et al. (54) found no association between nodule size or thyroglobulin level and malignancy risk in 137 surgically treated patients. However, they observed that malignancy was directly associated with suspicious US findings. These findings were similar to those of Maia et al. (56), who assessed the correlations among the cytological variables of the Bethesda system and the clinical, ultrasound, and scintigraphic data from patients with thyroid nodules with indeterminate cytology. Malignancy was found in 68.4% of the nodules with suspicious US characteristics vs. 14.8% of those with normal US findings. After the multivariate analysis, border irregularity as observed by US and Bethesda IV category were able to accurately predict malignancy in 76.9% of the thyroid nodules with indeterminate cytology.

evaluating functional nodules (9,22). Hyperfunctioning nodules are almost always benign, while non-functioning nodules carry estimated malignancy risk rates of 10 to 20% (8,9,22). Additionally, scintigraphy is indicated for determining the functional status of nodules with indeterminate cytology; the goal is to detect hot nodules that are probable follicular adenomas and to differentiate between the nodules in multinodular goiters (43). Due to improved nuclear imaging methods, studies using dynamic nuclear magnetic resonance (NMR) imaging have become increasingly frequent. Gupta et al. (44) recently studied the impact of NMR spectroscopy techniques on the detection of thyroid follicular neoplasms. Choline peaks were observed in eight of the analyzed cases of follicular carcinoma, with a sensitivity of 100% and a specificity of 94%. Due to the limited follow-up duration and small sample size, the method still needs validation in larger studies. In contrast, Kim et al. (45) did not find FDG-PET to be satisfactory for defining malignancy in the thyroid nodules of 50 patients; therefore, it is still considered to be a low-efficacy method.

ULTRASOUND OF THYROID NODULES Cervical ultrasound is the method of choice for studying thyroid nodules, and it enables the evaluation of the size, location, and characteristics suggestive of malignancies (Figure 1) (8,9,14,22,46-48). According to Leenhardt et al. (49), hypoechogenicity has a moderate positive predictive value (50 to 63%) for malignancy in thyroid nodules, with high sensitivity (75%) and specificity (61 to 83%) for US examination. Li et al. (50) reviewed the US features of 115 nodules in 104 patients with PTC and found that microcalcifications, central flow, and irregular borders were directly associated with malignant thyroid nodules. Gonzalez-Gonzales (51) studied the US characteristics of 341 thyroid nodules and found that microcalcifications were the only variable that was significantly associated with malignancy. Moon et al. (32) analyzed 1083 thyroid nodules and found that central flow is the most common distinction between benign and malignant nodules. Of the 1083 nodules studied,

Figure 1 - Ultrasound parameters suggestive of malignancy in thyroid nodules. Adapted from Lew et al. (14).

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Physician experience is quite important for performing this procedure, and US-guided FNAB is preferable. Similarly, pathologist experience in interpreting the aspirated material can guide the therapeutic approach. The procedure is relatively simple, quick, safe, low-cost, and devoid of significant complications (8,9,22,40). Choi et al. (73) found that 16.1% of 3.767 FNAB-US samples were inadequate, largely due to the lack of physician experience, a predominance of cystic lesions, and the presence of macrocalcifications. Additionally, Akgu¨l et al. (74) found no relationship between malignancy and nodule diameter or clinical (age, gender, and functional gland status) and ultrasound aspects in cases with inadequate cytology results. The authors found a 12.6% rate of malignancy in nodules with unsatisfactory cytological results. Regarding the cytological variables, an indeterminate or "follicular tumor’’ diagnosis was a significant problem when attempting to identify malignancies. The authors (9) defined four possible cytopathological results: benign, malignant, suspicious for malignancy (follicular neoplasm or Hu¨rthle cell carcinoma), or non-diagnostic. Thus, they estimated that samples with indeterminate results ("follicular tumor") represented approximately 15 to 30% (8,9,22) of their cases. Given that 70 to 80% of the "undetermined" lesions were eventually classified as benign in the final histological analysis (8,16,22,23,43,46,47), surgery recommendations in these case are problematic (8,23,27,43,46,47). The Bethesda classification system was created to guide cytopathological diagnoses and to help identify important correlations with malignancy in the final histological study. It consists of a six-category classification system associated with increased risk of malignancy and is based on a cytohistological analysis of 3207 FNAB samples from 2468 patients (Figure 2) (75). This classification system ensures the uniformity of information shared among pathologists, clinicians, and surgeons, and it provides better correlations between malignancy and cytological results, thus enabling more appropriate management. Given these objectives, several studies have been conducted regarding the cytological parameters that determine malignancy. According to Kelman et al. and Goldstein et al., the presence of cellular atypia in indeterminate cytology nodules indicates a greater likelihood of malignancy (76-78). Lubitz et al. (79) determined that nine of the 17 cytological characteristics examined in a study of 144 patients were associated with malignancy. In their multivariate analysis, only the presence of vascular transgressions and nuclear cracks were correlated with malignancy in the nodules investigated. Yehuda et al. (80) studied the predictive value of certain cytological variables, including "atypia", when predicting thyroid nodule malignancies in 111 patients and found a 56% malignancy rate in the final histological analysis. Micro-nucleoli, irregular nuclear contours, and dense chromatin were the most frequent characteristics noted in the malignant tumors, and there was an 83% probability of malignancy when these three characteristics were present. However, cellular atypia was present in 66% of the malignant nodules and in 78% of the benign cases, a difference that was not significant. Kato et al. (81) studied the specificity of 4 cytological variables indicative of "atypia" for predicting malignancy in

Stojadinovic et al. (55) studied 216 patients with thyroid nodules who were examined by US and electrical impedance (EIS) scanning prior to FNAB and thyroidectomy. A Bayesian network model successfully predicted malignancy based on the EIS technique. The model’s positive and negative predictive values were 83 and 79%, respectively. These studies require the use of this technique on a large scale with elaborate protocols and long-term follow-up to confirm their effectiveness and practicality. Combinations of ultrasound characteristics and clinical, laboratory, and cytological markers have frequently been examined in studies that aim to establish prediction models for thyroid malignancies (43,46,47,52-55,57-59). According to the UICC/AJCC, a classification system based on the pTNM parameters and age at diagnosis should be used to categorize the severity of all types of tumors, including thyroid cancer (60,61). Age is one of the criteria in this system, which uses 45 years of age as the cutoff point. This cut-off point was corroborated by Banks et al. (36) and Baier et al. (52). Although the rate of thyroid nodules is 5 to 11 times higher in females, the annual incidence of thyroid cancer in the United States is approximately 1.2 to 2.6 per 100,000 males and 2.0 to 3.8 per 100,000 females (7,8,10,52,62,64). While some authors believe that males have a 2- to 3-fold greater risk of thyroid nodule malignancy, caution should be exercised in the interpretation of this result (60,65) because other studies have not demonstrated such a difference (56,66,67). Several studies have shown the importance of age and male gender as prognostic markers for thyroid cancer, regardless of the ultrasound characteristics (9,62,63,68,69). Alves et al. (70) studied clinical, scintigraphic, ultrasound, and cytological predictors and observed that aspiration cytology yielded better results (sensitivity of 94% and specificity of 97%) than scintigraphy (sensitivity of 89% and specificity of 21%) or US (sensitivities ranging from 60 to 100% and specificities ranging from 25 to 69%). Another study has proposed a risk analysis based on patient age (older than 50 years), nodule size (2.5 cm), and cytological criteria (nuclear atypia and indefinite or suspicious cytological results) (36). For nodules with diameters less than 2.5 cm, the risk of malignancy was increased by 53% for each 1-cm decrease beginning at 2.5 cm. For larger nodules, the risk increased by 39% for each 1-cm increase. The patients with cytology results suspicious for papillary thyroid carcinoma had the greatest risk of malignancy. Maia et al. (71) evaluated the risk factors for malignancy in 143 patients with thyroid nodules. The FNAB sensitivity and specificity for malignancy were 82.8 and 97.7%, respectively. The age at diagnosis was an independent risk factor for malignancy, with a cutoff point of 38.5 years. The multivariate model showed that age .39 years, nodule size $2 cm, microcalcifications, and border irregularity based on ultrasound study were predictive factors for malignancy, with a combined accuracy of 81.7%.

FINE-NEEDLE ASPIRATION CYTOLOGY FNAB still remains the most important method for detecting malignancy in the management and monitoring of thyroid nodules. It has a high sensitivity (65 to 98%) and specificity (72 to 100%) (8,54,56,72), and it has a falsepositive rate for cancer detection of 0 to 7% and a falsenegative rate of 1 to 11% (8,54).

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Figure 2 - The Bethesda cytological classification system and its correlation with the risk of thyroid nodule malignancy. Adapted from Theoharis et al. (60).

results by a specialized cytopathologist with experience in thyroid studies resulted in a 10.9% reduction in the cases classified as Bethesda category III or IV, most of which were reclassified as benign cytology (category II); these results were confirmed by the post-surgical treatment. Davidov et al. (83) evaluated the Bethesda classification data of 250 patients who had their FNAB results reviewed by a second pathologist. There was diagnostic agreement between the first analysis and the second opinion in 66% of the cases. The highest concordance rate occurred in the malignant cytology group (categories V and VI), while the rate was only 37% in the indeterminate cytology group (categories III and IV). The second opinion increased the FNAB diagnostic accuracy by 14% and reduced the surgery rate by 25%. Such results demonstrate the importance of cytological review by a pathologist experienced in thyroid surgery recommendations for patients with indeterminate cytology (82,83).

466 surgically treated patients with cytologically indeterminate thyroid nodules. The "atypia" FNAB diagnosis was associated with a 42% risk of malignancy. This risk was 7% when there were no atypical features and 81% when there were four or more. When irregularity and nuclear inclusions were present, there was a 79.3% probability of malignancy and 98% specificity, which is similar to the findings of Yehuda (65). In summary, several cytological, clinical, and laboratory parameters have been studied as predictors of malignancy in thyroid nodules, especially in nodules with indeterminate cytology (Figure 3). Maia et al. (82) evaluated the correlation between the cytological variables of the Bethesda system and clinical, sonographic, and scintigraphic data on indeterminate thyroid nodules. In a sample of nodules with a 25% malignancy rate, category IV of the Bethesda system was an independent predictor of malignancy. A blind review of the cytology

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Figure 3 - Malignancy parameters in nodules with indeterminate cytology. Adapted from Banks et al. (50) and Yehuda et al. (65).

48% of the 120 papillary carcinoma cases evaluated and occured more frequently in the classic PTC cases (66%) than in the follicular PTC variant (21%). Furthermore, there was a strong association between the BRAF (V600E) mutation and extra-thyroid invasion, lymph node metastasis, and recurrence risk, indicating that the mutation is an important prognostic marker for classic PTC. According to Fadda et al. (87), it is possible to identify two risk categories (high and low) in nodules with indeterminate cytology (follicular neoplasms) based on HBME-1 and Gal-3 expression. Indeterminate cytology was present in 50 of 120 surgically treated cases. In these 50 indeterminate tumors, a positive immunohistochemical panel was observed in 76.9% of the cases with malignant nodules in the final histology, and a negative panel (no positive markers) was observed in almost all (96.8%) of the benign cases. These data were corroborated by Kang et al. (88) in an analysis of the BRAFV600E mutation in (preoperative) FNAB samples from 200 surgically treated thyroid nodules. The mutation was present in 63.3% of the malignant cases with initially indeterminate cytology. Therefore, for nodules with indeterminate cytology (Bethesda categories III and IV), negative tumor markers (HBME-1 and Gal-3) in the FNAB sample suggests conservative management, and a positive immunohistochemical panel suggests surgical treatment.

TUMOR MARKERS OF THYROID CYTOLOGY (FNAB): INDICATIONS AND CLINICAL APPLICATIONS This discussion refers to the accuracy and specificity of methods and molecular markers of thyroid malignancy, as well as to the appropriate timing of the immunocytochemical analysis of FNAB samples. The appropriate indication of molecular immunocytochemical markers of malignancy increased with the diagnostic pitfalls of Bethesda categories III, IV, and V (20,24,25,84,85). Galectin 3 (Gal-3) immunodetection is one of the most widely studied markers for malignancy in follicular lesions with indeterminate cytology (20,24,25). Bartolazzi et al. (46) examined Gal-3 expression in 1009 thyroid lesion samples and 226 FNAB cytological results, which showed 98% sensitivity and 99% specificity to discriminate benign and malignant lesions. Pennelli et al. (85) corroborated these results by observing an 80% sensitivity and an 86% specificity in a group of one hundred indeterminate cytological nodules. The BRAF (V600E) mutation, which is characteristic of PTC, has provided greater diagnostic accuracy for nodules with indeterminate cytology and for nodules that are suspicious for malignancy (20,23,26). While researching the BRAF (V600E) mutation, Kim et al. (47) studied 1074 patients with thyroid nodules and observed an increase in the FNAB sensitivity from 67.5 to 89.6% and an increase in the accuracy from 90.9 to 96.6%. In another analysis, Nikiforov et al. (27) reviewed 470 cytology specimens from 328 patients for BRAF mutations, RAS mutations, RET/PTC markers, and PAX8/PPAR gamma mutations. BRAF mutations were the most common finding, and the presence of three mutations was predictive of a malignancy diagnosis in 97% of the confirmed cases. Cerruti et al. (86) analyzed four protein markers from cytology material (FNAB) to evaluate thyroid nodules with suspected malignancy. Greater diagnostic accuracy was observed when both proteins derived from chromosome 1 (chromosome 1 open reading frame 24, C1orf24) and membrane protein 1 (integral membrane protein 1, ITM1) were present. Additionally, the BRAF mutation (V600E) was verified in

FOLLOW-UP OF THYROID NODULES WITH INITIALLY BENIGN CYTOLOGICAL RESULTS Thyroid nodules with a benign diagnosis in the initial cytological evaluation have long been thought to require only cervical sonographic assessment for long-term follow up, regardless of the results of the US examination. Despite a false-negative rate that has been classically established at 5% (8,9), several authors have demonstrated the value of repeat FNAB studies for certain thyroid nodules with initially benign cytology (43,60,89-93). There is still controversy over what criteria should be used to select such nodules and over whether systematically repeating FNAB

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benign cytology that underwent repeated FNAB studies, which raised the malignancy diagnosis rate by 7%. Of these cases, 82.1% were identified by the third FNAB-US (there was a 13-month interval between the first and third procedures). The ultrasound analysis demonstrated that features suggestive of malignancy (microcalcifications, border irregularities, central flow, and hypoechogenicity features) were significantly more common in the malignant nodules group. Kwak et al. (94) found that 25% of nodules with initially benign cytology exhibited significant growth when examined in repeated FNAB studies. However, this group demonstrated an average malignancy rate of 1% compared with a rate of 20% obtained by ultrasound detection of malignant parameters. Other authors have contested this position and recommended against repeated FNAB studies due to their high specificity and to the low cost-effectiveness of repeating the procedure in large numbers of nodules that have been diagnosed as benign (98-100). Aguilar et al. (101) found no changes from the initial diagnosis in 99.5% of the 184 nodules they investigated, and only one (0.5%) was later diagnosed with a malignancy. Similarly, Mittendorf and McHenry (102) found that initially benign cytology findings remained unchanged in 86.7% of the cases with follicular lesions; malignancies were found in 6.7%. This discussion is important because it is part of the routine clinical monitoring of patients with suspicious US features and benign FNAB cytological results. Clinical US follow-up should be performed every 12 to 18 months (9,22,23); nonetheless, patients with initially benign cytological results and suspicious US findings have been found to have higher malignancy rates during repeated FNAB follow-up than patients without suspicions US findings (97). A consensus review by the American Association of Clinical Endocrinologists (AECA), the ATA and the European Thyroid Association revealed that 31% of the 166 specialists interviewed would order another FNAB 6 to 12 months after obtaining initially benign cytological results, regardless of the recommended guidelines (103), and that only 6% would request an immunohistochemical panel after indeterminate cytology. This indication has become increasingly consistent in HBME-1, Gal-3, and BRAFV600E immunohistochemistry studies (87,88). The current clinical accuracy of these clinical and laboratorial variables (ultrasound or scintigraphy, cytology, and possible repeated FNAB studies) as malignancy predictors for thyroid nodules is still controversial.

studies to minimize the number of false-negative results is justified. In solid-nodule cases (including mixed nodules with solid portions) where the growth is less than 20% of the diameter in two dimensions, the appropriate US follow-up interval may be as long as every 3 to 5 years (8,22,30,47). Thus, some aspects of managing nodules with initially benign cytology deserve further discussion. According to some authors, the risk of malignancy is lower for initially benign thyroid nodules without suspicious US characteristics (0.6%) than in those with US results that suggest malignancy (20.4%) (94). Of 122 surgically treated thyroid nodules, 23 (18.8%) of those with initially benign cytology were found to be malignant after being reaccessed by FNAB at an average interval of 15.5 months. The authors concluded that repeated FNAB studies of initially benign nodules with suspicious US results increases malignancy detection during follow-up. Kwak et al. (89) reviewed sonographic-cytological correlations in 568 patients to determine whether repeated FNAB studies are indicated for thyroid nodule follow-up. The authors found a high risk of malignancy (92 to 98%) in thyroid nodules that were classified as "malignant" or "suspected for malignancy’’, regardless of the US findings. For nodules with initially benign cytology, however, suspicious US findings correctly predicted malignancy in more than half of the cases (56.6% vs. 2.9%). Repeated FNAB studies revealed "suspected" or "malignant" cytology in 15 (93.8%) of the 16 thyroid carcinomas that were detected during the follow-up. The authors recommended repeated FNAB studies for nodules with initially benign cytology and suspicious US findings. Studies on the management of supposedly benign ("Thy 2") thyroid nodules using the "Thy 1-5" cytological classification system suggest performing an additional FNAB after 3 to 6 months for diagnostic confirmation and to reduce the false-negative rate, regardless of the clinical or ultrasound findings (13,23). Illouz et al. (95) analyzed 119 surgically treated thyroid nodules and found that systematically repeating FNAB studies detected 22.7% of the malignant nodules that were undiagnosed in the initial cytology. The authors recommend at least three FNAB studies to reduce the falsenegative rate and accurately diagnose malignancy. A retrospective analysis of more than ten thousand FNAB studies demonstrated that the procedure increased the diagnostic accuracy by 8% (from 90 to 98%) when it was sequentially performed (96). The use of repeated biopsies for initially benign nodules reduced the FN misdiagnosis rate from 5.2% to less than 1.3%. Orlandi et al. (92) studied 799 sequential, annual FNABUS studies with favorable results. The studies were performed on 302 patients over 2 to 12 years of follow-up. The authors concluded that FNAB monitoring could be discontinued after at least three benign cytology assessments when clinical suspicion was absent. Flanagan et al. (90) observed that repeating FNAB up to three times increased the sensitivity from 81.7 to 90.4% and reduced the FN rate by 6.7%. Sensitivity did not increase between the third and the fifth procedures in this study, suggesting that up to three systematic FNAB studies are sufficient for making clinical or surgical decisions about suspected malignancies. Similarly, Maia et al. (97) found a malignancy prevalence of 28.5% in nodules with initially

FINAL CONSIDERATIONS The literature from the last five years has revealed new prospects for and trends in the approach to the diagnosis of thyroid nodules, with greater emphasis on US review and investigations of cytological tumor markers. An US review combined with cytological data (including the Bethesda classification system) improves the accuracy and efficiency of thyroid nodule malignancy prediction in cases with indeterminate cytology, especially when reviewed by thyroid pathology experts. In most of the published studies, the use of ultrasound criteria to determine whether to perform repeated FNAB-US studies for nodules with initially benign cytology increased the diagnostic accuracy for malignancy over a mean follow-up of 12-18 months. Higher malignancy rates have been observed in

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12. Derwahl M. TSH receptor and Gs-mutations in the pathogenesis of toxic thyroid adenomas—a note of caution. J Clin Endocrinol Metab. 1996;81(8):2783-5, http://dx.doi.org/10.1210/jc.81.8.2783. 13. British Thyroid Association, Royal College of Physicians: British Thyroid Association Guidelines for the management of thyroid cancer. 2 nd edition. 2007 [http://www.british-thyroid-association.org/ Guidelines/]. 14. Lew JI, Rodgers SE, Solorzano CC. Developments in the use of ultrasound for thyroid caˆncer. Current Opinion in Oncology. 2010; 22(1):11-6, http://dx.doi.org/10.1097/CCO.0b013e3283337f16. 15. Mortensen JD, Woolner LB, Bennett WA. Gross and microscopic findings in clinically normal thyroid glands. J Clin Endocrinol Metab. 1955;15(10):1270-80, http://dx.doi.org/10.1210/jcem-15-10-1270. 16. Hegedus L. Clinical practice. The thyroid nodule. N Engl J Med. 2004; 351(17):1764-71, http://dx.doi.org/10.1056/NEJMcp031436. 17. 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initially benign nodules with suspicious US findings than in those evaluated using the widely used nodule growth criteria. The use of US malignancy criteria combined with Bethesda categories III or IV improves malignancy detection, and they should be considered to guide surgical decisions for this group of nodules. Determination of the clinical applicability of genetic and molecular markers in FNAB samples requires additional, consistent long-term studies. The initial results presented in this field of FNAB immunohistochemical markers, were satisfactory for making decisions about which patients required surgery or clinical-follow up in specific cases, especially those with indeterminate cytology. The BRAF V600E mutation and the simultaneous cytological expression of HBME-1, Gal-3, and CK-19 improve malignancy prediction and are good candidates for guiding surgical decisions for Bethesda category III and IV nodules. Malignancy prediction models are increasingly desirable for establishing early diagnoses and improving surgical decisions in specific patients, such as those with indeterminate or undiagnosed cytology thyroid nodules.

ACKNOWLEDGMENTS The CAPES (no 33003017065P0 - CLI´NICA ME´DICA – social demand) supplied a FCM-Unicamp post-graduation research grant to Maia FFR, and the Sa˜o Paulo Research Foundation (FAPESP) (process No. 2008/ 10183-7) supplied public research aid.

AUTHOR CONTRIBUTIONS Maia FF conducted the cytopathological review, ultrasound and database searches and participated in the design and statistical analysis. ZantutWittmann DW conceived of the study and participated in the design and coordination. All of the authors read and approved the final version of the manuscript.

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CLINICS 2012;67(8):955-958

DOI:10.6061/clinics/2012(08)16

REVIEW

Auditory stimulation and cardiac autonomic regulation Vitor E. Valenti,I Heraldo L. Guida,I Ana C. F. Frizzo,I Ana C. V. Cardoso,I Luiz Carlos M. Vanderlei,II Luiz Carlos de AbreuIII I

Universidade Estadual Paulista (UNESP), Faculdade de Filosofia e Cieˆncias, Departamento de Fonoaudiologia, Marı´lia/SP, Brazil. II Universidade Estadual Paulista (UNESP), Faculdade de Cieˆncias e Tecnologia, Programa de Po´s-Graduac¸a˜o em Fisioterapia, Presidente Prudente/SP, Brazil. III Faculdade de Medicina do ABC, Laborato´rio de Escrita Cientı´fica, Departamento de Morfologia e Fisiologia, Santo Andre´/SP, Brazil.

Previous studies have already demonstrated that auditory stimulation with music influences the cardiovascular system. In this study, we described the relationship between musical auditory stimulation and heart rate variability. Searches were performed with the Medline, SciELO, Lilacs and Cochrane databases using the following keywords: ‘‘auditory stimulation’’, ‘‘autonomic nervous system’’, ‘‘music’’ and ‘‘heart rate variability’’. The selected studies indicated that there is a strong correlation between noise intensity and vagal-sympathetic balance. Additionally, it was reported that music therapy improved heart rate variability in anthracycline-treated breast cancer patients. It was hypothesized that dopamine release in the striatal system induced by pleasurable songs is involved in cardiac autonomic regulation. Musical auditory stimulation influences heart rate variability through a neural mechanism that is not well understood. Further studies are necessary to develop new therapies to treat cardiovascular disorders. KEYWORDS: Auditory Stimulation; Autonomic Nervous System; Music. Valenti VE, Guida HL, Frizzo AC, Cardoso AC, Vanderlei LC, Abreu LC. Auditory stimulation and cardiac autonomic regulation. Clinics. 2012;67(8):955958. Received for publication on February 2, 2012; First review completed on March 4, 2012; Accepted for publication on March 25, 2012 E-mail: vitor.valenti@gmail.com Tel.: 55 14 3402-1300

on the adaptations of abnormal autonomic modulation and health impairments (11,12). HRV analysis has received attention because it is related to highly irregular fluctuations of the heart rate and allows for better discrimination between normal and abnormal physiology (13). Moreover, previous studies have indicated that music therapy has positive effects on HRV in ill patients (14,15). Elucidating physiological responses involved in musical auditory stimulation is important to investigate future therapies to prevent the development of cardiovascular disorders. Therefore, this study aimed to analyze existing data regarding the effects of musical auditory stimulation and heart rate variability to contribute to the understanding of this topic and to facilitate the development of new treatments based on auditory stimulation therapy with music.

INTRODUCTION A previous study from our group demonstrated that a portion of subjects with auditory deficit presented with cardiovascular disorders (1). The literature demonstrated that auditory stimulation with music is known to induce many psychological responses (2-4). However, the effects of music on physiological phenomena have not been as well studied. Auditory stimulation with music lowers the heart rate and blood pressure in humans (5) and spontaneously hypertensive rats (6), suggesting that music may affect cardiac autonomic function. Cardiovascular responses are important for evaluating a patient’s health (7-9). Heart rate variability (HRV) is a noninvasive method for investigating the autonomic nervous system (ANS), and it describes the oscillations of the intervals between consecutive heartbeats. This method is a conventionally accepted procedure to describe the fluctuations in the intervals between consecutive heartbeats (RR intervals), which are known to influence the sinusal node (10). HRV may be analyzed with linear and nonlinear methods, and changes in those patterns provide information

METHODS Search strategy and selection Literature searches were performed between September 2011 and December 2011. The Medline (via PubMed), Lilacs and Cochrane databases were searched using the following subject keywords: ‘‘auditory stimulation’’, ‘‘autonomic nervous system’’, ‘‘music’’ and ‘‘heart rate variability’’. These words were defined by the Health Sciences Descriptors (DeCS) and their corresponding English versions, the Medical Subject Headings (MeSH). The studies were selected by a reviewer and supervised by a senior reviewer. Based on the titles and abstracts, we

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.

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excluded manuscripts that were not clearly related to the subject of the review. Subsequently, all of the selected titles and abstracts were submitted to a final evaluation, which considered the inclusion criteria. The reference lists were independently checked to identify studies of possible relevance that were not found in the electronic search. We excluded literature reviews and studies that presented no abstract or full text in English. As inclusion criteria, we considered clinical trials and basic studies published between 2000 and 2010 that investigated the effects of musical auditory stimulation on the ANS.

startle reflex is 110 dB, and this intensity is much louder than environmental noise. However, cardiac accelerative responses that habituated over trials were observed in subjects exposed to repeated 60 dB and 110 dB white-noise stimuli (21). The responses were regarded as a startle and defense response in humans or a fight/flight reaction in animals. The rise in the blood pressure and heart rate in response to acoustic startle stimuli indicates an autonomic function responding to the acoustic stimuli (22). Furthermore, cortical centers and subcortical processing centers were thought to be involved in the cardiovascular and hormonal responses to long-term stress activation by environmental noises, even though the noise intensity was as low as 53 dB (23). Musical auditory stimulation was also investigated during stress situations (16). Another study cited in our review (16) tested whether physiological stress recovery was faster during exposure to pleasant nature sounds than noise. As a main finding, they suggested that nature sounds facilitate recovery from sympathetic activation after a psychological stressor. The mechanisms underlying the faster recovery could be related to positive emotions (pleasantness) evoked by the nature sound, as suggested by previous research using non-audio film stimuli (24). Other perceptual attributes may also influence recovery. In the study of Alvarsson et al. (16), ambient noise was perceived as less familiar than other sounds, presumably because it contained no identifiable sources. One may speculate that this lack of information might have caused increased mental activity and thereby an increased skin conductance level compared with the nature sound. An effect of the sound pressure level may be observed in the difference between loud and quiet noise. Moreover, this difference is in line with previous psychoacoustic research (25) and is not a surprising finding considering the large difference (30 dBA) in the sound pressure level. Auditory stimulation therapy with music was investigated in anthracycline-treated patients (15). Anthracycline is a compound known to induce cardiovascular disorders (15). Chuang and coworkers indicated that long-term music therapy improved heart rate variability in anthracyclinetreated breast cancer patients (15). The findings of a previous study also suggest that the parasympathetic nervous system is activated by music therapy and appears to protect against congestive heart failure events in elderly patients with cerebrovascular disease and dementia by reducing the levels of both epinephrine and norepinephrine (26). Therefore, music therapy intervention may also help breast cancer patients control the progression and relieve symptoms of cardiac damage, which is a result of treatment

RESULTS The electronic search yielded a total of 1,751 references. Among these references, the first elimination round resulted in the exclusion of 1,632 titles and abstracts that were not clearly related to the subject of the review. The titles of the remaining 119 abstracts were submitted to a final evaluation that accounted for the inclusion criteria. An investigation of the reference lists confirmed the absence of relevant documents. Summaries of the five studies analyzed (6,16-19) were selected. Table 1 shows the levels of variability and the main results and conclusions of the studies included in this update.

DISCUSSION In general, the analysis of texts selected for this review indicated that harmonic music is able to improve cardiac autonomic regulation. The literature on the effect of music on ANS activity in healthy subjects is quite large. In contrast, the literature on how musical auditory stimulation affects individuals with cardiovascular dysfunction is less developed. In this review, we reported published studies on the effects of musical auditory stimulation on heart rate variability. The intensity of musical auditory stimulation is an important issue. In Lee et al. (19), white noise exposure above 50 dB enhanced sympathetic activity. They also found a strong correlation between the LF/HF ratio (low frequency-high frequency ratio) and noise intensity. The LF/HF ratio corresponds to the sympathetic-vagal balance (20). Thus, noise intensity was indicated to influence heart rate variability. The cardiovascular responses to sound may be conducted through many pathways, and one example is the startle response mediated by a brainstem circuit. The acoustic startle reflex, a well-known effect of loud sounds on the cardiovascular system, is described as the abrupt response of the heart rate and blood pressure to a sudden loud sound stimulation. The typical intensity used to elicit a

Table 1 - Main studies exploring the effects of auditory stimulation on cardiac autonomic regulation. LF/HF ratio: Low frequency/high frequency ratio; dB: decibel. Authors and year

Main conclusions

Lee et al., 2010 (6)

White noise exposure over 50 dB increases sympathetic activity, and there is a strong correlation between the LF/HF ratio and the noise intensity. Sympathetic activation induced by psychological stress recovers more quickly during exposure to pleasant nature sounds than to unpleasant noise. Long-term music therapy improves heart rate variability in anthracycline-treated breast cancer patients. Music reduces renal sympathetic nerve activity and blood pressure through the auditory pathway, the hypothalamic suprachiasmatic nucleus, and histaminergic neurons. Pleasure in response to music induces dopamine release in the striatal system.

Alvarsson et al., 2010 (17) Chuang et al., 2011 (16) Nakamura et al., 2007 (18) Salimpoor et al., 2011 (19)

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with anthracycline-containing chemotherapy. As a main conclusion, Chuang et al. (15) suggested that regular music therapy appears to be useful for promoting autonomic function, although further research is necessary to determine whether more (or more frequent) sessions of music therapy intervention can promote and maintain autonomic function after music therapy is stopped. To study the effects of musical auditory stimulation on cardiovascular responses in more detail, a previous investigation (17) studied the neural mechanism involved in this process in rats. A very elegant study performed by Nakamura et al. (17) indicated that in rats, musical auditory stimulation reduces renal sympathetic nerve activity and blood pressure through the auditory pathway, the hypothalamic suprachiasmatic nucleus, and histaminergic neurons. Moreover, the authors suggested that only certain types of music affect renal sympathetic activity and blood pressure in rats. Animals with bilateral lesions in the auditory cortex may discriminate a simple sound, suggesting that there is another auditory sensing pathway that is not mediated by the auditory cortex (27), but lesions of the cochleae or the auditory cortex eliminated music-induced changes in renal sympathetic activity and blood pressure (17), indicating that the changes to renal sympathetic activity and blood pressure depended on signaling through the auditory system. In the same context, a recent investigation presented the first direct evidence that the intense pleasure experienced when listening to music is associated with dopamine activity in the mesolimbic reward system, including both the dorsal and ventral striatum (18). One explanation for this phenomenon is that it is related to the enhancement of emotions (28). The emotions induced by music are evoked, among other things, by temporal phenomena, such as expectations, delay, tension, resolution, prediction, surprise and anticipation (29). Indeed, Salimpoor et al. (18) found a temporal dissociation between distinct regions of the striatum while listening to pleasurable music. The combined psychophysiological, neurochemical and hemodynamic procedure used revealed that peaks of autonomic nervous system activity, which reflect the experience of the most intense emotional moments, are associated with dopamine release in the nucleus accumbens. This region has been implicated in the euphoric component of psychostimulants, such as cocaine (30), and is highly interconnected with limbic regions that mediate emotional responses, such as the amygdala, hippocampus, cingulate and ventromedial prefrontal cortex (31). In contrast, immediately before the climax of emotional responses, there was evidence for relatively greater dopamine activity in the caudate. This subregion of the striatum is interconnected with the sensory, motor and associative regions of the brain (31) and has typically been implicated in the learning of stimulusresponse associations (31) and in mediating the reinforcing qualities of rewarding stimuli, such as food (32). In summary, in this review, we presented important studies that aimed to clarify the effects of musical auditory stimulation on heart rate variability. The potential of using HRV induced by a musical auditory stimulus as a clinical indicator for evaluating and identifying health impairments involving autonomic changes is promising. This technique could be used as a tool for the early diagnosis and prognosis of autonomic dysfunction in subjects exposed to intense sounds for long periods. There are many potential clinical applications of this method in individuals with this condition.

ACKNOWLEDGMENTS Our groups thanks the Faculdade de Filosofia e Cieˆncias da Universidade Estadual Paulista, FFC/UNESP-Marı´lia, for help performing our studies.

AUTHOR CONTRIBUTIONS All of the authors participated in the revision of the manuscript. All of the authors determined the design, interpreted the text and drafted the manuscript. All of the authors read and approved the version submitted for publication.

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CLINICS 2012;67(8):959-962

DOI:10.6061/clinics/2012(08)17

RAPID COMMUNICATION

Correlation of disability and pain with postural balance among women with chronic low back pain Guilherme Carlos Brech, Silvia Ferreira Andrusaitis, Gabriela Faller Vitale, Ju´lia Maria D’Andre´a Greve Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo (HCFMUSP), Laboratory for Motion Study from the Orthopedics and Traumatology Institute (IOT), Sa˜o Paulo/SP, Brazil. Email: guilhermebrech@yahoo.com.br / guibrech@gmail.com Tel.: 5511 2661-6041

After the volunteers had been given an explanation of the study and had signed an informed consent, they were evaluated in accordance with an assessment protocol (8).

INTRODUCTION Low back pain is one of the most common musculoskeletal complaints and, from a public health perspective, is the most costly (1,2). In most cases, it is benign and disappears within six weeks, but approximately 20% of individuals with lumbar pain do not show any improvement in their condition, which may progress to chronic low back pain. This chronic condition is difficult to manage and is correlated with many mechanical alterations (3). Maintenance of postural balance under static or dynamic conditions is essential for any functional activity (4). In individuals with chronic low back pain, the degree of control may change such that performing daily tasks becomes compromised and the chronic nature of the disease is sustained (4-7). The mechanisms that lead to such abnormalities of postural control remain unclear. Pain is an important factor in mechanical and neural alterations, but the extent to which it influences postural balance still cannot be determined. One of the hypotheses of this study is that the greater the lumbar pain and physical disability, the worse the patient’s maintenance of both static and dynamic posture. Thus, the aim of this study was to investigate the correlations between lumbar pain and the degree of disability and postural balance among women with chronic low back pain.

Assessment protocol The assessment protocol was administered by two experienced evaluators with training in handling the assessment instruments. All volunteers gave responses to the Oswestry Disability Index (ODI) questionnaire, using the version that was translated and validated for the Portuguese language (9), and the visual analogue scale (VAS) for pain (10) with regard to the daily frequency and intensity of their low back pain. Both the intensity and frequency of pain were represented by a straight line of 10 cm; 0 represented no pain, and 10 represented the worst and most frequent pain imaginable (10). In addition to the questionnaire and the pain scale, four balance tests were conducted on the Balance MasterH force platform system (Neurocom International, Inc. Clackamas, Oregon, USA). The tests performed included the following:

Clinical test of sensory interaction in balance Static balance was assessed by means of the modified clinical test of sensory interaction in balance. This consists of assessing body sway under four sensory conditions while the individual is on the force platform: eyes open and closed on a stable surface and eyes open and closed on an unstable surface. Each condition was repeated three times for ten seconds each, and the arithmetic mean of each of the attempts was used. The test measured the individual’s speed of movement from the pressure center in degrees per second. For this test, the force platform was used with four coupled sensors. Diminution of the size of this variable was considered to be a positive outcome. The variables studied were the mean sway speeds with eyes open (EO) and eyes closed (EC) on a stable surface and an unstable surface. The mean sways in the anteroposterior direction and mediolateral direction were also assessed under the same conditions. The next three tests were performed with the aim of assessing functional limitations in activities of daily living.

METHODS Ten sedentary female volunteers aged 30 to 55 years presenting with nonspecific chronic low back pain were studied between April 2008 and April 2009. These women did not have any significant radiological abnormalities and did not have any neurological impairment. For patients to be included in the study, they needed to be free from vestibular abnormalities and musculoskeletal disorders of the hips and lower limbs. The exclusion criteria were as follows: unable to do the evaluation or worsening of the symptoms during the tests. This study was conducted with approval from our institution’s ethics committee (no. 1248/07).

Single-leg test

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.

This test was performed with the subject standing on one leg on the force platform under four conditions: with eyes open and closed, for the left and right legs. Like in the Clinical test of sensory interaction in balance, each condition was

No potential conflict of interest was reported.

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The tests were conducted with a significance level of 5%.

repeated three times for ten seconds each, and the arithmetic mean of each of the attempts was used. The variables evaluated in this protocol were the mean sway speeds under EO and EC conditions on the left and right legs.

RESULTS The volunteers’ mean age was 46.2 years. The mean intensity and frequency of pain, as determined by the VAS, were 4.9¡3.1 and 6.5¡2.3, respectively; the mean degree of disability measured by the ODI was 14.5¡7.1. The patients were not taking any medication or on any treatment plan at the time of the study. Table 1 shows that the ODI results were inversely correlated with the mean sway speed on a stable surface with eyes open (r = -0.631 and p = 0.037). Tables 2 to 4 show that there were no correlations between the intensity and frequency of pain and the degree of disability for the balance measurements of the Step upand-over, Single-leg and Sit-to-stand tests (p.0.05).

Sit-to-stand test The sit-to-stand transfer test was performed on a platform with the individual initially sitting on a bench (height of 30 cm) without a backrest and with the knees flexed at 90 ˚ and the feet separated by 10 cm in relation to the heels. The arms were kept at the sides of the body throughout the test. The subjects were instructed to stand up safely and quickly. Three repetitions of the movement were made at intervals of 30 seconds between each attempt. The parameters measured were the mean weight transfer time, mean rising rate and sway speed while rising.

Step up-and-over

DISCUSSION

For this test, the subjects were instructed to go up a 10-cm high step, putting only one foot on the step. The other foot was expected to go directly over the step and down onto the platform without contacting the step. When both feet reached the platform, after going up the step, the subjects were instructed to remain as stationary as possible. Three attempts were made for each leg, starting with the left leg. The variables evaluated in this protocol were the mean weight transfer index, mean movement time and mean impact index. In all of the balance tests performed in this study, the subjects could only make three attempts to perform each movement in each test. If they were unable to successfully perform the test after three incomplete attempts, the test was excluded.

Chronic low back pain may be related to many causes, but poor neuromuscular control has been identified as an important factor in the occurrence and perpetuation of this musculoskeletal dysfunction (6,12). Nonetheless, no conclusion can yet be reached regarding the extent to which pain may be a determinant for postural control performance. One of the hypotheses put forward in this study was that greater lumbar pain and physical disability would correlate with worse functional performance in terms of both static and dynamic posture maintenance. Thus, ten patients with chronic low back pain were selected to undergo four tests on the Balance MasterH force platform system. Two of these tests evaluated static posture (the Clinical test of sensory interaction in balance and the Single-leg test), and two evaluated functional activities (the Step up-and-over test and the Sit-to-stand test). The results obtained from this study did not confirm that there was a correlation between pain and poorer maintenance of postural balance. The mean sway speed on a stable surface with eyes open presented an inverse correlation with the degree of disability (ODI) (r = -0.631 and p = 0.037); thus, the greater the value obtained in the ODI, the lower the sway speed. In principle, these data may signify good test performance given that there was less

Statistical analysis Descriptive analyses (means and standard deviations, SD) were produced in relation to the following sample characteristics: age, VAS and ODI. To verify that the data distribution was normal, the Kolmogorov-Smirnov test was used. Pearson correlations (weak, 0 to 0.3; moderate, 0.3 to 0.7 and strong, .0.7) were calculated between the VAS for pain intensity and frequency and the ODI of balance measurements (11).

Table 1 - Correlation between intensity and frequency of pain and degree of disability with the clinical test of sensory interaction in balance test. Correlation Mean-Firm-EO Mean-Firm-EC Mean-Foam-EO Mean-Foam-EC Firm-EO-Mean-X Firm-EO-Mean-Y Firm-EC-Mean-X Firm-EC-Mean-Y Foam-EO-Mean-X Foam-EO-Mean-Y Foam-EC-Mean-X Foam-EC-Mean-Y

r r r r r r r r r r r r

(p-value) (p-value) (p-value) (p-value) (p-value) (p-value) (p-value) (p-value) (p-value) (p-value) (p-value) (p-value)

VAS Intensity

VAS Frequency

ODI

-0.236(0.484) -0.188 (0.581) -0.486 (0.130) -0.145 (0.671) -0.231(0.494) 0.097(0.777) -0.113(0.741) 0.069(0.840) 0.135(0.693) 0.072(0.832) 0.181(0.594) 0.313(0.349)

-0.422(0.196) -0.232(0.492) -0.255(0.449) -0.305(0.361) -0.060(0.862) 0.390(0.236) -0.094(0.783) 0.392(0.233) -0.377(0.253) 0.391(0.235) -0.417(0.203) 0.326(0.328)

-0.631(0.037*) -0.386(0.241) -0.588(0.057) -0.056(0.870) -0.339(0.308) -0.191(0.574) -0.188(0.579) -0.170(0.618) 0.129(0.706) -0.323(0.332) 0.277(0.410) -0.067(0.844)

VAS: visual analogue pain scale; ODI: Oswestry Disability Index; Mean: mean sway speed; EO: eyes open; EC: eyes closed; Firm: stable surface; Foam: unstable surface; Mean-Y: mean sway in the anteroposterior plane; Mean-X: mean sway in the mediolateral plane. * p,0.05.

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Back pain: Disability, pain and balance Brech GC et al.

Table 2 - Correlations between intensity and frequency of pain, degree of disability and balanced measurements in the Sit-to-stand test. Correlation Lift-Up Index-LMean Lift-Up Index-RMean Movement Time-LMean Movement Time-RMean Impact Index-LMean Impact Index-RMean

r r r r r r

(p-value) (p-value) (p-value) (p-value) (p-value) (p-value)

VAS Intensity

VAS Frequency

ODI

-0.092(0.788) -0.042(0.903) -0.315(0.345) -0.308(0.356) -0.232(0.492) 0.008(0.982)

-0.182(0.591) -0.281(0.402) 0.235(0.486) 0.343(0.302) -0.363(0.273) -0.312(0.351)

0.020(0.953) 0.154(0.650) -0.318(0.341) -0.255(0.449) -0.136(0.690) -0.033(0.923)

VAS: visual analogue pain scale; ODI: Oswestry Disability Index; Lift-Up Index Mean: mean weight transfer index; Movement Time-Mean: mean movement time; Impact Index-Mean: mean impact index; L: left leg; R: right leg.

sway while maintaining the posture. This result is concordant with previous results from similar studies. Henry et al. (6) found that patients with low back pain presented with smaller magnitudes of peak movement from the pressure center and larger peak movements from the center of mass compared with individuals without a history of lumbar pain. These authors concluded that their results represented a choice made by these individuals with low back pain that involved a strategy for using their ankles to compensate for their balance. Another strategy to correct postural balance among individuals without lumbar pain requires activation of the hip and lumbar spine muscles. In individuals with chronic low back pain, these muscles are activated (12). These findings were supported by the results from the study by Brumagne et al. (7), who determined that individuals with low back pain presented less variability in their choice of strategies. They observed that strategies using the ankles were most commonly used because of the inefficacy of their trunk muscles. Although the equipment used in this study did not have tools to confirm these balance compensation strategies, it can be presumed that the same events occurred among the patients in this study. Their smaller movements from the pressure center were due to a strategy for using the ankles, which was a more effective strategy than using their hips to maintain their balance, thereby resulting in movement through a smaller area. Among patients with chronic low back pain, this might be more effective while maintaining balance if the trunk and hip muscles were less active. Future studies must be performed to evaluate these suppositions. The other balance measurements from the Step up-andover, Single-leg and Sit-to-stand tests did not present any correlations with the intensity or frequency of pain as assessed through the VAS or with the degree of disability as assessed through the ODI. It has been shown that individuals with chronic lumbar pain have abnormal postural balance in

comparison with individuals without a history of pain, especially under conditions that require greater postural demands. Mientjes et al. (13) and Della Volpe et al. (4) did not observe any notable abnormalities in static balance among individuals with lumbar back pain; however, when these individuals were subjected to more challenging postures, such as with their eyes closed and staying upright on an unstable surface, the individuals with low back pain presented greater sway than the control group. Alternatively, in a systematic review study, Ruhe et al. (3) found that there was no correlation between pain intensity and the magnitude of excursion from the pressure center. This study had certain limitations, especially with regard to the equipment used and the small sample size. The Balance MasterH system is not considered to be the gold standard for assessing postural balance, but it is capable of assessing functional balance, thereby reproducing activities of daily living. Regarding the small sample size, although the incidence of chronic low back pain is high, its multifactorial nature means that there will be subgroups of patients with different characteristics within a given group of individuals presenting with pain. Thus, these subgroups should not be grouped within the same study (14). We sought to demarcate the most homogenous study group possible. Considering the results encountered, new studies should be performed using assessment methods of greater specificity, including the use of a force platform. The findings may possibly be correlated with trunk and ankle muscle activity using surface electromyography during tests that are more challenging to postural balance. The intensity and frequency of lumbar pain were not correlated with postural balance in women with chronic low back pain. Regarding postural balance, the only significant correlation identified was between the degree of disability and the sway speed on a stable surface with eyes open.

Table 3 - Correlations between intensity and frequency of pain, degree of disability and balance measurements in the Sit-to-stand test. Correlation WT Transfer-Mean Rising Index-Mean COG Sway Vel-Mean

r (p-value) r (p-value) r (p-value)

VAS Intensity

VAS Frequency

ODI

-0.513(0.106) -0.245(0.468) 0.361(0.275)

-0.595(0.054) -0.424(0.193) 0.476(0.139)

-0.288(0.391) -0.266(0.430) 0.241(0.476)

VAS: visual analogue pain scale; ODI: Oswestry Disability Index; WT Transfer-Mean: mean weight transfer; Rising Index-Mean: mean rising rate; COG Sway Vel-Mean: sway speed while rising.

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4. Della Volpe R, Popa T, Ginanneschi F, Spidalieri R, Mazzochio R, Rossi A. Changes in coordination of postural control during dynamic stance in chronic low back pain patients. Gait Posture. 2006;24(3):349-55, http:// dx.doi.org/10.1016/j.gaitpost.2005.10.009. 5. Kuukkanen TM. An experimental controlled study on postural sway and therapeutic exercise in subjects with low back pain. Clinical Rehabilitation. 2000;14(2):192-202, http://dx.doi.org/10.1191/026921500667300454. 6. Henry SM, Hitt JR, Jones SL, Bunn JY. Decreased limits of stability in response to postural perturbations in subjects with low back pain. Clin. Biomech (Bristol, Avon). 2006;21(9):881-92, http://dx.doi.org/10.1016/ j.clinbiomech.2006.04.016. 7. Brumagne S, Janssens L, Knapen S, Claeys K, Suuden-Johanson E. Persons with recurrent low back pain exhibit a rigid postural control strategy. Eur Spine J. 2008;17(9):1177-84, http://dx.doi.org/10.1007/ s00586-008-0709-7. 8. Andrusaitis SF, Brech GC, Vitale GF, Greve JMDA. Trunk stabilization among women with chronic lower back pain: a randomized, controlled, and blinded pilot study. Clinics. 2011;66(9):1645-50, http://dx.doi.org/ 10.1590/S1807-59322011000900024. 9. Vigatto R, Alexandre NMC, Correa Filho HR. Development of a Brazilian Portuguese Version of the Oswestry Disability Index. Spine. 2007;32(4): 481-6, http://dx.doi.org/10.1097/01.brs.0000255075.11496.47. 10. Dolan P, Greenfield K, Nelson R, Nelson I. Can exercise therapy improve the outcome of microdiscectomy? Spine (Phila Pa 1976). 2000;25(12):152332, http://dx.doi.org/10.1097/00007632-200006150-00011. 11. Kirkwood B, Sterne J. Essential medical statistics. 2nd ed. Oxford: Blackwell Science; 2003. 12. Mok NW, Brauer SG, Hodges PW. Hip strategy for balance control in quiet standing is reduced in people with low back pain. Spine (Phila Pa 1976). 2004;29(6):E107-12, http://dx.doi.org/10.1097/01.BRS.0000115134.97854. C9. 13. Mientjes MIV, Frank SJ (1999) Balance in chronic low back pain patients compared to healthy people under various conditions in upright standing. Clin Biomech (Bristol, Avon). 1999;14(10):710-6. 14. Dankaerts W, O’Sullivan P. The validity of O’Sullivan’s classification system (CS) for a sub-group of NS-CLBP with motor control impairment (MCI): overview of a series of studies and review of the literature. Manual Therapy. 2011;16(1):9-14, http://dx.doi.org/10.1016/j.math.2010.10.006.

Table 4 - Correlations between intensity and frequency of pain, degree of disability and balance measurements at the Step up-and-over test. Correlation Mean-EO-L Mean-EC-L Mean-EO-R Mean-EC-R

r r r r

(p-value) (p-value) (p-value) (p-value)

VAS Intensity

VAS Frequency

ODI

-0.315(0.345) 0.166(0.626) -0.145(0.670) 0.388(0.238)

-0.419(0.200) 0.531(0.093) -0.394(0.230) 0.540(0.086)

-0.556(0.075) 0.008(0.981) -0.600(0.051) 0.135(0.693)

VAS: visual analogue pain scale; ODI: Oswestry Disability Index; Mean: mean sway speed; EO: eyes open; EC: eyes closed; L: left leg; R: right leg.

AUTHOR CONTRIBUTIONS Brech GH, Andrusaitis SF and Vitale GF performed the data collection, analysis and preparation of the manuscript. D’Andre´a Greve JM was supervisor and editor of the manuscript.

REFERENCES 1. Holm S, Indahl A, Solomonow M. Sensorimotor control of the spine. J Electromyogr Kinesiol. 2002;12(3):219-34, http://dx.doi.org/10.1016/ S1050-6411(02)00028-7. 2. Yahia A, Jribi S, Elleuch M, Baklouti S, Elleuch MH. Evaluation of the posture and muscular strength of the trunk and inferior members of patients with chronic lumbar pain. Joint Bone Spine. 78(3):291-297. 3. Ruhe A, Fejer R, Walker B. Center of pressure excursion as a measure of balance performance in patients with non-specific low back pain compared to healthy controls: a systematic review of the literature. 2011;20(3):358-68.

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DOI:10.6061/clinics/2012(08)18

RAPID COMMUNICATION

Malignant peripheral nerve sheath tumors: clinicopathological aspects, expression of p53 and survival Karin S. G. Cunha,I,II Anabela C. Caruso,IV Paulo A. S. de Faria,IV Licı´nio E. da Silva,I Andre´a R. C. Pires,I Mauro Geller,III,V,VI Vaˆnia S. Lopes,I Rodrigo S. de Moura-NetoVII I

Universidade Federal Fluminense (UFF), School of Medicine, Post-graduate Program in Pathology, Niteroi/RJ, Brazil. II Universidade Federal Fluminense (UFF), Nova Friburgo University Pole, School of Dentistry, Nova Friburgo/RJ, Brazil. III Carlos Chagas Institute, Post-graduation Program of Medicine, Rio de Janeiro/RJ, Brazil. IV Instituto Nacional do Caˆncer (INCA), Pathology Division, Rio de Janeiro/RJ, Brazil. V Tereso´polis Medical School (UNIFESO), Department of Microbiology and Immunology, Tereso´polis/RJ, Brazil. VI Universidade Federal do Rio de Janeiro (UFRJ), Martaga˜o Gesteira Institute of Puericulture and Pediatrics, Department of Clinical Genetics, Rio de Janeiro/RJ, Brazil. VII Universidade Federal do Rio de Janeiro (UFRJ), Department of Botany, Biology Institute, Rio de Janeiro/RJ, Brazil. Email: karingcunha@gmail.com Tel.: 55 21 32531990/26299128

INTRODUCTION

Case Selection MPNSTs diagnosed from 1996-2005 were obtained from the pathology files of INCA. The following inclusion criteria were used: available medical records and preserved paraffin blocks from the resected primary tumor with a sufficient quantity of material (in patients submitted to radiotherapy and/or chemotherapy prior to primary tumor resection, the biopsy material was used if it had the same histological grade as the resected material). Tumors with one of the following features were included: arose within a peripheral nerve; arose during the transition from a benign neural tumor; developed in a NF1 patient and exhibited the same histological features of most MPNSTs originating from a nerve; and developed in a nonNF1 patient, exhibited the same histological features as most MPNSTs and expressed S-100 (Dako Corp., Carpinteria, CA, USA, 1:4,000) and/or CD57 (clone TB01; Dako, 1:50). All samples were immunoreactive for anti-vimentin (clone V9; Dako, 1:800) and negative for anti-cytokeratin (clone AE1/ AE3; Dako, 1:400), anti-melanosome, (clone HMB-45, 1:200), anti-actin (smooth muscle; clone 1A4; Dako, 1:250), anti-actin/ muscle (clone HHF35; Dako, 1:1,000), and anti-desmin (clone D33; Dako, 1:100) antibodies, except the malignant triton tumors, which exhibited anti-actin/muscle and desmin immunopositive areas. The immunohistochemistry (IHC) was performed after reviewing the H&E sections. Plexiform neurofibromas diagnosed from 1996-2005 were obtained from the pathology files of INCA. The following inclusion criteria were used: available medical records, preserved paraffin blocks with sufficient quantity of material and presence of heterogeneous and diffuse expression of S-100 protein. The other selected neurofibromas had been used in two previous studies (11,12). The diagnoses of all the MPNSTs and neurofibromas were confirmed by two pathologists.

Malignant peripheral nerve sheath tumors (MPNSTs) are rare and highly aggressive neoplasms, representing only 5% of soft tissue sarcomas (1,2). Approximately half of MPNST cases occur in association with neurofibromatosis type 1 (NF1) (3). MPNSTs may appear de novo or develop from the malignant transformation of a benign neural neoplasm, generally a plexiform neurofibroma (1). Solitary (unassociated with NF1) and localized (or discrete; multiple in NF1) neurofibromas do not have malignant transformation potential (1,3). NF1 loss of heterozygosity (LOH) has been demonstrated in NF1-associated and sporadic MPNSTs. Although NF1 LOH is believed to be sufficient for neurofibroma development, MPNST pathogenesis has been suggested to be a multistage process that includes other molecular alterations (4,5). TP53 mutations have been found in a subgroup of MPNSTs, indicating that a p53-mediated pathway is involved in their development (5,6). Some clinicopathological features (e.g., the presence of NF1, high histological grade, necrosis, and rhabdomyoblastic differentiation) have been indicated to be important factors for lower survival in MPNST cases in some studies but not in others (2,7–10). The clinical significance of p53 expression in MPNSTs is also a controversial issue. We aimed to study p53 expression in MPNSTs and investigate its impact, as well as the impacts of the clinicopathological features of MPNSTs, on the survival rates. We also compared p53 expression in MPNSTs with their clinicopathological features and with p53 expression in neurofibromas.

MATERIALS AND METHODS The Ethical Committee of the National Institute of Cancer (INCA), RJ, Brazil, approved this study.

Histological Analysis of Malignant Peripheral Nerve Sheath Tumors

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.

Tables 1 and 2 show the clinical and pathological features, respectively, of the MPNSTs analyzed in this research. The tumors were classified as low- or high-grade according to the Armed Forces Institutes of Pathology criteria (1).

No potential conflict of interest was reported.

963


27 44 29 23 28 32 34 40 20 53 21 23 42

68 19 23 34 60 40 78 24 45 63 85 72 80 30 41

1 2 3 4 5 6 7 8 9 10 11 12 13

14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Patient

Age at diagnosis

964

M M F F F F F M M M M F F F M

F F F F F F F F F F F M F

Gender

W W W B B B W W B B W W W B B

— W — B W W W W — B W W W

Race

No No Yes Yes Yes No No Yes Yes Yes No No No No Yes

Yes No Yes Yes Yes No No No Yes Yes Yes Yes No

NF1

Right upper limb Right upper limb Sacrum and spine Abdomen Right lower limb Thorax Face sinus Right upper limb Abdomen Supraclavicular Head (temporal) Left lower limb Right foot Thorax Right lower limb

Lumbar Left elbow Right flank Pelvis Right flank Left lower limb Right lower limb Breast Lumbar Left shoulder Left lower limb Left lower limb Pelvis

Site

— — — No Yes Yes No — -— Yes Yes Yes Yes Yes —

No No No No Yes Yes Yes No — Yes Yes — No

Free surgical margins Yes No No No No No No No No No No No No Yes No No Yes No No Yes No Yes No No No Yes No No

Resection + Rxt Rxt Rxt Resection Resection Resection + Rxt Resection + Rxt Biopsy + Ct Resection Resection Resection Resection + Rxt Resection Resection + Rxt Rxt

Local recurrence

Resection Resection + Rxt Resection + Rxt Resection Resection Resection Resection Resection + Rxt Rxt Resection Resection Ct Resection + Rxt

Treatment

Table 1 - Clinical data of the patients with malignant peripheral nerve sheath tumors.

No Yes Yes No Yes No No Yes Yes No No Yes No Yes Yes

No No No No No Yes No No No No No Yes Yes

Metastasis

Bone Cervical region

Lung

Lung Lung

Lung

Lung Lung

Lung Liver and pancreas

Lung

Site of metastasis

No No Yes No No No Yes Yes Yes Yes Yes Yes Yes No Yes

No No Yes Yes No No No No Yes Yes No Yes Yes

Death

9.50 16.10 7.73 10.10 40.80 61.10 77.90 12.80 14.70 60.30 20.20 42.00 13.40 35.70 7.20

21.60 13.60 12.47 4.87 76.13 12.10 44.77 96.90 1.83 10.87 67.50 2.73 11.30

Overall survival (months)

7.00 .00* 7.67 5.43 22.83 61.10 50.20 6.60 13.07 60.30 20.20 39.93 8.10 4.67 6.97

18.23 13.60 12.47 4.87 76.13 .00* 44.77 96.90 1.83 10.87 67.50 .00* 9.60

Disease-free survival (months)

Malignant peripheral nerve sheath tumors Cunha KSG et al. CLINICS 2012;67(8):963-968


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Malignant peripheral nerve sheath tumors Cunha KSG et al.

Table 2 - Pathological data of the malignant peripheral nerve sheath tumors. Case number

Size (cm)

Grade

Presence of heterologous differentiation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

4.6 17.0 15.0 21.0 7.0 21.0 10.0 16.0 — 28.0 17.0 — 13.0 — — — 13.0 12.0 6.0 — — 23.0 5.0 10.0 13.0 3.0 11.0 —

high high high high low high high low high high high high high high low high high high low low high high high low low high high high

no rhabdomyosarcomatous areas no rhabdomyosarcomatous areas chondrosarcomatous areas no no no no no no no chondrosarcomatous areas no epithelioid no epithelioid chondrosarcomatous area no no no no chondroma area no no no no no

Necrosis

Mitotic index (mitotic figures in 10 high-power fields)*

p53 PI values

no yes yes yes no yes no no no yes yes yes yes no no yes yes no no no yes yes no no no no no yes

7.0 (s.d. = 2.0) 9.3 (s.d. = 1.5) 5.3 (s.d. = 1.2) 17.0 (s.d. = 2.0) 3.7 (s.d. = 1.5) 7.0 (s.d. = 3.5) 9.0 (s.d. = 2.0) 0.3 (s.d. = 0.6) 18.0 (s.d. = 4.0) 19.0 (s.d. = 1.0) 13.0 (s.d. = 2.6) 10.7 (s.d. = 1.5) 10.3 (s.d. = 5.7) 10.3 (s.d. = 4.0) 3.0 (s.d. = 0.0) 7.0 (s.d. = 2.6) 7.3 (s.d. = 1.2) 6.0 (s.d. = 2.0) 2.3 (s.d. = 1.5) 3.0 (s.d. = 1.0) 7.0 (s.d. = 2.0) 13.3 (s.d. = 3.2) 7.0 (s.d. = 2.0) 3.7 (s.d. = 1.5) 3.7 (s.d. = 1.2) 6.7 (s.d. = 0.6) 13.0 (s.d. = 3.0) 8.0 (s.d. = 1.0)

0.00070 0.00 0.00020 0.01597 0.00010 0.00 0.00 0.00090 0.00160 0.00050 0.00280 0.00040 0.00 0.00 0.00 0.00170 0.00 0.00 0.00200 0.00030 0.00 0.00060 0.00010 0.01610 0.00320 0.00050 0.00640 0.00

— information not available (the tumor resection was performed at another institution); PI, positivity values *, in each case, at least 30 fields were analyzed, and the mean value of the sum of the mitotic figures in 10 high-power fields is shown in the table.

The epithelioid MPNSTs included in this study were composed predominantly of epithelioid cells and exhibited spindle cells identical to those of conventional MPNSTs. The malignant triton tumors had areas of cells with rhabdomyoblast morphology, which expressed desmin and/or actin/muscle.

index (PI), which was defined as the p53-positive area divided by the tissue area. The tumors were classified as having low or high PIs (cut-off value = 0.0020). The intensity of the p53 expression (weak, moderate, or intense) was also evaluated.

Construction of Tissue Microarray Paraffin Blocks

Statistical Analysis

Two tissue microarray (TMA) paraffin blocks containing samples from all tumors were constructed. Five morphologically representative regions of each tumor were marked with a colored pen on the glass slides of H&E sections. Areas of necrosis and severe inflammatory infiltration were avoided. From each corresponding original paraffin block, five tissue cores (1.1 mm in diameter) were sampled from the marked areas in the donor block and mounted onto a recipient paraffin block, using the alternative method for the manual construction of TMAs (13).

Clinicopathological and immunohistochemical variables were compared using the chi-squared, Fisher’s exact, Student’s t-, and Mann-Whitney U tests. The KaplanMeier method was used to evaluate the survival curves. The statistical significance of the clinicopathological variables was determined with the log-rank test. Multivariate analysis was performed using the Cox regression model. SPSS software v.11 was used for the statistical analyses. Differences were considered significant if p,0.05.

Immunohistochemistry

RESULTS

Sections of 3 mm in thickness were cut and collected on silane-coated slides. After dewaxing, the p53 protein expression was assessed by IHC (anti-p53 antibody; clone DO-7; Dako Corporation, 1:100) using a protocol described elsewhere (14). A metastatic carcinoma was used as the positive control, and the omission of the primary antibody was used to establish the negative control. The quantification of the p53 staining was performed with computerized digital image analysis (Image-Pro Plus software v4.5; Media Cybernetics), as previously described (14). The quantification was expressed as the positivity

Twenty-eight MPNSTs and thirty-eight neurofibromas were included in this study. Figures 1A–1C show some examples of MPNST included in the study (malignant triton tumor and MPNST with chondrosarcoma differentiation). The overall and disease-free five-year survival rates of the MPNST patients were 46 and 39%, respectively. The data regarding the p53 expression in the MPNSTs and neurofibromas are described in Tables 2 and 3. p53 expression was more common in the MPNSTs than in the

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In the p53-positive MPNSTs, there was no correlation of high PI with the clinicopathological variables (MannWhitney test): NF1 (p = 0.179), local recurrence (p = 0.643), metastasis (p = 0.353), high grade (p = 0.208), high mitotic index (p = 0.156), and necrosis (p = 0.387). The epithelioid variant (p = 0.036) was the only significant variable in the log-rank analysis of disease-free survival (Table 4). The Cox regression analysis showed that necrosis (p = 0.024) was an independent prognostic factor for lower overall survival (p = 0.007) (Table 5).

DISCUSSION The five-year overall survival rate of patients with MPNSTs has been reported to range from 23% to approximately 50% (2,15–17), similar to the rate of 46% found in this study. In our study, seven patients censored in the overall survival calculation had recurrence or metastasis, and three patients were lost to follow-up when the disease became terminal. Their deaths most likely went unobserved; therefore, their overall survival time may have been overestimated. This issue is usually a bias inherent in retrospective studies that intend to measure overall survival. Although some authors have demonstrated the correlation of various clinicopathological parameters with the biological behavior of the MPNSTs, there is no consensus with regard to the importance of these factors in the prognosis of MPNST (2,7–10). In our study, the presence of necrosis was an independent predictor of mortality. In the present work, only one (2.6%) neurofibroma (plexiform neurofibroma) expressed p53. Other studies have also demonstrated that p53 expression is rare or absent in neurofibromas (18–21). In contrast, the majority (64.3%) of MPNSTs expressed p53. Our results are similar to those of previous studies that used the same anti-p53 antibody clone, and the results of these studies showed that p53 expression in the MPNSTs varies from 42 to 100% (6,9,16,18–20,22). To our knowledge, there are only two previous studies that used TMA technology to investigate p53 expression in MPNSTs (16,22). In some studies (9,20,21), an association between p53 expression and histological grade was observed, which differs from our research and that of others (18,19). One explanation for these divergent results could be the different criteria adopted for histological gradation. Although the United States National Institutes of Cancer and Fe´de´ration Nationale des Centres de Lutte Contre le Cancer (FNCLCC) grading systems are the most commonly used systems for sarcomas, the histological grading systems for sarcomas have no prognostic value for some histological subtypes, including MPNSTs (23). Therefore, we preferred to use a

Figure 1 - Malignant triton tumor: case 4 (A and B). A. Area of differentiation in the rhabdomyosarcoma (arrows); H&E, 40x. B. Cells immunopositive for desmin; immunohistochemistry (diaminobenzidine), 40x. C. Malignant peripheral nerve sheath tumor with heterologous differentiation in a chondrosarcoma: case 13; H&E, 20x. D. p53-immunopositive plexiform neurofibroma (arrow); immunohistochemistry (diaminobenzidine), 40x. E. Malignant peripheral nerve sheath tumor immunoreactive for p53: case 24; immunohistochemistry (diaminobenzidine), 40x. F. Malignant peripheral nerve sheath tumor immunoreactive for p53: case 4; immunohistochemistry (diaminobenzidine), 40x. OBS: positive cells in brown.

neurofibromas (x2 p,0.001). The only p53-positive neurofibroma (plexiform neurofibroma) presented weak nuclear immunoreactivity. In the MPNSTs, the nuclear p53 expression varied from weak to intense. Figures 1D–1F show the expression of p53 in the plexiform neurofibroma and in two MPNSTs. The p53 expression was not correlated with any of the following clinicopathological features of MPNSTs (Fisher’s exact test): NF1 (p = 0.433), local recurrence (p = 0.642), metastasis (p = 0.125), high grade (p = 0.364), high mitotic index (p = 0.278), and necrosis (p = 0.249).

Table 3 - Immunohistochemical data of neurofibromas and malignant peripheral nerve sheath tumors. Group Neurofibromas Plexiform neurofibromas Neurofibromas (from NF1 and non-NF1 patients) MPNSTs

% of positive cases (n)

Mean PI

Standard deviation

Median PI

PI min/max

IQ range

2.6% (n = 1) 6.3% (n = 1) 0.0% (n = 0)

0.0005 0.0005 —

— — —

— — —

0.0000/0.0005 0.0000/0.0005 —

— — —

64.3% (n = 18)

0.001931

0.0042199

0.000350

0.0000/0.01597

0.0017

PI, positivity index; IQ, interquartile; MPNSTs, malignant peripheral nerve sheath tumors.

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Table 4 - Results of the influence of the clinicopathological variables on the overall and disease-free survival in patients with malignant peripheral nerve sheath tumors. Variables Gender Female Male NF1 Yes No Location Trunk (yes/no) Limbs (yes/no) Lower limb (yes/no) Upper limb (yes/no) Head and neck (yes/no) Treatment performed Surgical resection + radiotherapy Other type of treatment Size of the tumor #5 cm .5 cm Free surgical margins Yes No Local Recurrence Yes No Metastases Yes No Histological Grade High Low Mitotic index $ 10 mitoses/10 high-power fields , 10 mitoses/10 high-power fields Necrosis Yes No Epithelioid Variant Yes No Malignant Triton Tumor Yes No Chondrosarcoma differentiation Yes No P53 protein expression Positive Negative Low PI High PI

Total SV (months)

Total SV (p log-rank)

54.3 23.5

Disease-free SV (months)

Disease-free SV (p log-rank)

0.109

47.3 19.3

0.118

31.6 59.9

0.044

31.4 41.6

0.959

49.4/42.2 37.9/47.0 37.7/46.4 12.2/46.1 49.0/48.9

0.955 0.937 0.980 0.912 0.977

47.3/27.4 25.0/50.5 21.9/50.8 11.4/40.1 50.2/39.7

0.390 0.199 0.108 0.679 0.531

35.9 60.0

0.184

35.886 55.629

0.282

44.7 57.6

0.716

28.9 55.4

0.610

49.7 55.1

0.925

48.57 41.5

0.516

46.0 60.6

0.606

54.4 23.4

0.135

31.7 68.6

0.045

23.94 65.28

0.095

24.0 53.7

0.051

21.54 44.77

0.386

19.1 62.4

0.003

23.95 50.23

0.138

* 44.49

*

6.21 43.98

0.036

9.2 46.3

0.566

18.42 42.26

0.958

54.5 43.8

0.511

46.17 37.89

0.356

43.8 50.9 38.9 43.5

0.942 429

49,1 42.6 46.9 49.4

0.418 0.502

SV, survival; PI, positive index; *, the value could not be calculated because all cases were censored.

simple system in which MPNSTs are classified into two grades: low and high (1). We could not observe any influence of p53 expression on survival rates, similar to the results of a previous study (24). In contrast, other researchers showed that p53 expression was an important predictive factor for lower survival rate (16,19). In some studies (16,22), p53 expression was more common in neurofibromas associated with NF1 than in those not associated with NF1, but other studies (20) did not observe this association, similar to our results. There are a few possible explanations for these divergent results. First,

some authors considered all cases that had any immunopositive cells as being positive, whereas others established cut-off points, varying from 3 to 10% of positive cells (9,20– 22,24). Another important factor is that all previous studies used conventional pathologist-based manual scoring to quantify the p53 staining, which increases the inter-observer and intra-observer variabilities. Our study was the first to use computerized image analysis to calculate the p53 expression. Moreover, technical considerations, such as the storage time of the tissue sections on glass slides, can influence p53 immunoreactivity.

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Malignant peripheral nerve sheath tumors Cunha KSG et al.

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7. Kar M, Deo SVS, Shukla NK, Malik A, DattaGupta S, Mohanti BK, et al. Malignant peripheral nerve sheath tumors (MPNST)-clinicopathological study and treatment outcome of twenty-four cases. World J Surg Oncol. 2006;4:55, http://dx.doi.org/10.1186/1477-7819-4-55. 8. Angelov L, Davis A, O’Sullivan B, Bell R, Guha A. Neurogenic sarcomas: experience at the University of Toronto. Neurosurgery. 1998;43(1):56-64; discussion 64-65. 9. Zhou H, Coffin CM, Perkins SL, Tripp SR, Liew M, Viskochil DH. Malignant peripheral nerve sheath tumor: a comparison of grade, immunophenotype, and cell cycle/growth activation marker expression in sporadic and neurofibromatosis 1-related lesions. Am J Surg Pathol. 2003;27(10):1337-45, http://dx.doi.org/10.1097/00000478-20031000000006. 10. Evans DGR, Baser ME, McGaughran J, Sharif S, Howard E, Moran A. Malignant peripheral nerve sheath tumours in neurofibromatosis 1. J Med Genet. 2002;39(5):311-4, http://dx.doi.org/10.1136/jmg.39.5.311. 11. Cunha KSG, Barboza EP, Fonseca EC da. Identification of growth hormone receptor in plexiform neurofibromas of patients with neurofibromatosis type 1. Clinics (Sao Paulo). 2008;63(1):39-42, http:// dx.doi.org/10.1590/S1807-59322008000100008. 12. Cunha KSG, Barboza EP, da Fonseca EC. Identification of growth hormone receptor in localised neurofibromas of patients with neurofibromatosis type 1. J. Clin. Pathol. 2003;56(10):758-63, http://dx.doi.org/ 10.1136/jcp.56.10.758. 13. Pires ARC, Andreiuolo F da M, de Souza SR. TMA for all: a new method for the construction of tissue microarrays without recipient paraffin block using custom-built needles. Diagn Pathol. 2006;1:14, http:// dx.doi.org/10.1186/1746-1596-1-14. 14. Cunha KSG, Caruso AC, Gonc¸alves AS, Bernardo VG, Pires ARC, da Fonseca EC, et al. Validation of tissue microarray technology in malignant peripheral nerve sheath tumours. J Clin Pathol. 2009;62(7):629-33, http:// dx.doi.org/10.1136/jcp.2008.063081. 15. Wong WW, Hirose T, Scheithauer BW, Schild SE, Gunderson LL. Malignant peripheral nerve sheath tumor: analysis of treatment outcome. Int J Radiat Oncol Biol Phys. 1998;42(2):351-60, http://dx.doi.org/ 10.1016/S0360-3016(98)00223-5. 16. Brekke HR, Kolberg M, Skotheim RI, Hall KS, Bjerkehagen B, Risberg B et al. Identification of p53 as a strong predictor of survival for patients with malignant peripheral nerve sheath tumors. Neuro-oncology. 2009;11(5):514-28. 17. Hruban RH, Shiu MH, Senie RT, Woodruff JM. Malignant peripheral nerve sheath tumors of the buttock and lower extremity. A study of 43 cases. Cancer. 1990;66(6):1253-65. 18. Kindblom LG, Ahlde´ n M, Meis-Kindblom JM, Stenman G. Immunohistochemical and molecular analysis of p53, MDM2, proliferating cell nuclear antigen and Ki67 in benign and malignant peripheral nerve sheath tumours. Virchows Arch. 1995;427(1):19-26. 19. Halling KC, Scheithauer BW, Halling AC, Nascimento AG, Ziesmer SC, Roche PC et al. p53 expression in neurofibroma and malignant peripheral nerve sheath tumor. An immunohistochemical study of sporadic and NF1-associated tumors. Am J Clin Pathol. 1996;106(3):2828. 20. Liapis H, Marley EF, Lin Y, Dehner LP. p53 and Ki-67 proliferating cell nuclear antigen in benign and malignant peripheral nerve sheath tumors in children. Pediatr Dev Pathol. 1999;2(4):377-84, http://dx.doi.org/ 10.1007/s100249900138. 21. McCarron KF, Goldblum JR. Plexiform neurofibroma with and without associated malignant peripheral nerve sheath tumor: a clinicopathologic and immunohistochemical analysis of 54 cases. Mod Pathol. 1998;11(7): 612-7. 22. Sabah M, Cummins R, Leader M, Kay E. Immunoreactivity of p53, Mdm2, p21(WAF1/CIP1) Bcl-2, and Bax in soft tissue sarcomas: correlation with histologic grade. Appl Immunohistochem Mol Morphol. 2007;15(1):64-9. 23. Fletcher CD, Unni KK, Mertens F. World Health Organization classification of tumours. Pathology and genetics of tumours of soft tissue and bone. Lyon: IARC Press. 2002;47:91. 24. Watanabe T, Oda Y, Tamiya S, Kinukawa N, Masuda K, Tsuneyoshi M. Malignant peripheral nerve sheath tumours: high Ki67 labelling index is the significant prognostic indicator. Histopathology. 2001;39(2):187-97, http://dx.doi.org/10.1046/j.1365-2559.2001.01176.x.

Table 5 - Results of the Cox regression model for significant variables in the univariate analysis of overall survival. Steps

1

2

3 4

Co-variables

Presence of NF1 Histological Grade Necrosis Mitotic Index Presence of NF1 Necrosis Mitotic Index Necrosis Mitotic Index Necrosis

p-value

Exp (B)

95% CI for Exp (B) lower

upper

0.497 0.846 0.276 0.370

0.627 1.233 0.426 1.076

0.163 0.161 0.092 0.917

2.413 9.283 1.975 1.262

0.465 0.255 0.289 0.143 0.226 0.007

0.609 0.413 1.083 0.343 1.093 0.199

0.161 0.090 0.935 0.082 0.947 0.062

2.301 1.897 1.254 1.434 1.261 0.639

Necrosis was an important prognostic factor for lower overall survival, and the epithelioid variant was an important prognostic factor for shorter disease-free survival. p53 expression was not associated with any clinicopathological features and did not have an impact on the survival rates of the MPNST patients. p53 expression was rare in the neurofibromas and common in the MPNSTs, showing that the p53 pathway most likely plays an important role in the tumorigenesis of MPNSTs.

AUTHOR CONTRIBUTIONS Cunha KS designed and conducted the research, analyzed the data, wrote the paper and had primary responsibility for the final content. Faria PA, Geller M and Moura-Neto RS designed the research and wrote the paper. Lopes VS designed and conducted the research. Caruso AC and Pires AR conducted the research. Silva LE analyzed the data. All authors read and approved the final version of the manuscript.

REFERENCES 1. Scheithauer BW, Woodruff JM, Erlandson RA. Primary malignant tumors of peripheral nerve. Tumors of the peripheral nervous system. Washington, DC: Amer Registry of Pathology; 1999. p.303-72. 2. Ducatman BS, Scheithauer BW, Piepgras DG, Reiman HM, Ilstrup DM. Malignant peripheral nerve sheath tumors. A clinicopathologic study of 120 cases. Cancer. 1986;57(10):2006-21, http://dx.doi.org/10.1002/10970142(19860515)57:10,2006::AID-CNCR2820571022.3.0.CO;2-6. 3. Friedman JM, Gutmann DH, Maccollin M, Riccardi VM. Neurofibromatosis: phenotype, natural history, and pathogenesis. Johns Hopkins University Press; 1999. 4. Serra E, Puig S, Otero D, Gaona A, Kruyer H, Ars E, et al. Confirmation of a double-hit model for the NF1 gene in benign neurofibromas. Am J Hum Genet. 1997;61(3):512-9, http://dx.doi.org/10.1086/515504. 5. Karube K, Nabeshima K, Ishiguro M, Harada M, Iwasaki H. cDNA microarray analysis of cancer associated gene expression profiles in malignant peripheral nerve sheath tumours. J Clin Pathol. 2006;59(2):1605, http://dx.doi.org/10.1136/jcp.2004.023598. 6. Mawrin C, Kirches E, Boltze C, Dietzmann K, Roessner A, SchneiderStock R. Immunohistochemical and molecular analysis of p53, RB, and PTEN in malignant peripheral nerve sheath tumors. Virchows Arch. 2002;440(6):610-5, http://dx.doi.org/10.1007/s00428-001-0550-4.

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DOI:10.6061/clinics/2012(08)19

RAPID COMMUNICATION

The long-term impact of a program to prevent central line-associated bloodstream infections in a surgical intensive care unit Adriana P. Paula, Priscila R. Oliveira, Erique P. Miranda, Cassia S. Felix, Clara B. Lorigados, Arlete M. Giovani, Ana Lucia L. Lima Hospital das Clinicas da Faculdade de Medicina da Universidade de Sa˜o Paulo/SP, Brazil. Email: priscilarosalba@gmail.com Tel.: 55 11 2661-6900

catheterization in a 10-bed surgical ICU located in a tertiary hospital specializing in orthopedic care in Sa˜o Paulo, Brazil. The intervention consisted of an 18-month program of weekly visits to the ICU by professionals from an infection control board. The visits consisted of the inspection of all patients with central lines and guidance for the ICU team on the correct techniques for inserting and maintaining these catheters, including the following: full-barrier precautions during the insertion of central venous catheters; the use of a fixation hub with suturing; hand washing before manipulating the device (e.g., for blood sampling and drug infusion); cleaning the skin with chlorhexidine; avoiding the femoral site if possible; and removing unnecessary catheters or those in poor condition. During these visits, all of the professionals belonging to the medical and nursing staff were approached individually to ensure that they had understood the instructions. A bloodstream infection was defined according to the CDC criteria [11]. CLABSIs were considered to be ICU related if detected at least 48 hours after admission to the unit or less than 48 hours after discharge from the unit. The CLABSI incidence rates were expressed as the total number of CLABSI episodes over the number of catheter days. The statistical analyses were performed using an ANOVA with a Bonferroni (post-hoc) correction. Statistical significance was defined as p,0.05. SPSS 10.0 for Windows (SPSS Statistics, IBM, Chicago, IL, USA) and Microsoft Excel 2007 (Microsoft Inc., Redmond, WA, USA) were used for data collection and statistical analyses.

INTRODUCTION Central line-associated bloodstream infections (CLABSIs) are an important type of healthcare-associated infection in intensive care units (ICUs) with high mortality rates and high healthcare costs (1). According to the World Health Organization (WHO), CLABSIs are also the most common cause of healthcare-associated infections of the bloodstream (2). In the United States, the median rate ranges from 1.8 to 5.2 per 1,000 catheter days, according to the Centers for Disease Control and Prevention (CDC) (3). Each year, 100,000 cases and 30,000 deaths occur among patients in ICUs (4). In limited-resource countries, CLABSI rates range from 1.6 to 44.6 cases per 1,000 central line days in adult and pediatric ICUs and from 2.6 to 60.0 cases per 1,000 central line days in neonatal ICUs. CLABSIs are associated with significant additional mortality, with an odds ratio ranging from 2.8 to 9.5 (5). The optimal intervention method for reducing the incidence of CLABSIs has not been definitively identified, but some studies have proven that many practical, low-cost, low-technology educational measures related to inserting and maintaining central lines not only may be successful and effective but can also be sustained (3,4,6–11). Within this scenario, the CDC has recommended that an education program be implemented for healthcare personnel (6). The objective of this study was to describe the long-term impact of a program for decreasing CLABSI rates in a surgical ICU in Sa˜o Paulo, Brazil.

RESULTS

METHODS

During the pre-intervention period (baseline), the mean incidence rate of CLABSI events per 1,000 catheter days observed was 15.85 (¡ standard deviation [SD]: 19.44; 95% CI: 6.19–25.52), ranging from 0 to 60.98 events. The median and interquartile range were 12.66 and 27.24, respectively (Table 1). During the intervention period, the mean incidence rate fell to 4.98¡7.11 (95% CI: 1.45–8.52), and the median reached 0 (interquartile range: 12.53), ranging from 0 to 22.30. This reduction trend was maintained in the post-intervention period, during which 3.91¡8.21 events (95% CI: -0.18–7.99) were observed, ranging from 0 to 31.58. A 68% reduction in the mean CLABSI rate occurred during the intervention, and a 75% decrease was observed after this period.

For this interrupted time-series analysis, we compared the CLABSI rates during an 18-month baseline period (from September 2005 to February 2007), an 18-month intervention period (from March 2007 to August 2008) and an 18-month post-intervention period (from September 2008 to February 2010) in a cohort of patients who received central line

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.

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The impact of a program to prevent CLABSI Paula AP et al.

CLINICS 2012;67(8):969-970

Table 1 - A comparison of the incidence rate and catheter days among the study periods.

Period Catheter days Mean (¡ standard deviation) Median (¡ interquartile range) Range Incidence rate (per 1,000 catheter days) Mean (¡ standard deviation) Median (¡ interquartile range) Range

Baseline

Intervention period

Period after intervention

18 months

18 months

18 months

98.44¡ 41.03 107¡77.75 36–156

99.78¡37.05 95¡56.75 44–186

87.72¡38.46 84¡49.5 34–163

15.85¡19.44 12.66¡27.24 0–60.98

4.98¡7.11 0¡12.53 0–22.30

3.91¡8.21 0¡31.58 0–31.58

There were statistically significant differences among all of the periods (p = 0.013) and between the first and second periods (p = 0.022) and first and third periods (p = 0.045) (Figure 1). The number of catheter days did not differ between any of the periods (p = 0.598, ANOVA).

staff compliance was not evaluated, either by a written test or by statistical analysis, because the infection control team was actively monitoring the ICU to ensure the accomplishment of the educational approach. Our program produced sustained long-term decreases in the CLABSI rate. This success shows the importance of interactions between infection control and ICU professionals for achieving the goal of reducing infections in this setting.

DISCUSSION CLABSIs are associated with high mortality and costs for healthcare systems around the world, and educational strategies are regarded as the cornerstone of infection control. This strategy has been supported by several studies (2,3,5–9), even in settings with a low baseline incidence of CLABSIs (6), although some studies have shown that noneducational measures have been confounders (4,9,10,11). A single study in Brazil found no statistically significant reduction in the incidence of CLABSIs despite good compliance with educational measures, most likely due to the use of an open-infusion system (7). In this study, we achieved lower CLABSI rates through educational measures without increasing costs to the hospital. In this approach, every health professional was approached individually regarding the insertion technique and maintenance of central venous catheters. Thus, each professional felt valued and motivated, which contributed toward the success of the intervention. Importantly, even after the intervention, the rates remained lower than the baseline. Provonost et al. (3) found a sustained reduction of up to 66% in CLABSI rates after 18 months of follow-up in a larger study with an educational intervention based on a similar approach, although their baseline incidence was lower than in the present study. The present study has limitations. The absence of randomization, the involvement of just one service and the absence of detailed statistical intervention analyses make it difficult to establish a causal connection between the intervention and the reduced CLABSI rates, although the long-term follow-up reduced any effect from seasonal trends. The possibility of CLABSI underreporting was minimized through the active surveillance of signs and symptoms, microbiological data and the prescribing of antibiotics in the ICU, although the microorganisms identified were not reported in this study. Furthermore,

REFERENCES 1. Weber DJ, Rutala WA. Central line-associated bloodstream infections: prevention and management. Infect Dis Clin North Am. 2011;25(1):77102, http://dx.doi.org/10.1016/j.idc.2010.11.012. 2. World Health Organization. Preventing bloodstream infections from central line venous catheters. 2012. Avaiable in: http://www.who.int/ patientsafety/implementation/bsi/en/index.html. 3. Pronovost P, Needham D, Berenholtz S, Sinopoli D, Chu H, Cosgrove S, et al. An intervention to decrease catheter-related bloodstream infections in the ICU. N Engl J Med. 2006;355(26):2725-32. 4. Weber DJ, Brown VM, Sickbert-Bennett EE, Rutala WA. Sustained and Prolonged Reduction in Central Line–Associated Bloodstream Infections as a Result of Multiple Interventions. Infect Control Hosp Epidemiol 2010;31(8):875-7, http://dx.doi.org/10.1086/655438. 5. Rosenthal VD. Central line-associated bloodstream infections in limitedresource countries: a review of the literature. Clin Infect Dis. 2009;49(12):1899-907, http://dx.doi.org/10.1086/648439. 6. Parra AP, Mena´rguez MC, Granda MJP, Tomey MJ, Padilla B, Bouza E. A Simple Educational Intervention to Decrease Incidence of Central Line– Associated Bloodstream Infection (CLABSI) in Intensive Care Units with Low Baseline Incidence of CLABSI. Infect Control Hosp Epidemiol. 2010;31(9):964-7, http://dx.doi.org/10.1086/655841. 7. Santana SL, Furtado GH, Wey SB, Medeiros EA. Impact of an education program on the incidence of central line-associated bloodstream infection in 2 medical-surgical intensive care units in Brazil. Infect Control Hosp Epidemiol. 2008;29(12):1171-3, http://dx.doi.org/10.1086/ 591862. 8. Marra AR, Cal RG, Dura˜o MS, Correa L, Guastelli LR, Moura DF Jr, et al. Impact of a program to prevent central line-associated bloodstream infection in the zero tolerance era. Am J Infect Control. 2010;38(6):434-9, http://dx.doi.org/10.1016/j.ajic.2009.11.012. 9. Guerin K, Wagner J, Rains K, Bessesen M. Reduction in central lineassociated bloodstream infections by implementation of a postinsertion care bundle. Am J Infect Control. 2010;38(6):430-3, http://dx.doi.org/ 10.1016/j.ajic.2010.03.007. 10. Ong A, Dysert K, Herbert C, Laux L, Granato J, Crawford J, et al. Trends in central line-associated bloodstream infections in a trauma-surgical intensive care unit. Arch Surg. 2011;146(3):302-7, http://dx.doi.org/ 10.1001/archsurg.2011.9. 11. Garner JS, Jarvis WR, Emori TG, Horan TC, Hughes JM. CDC definitions for nosocomial infections, 1988. Am J Infect Control. 1988;16(3):128-40, http://dx.doi.org/10.1016/0196-6553(88)90053-3.

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DOI:10.6061/clinics/2012(08)20

TECHNICAL NOTE

An innovative model of autologous anorectal transplantation with pudendal nerve reconstruction Flavio Henrique Ferreira Galvao, Victor Edmund Seid, Daniel Reis Waisberg, Ruy Jorge Cruz-Jr, Hugo Hirano, Sergio Catanozi, Eleazar Chaib, Luiz Augusto Carneiro D’Albuquerque Faculdade de Medicina da Universidade de Sa˜o Paulo, Department of Gastroenterology, Division of Transplantation, Laboratories of Medical Investigation (LIM37 and LIM10), Sa˜o Paulo/SP, Brazil. Email: fgalvao@usp.br Tel.: 55 11 3061 8319

Figure 1 - (A) An anorectal graft showing the inferior mesenteric artery (IMA) and vein (IMV) and the rectum (R). (B) The infrarenal aorta (Ao) and vena cava (IVC) were cross-clamped at the site of anastomosis. (C) The anastomoses between the Ao and IMA and between the IVC and IMV. (D) The anorectal graft following reperfusion. (E) Pudendal nerve anastomosis (arrow head). (F) Anal anastomosis in the perineum.

However, there is a lack of pre-clinical large animal models to study the potential benefits of this procedure. In this paper, we describe an innovative surgical technique of anorectal autotransplantation, which allows for physiological studies without the immunologic effects related to allotransplantation. The technique was initially performed in three swine weighing 25-35 Kg. After general anesthesia, an abdominal and perineal incision was performed in which the rectum, anal sphincter, and skin surrounding the anus were procured en bloc. The graft’s vascular pedicle contained the inferior mesenteric artery (IMA) with a patch of the aorta and inferior mesenteric vein (IMV), which was divided near the splenic vein (Figure 1A). The pudendal nerve was carefully isolated and transected. The graft was removed and immediately flushed with 250 ml of cold heparinized preservation solution through a catheter inserted in the IMA. The graft was then placed in a cooler with cold solution without heparin for a total of 20 minutes.

Fecal incontinence is an important public health problem that has a significant impact on the quality of life of individual patients. The prevalence of fecal incontinence in the United States ranges from 2 to 17%, but this may be an underestimation (1). The current treatments for severe fecal incontinence are often complex and have disappointing results (1). Anorectal transplantation may be a definitive method for restoring normal sphincter function in these patients. We and others have previously investigated anorectal transplantation in rats with promising results (2-4).

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.

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CLINICS 2012;67(8):971-972

The infrarenal aorta and vena cava were isolated and crossclamped, and the graft was implanted by arterial and venous anastomoses between the IMA and the aorta (Figure 1B) and the IMV and vena cava (Figure 1C). After reperfusion (Figure 1D), the entire graft was placed inside the abdominal cavity, and the pudendal nerve was reconstructed (Figure 1E). Finally, the anus was placed in its original position, and the perineum was closed (Figure 1F). Digestive tract continuity was reestablished by an end-to-end colorectal anastomosis. The abdominal cavity was closed, and the animals were observed for an additional two hours. At the end of the experiment, the graft was removed for histological analysis. The average times for graft recovery and implantation (including vascular, nervous and GI reconstruction) were 57 and 99 minutes, respectively. No problems regarding graft perfusion were observed at the end of the experiment. Histological graft assessment showed normal aspects in one graft and mild ischemia-reperfusion injury in two. To the best of our knowledge, there is only one report of a pre-clinical model of anorectal transplantation (5). Those authors described the donor and recipient surgical technique in four swine allotransplantations, with a 25% rate of technical failure after a 24-hour observation period. The experimental model described in this paper mimics the surgical stress and potential effects of graft denervation of allotransplantation but precludes, for obvious reasons, the antigen-mediated immune response. In addition, the technical modifications were associated with a significant reduction in the surgical time and cost. Therefore, anorectal autotransplantation in swine is a feasible and relatively simple procedure that may be a relevant technique to study regeneration, anorectal physiology, and the potential benefits of this unique type of transplantation without the interference of immune-mediated effects.

ACKNOWLEDGMENTS This study was supported by Laboratory of Medical Investigation (LIM37), University of Sa˜o Paulo School of Medicine.

AUTHOR CONTRIBUTIONS Galvao FH conceived and designed the study, provided the hypothesis, performed the operative procedure, was responsible for the data analysis and interpretation, manuscript writing and approval of the manuscript final version. Seid VE conceived and designed the study, provided technical help with the operative procedure, was responsible for the data analysis, interpretation of results and approval of the manuscript final version. Waisberg DR conceived and designed the study, provided technical help with the operative procedure, was responsible for the data analysis and interpretation of results. Cruz Jr RJ conceived and designed the study, provided advice and consultation, was responsible for the manuscript writing, critical review and approval of the manuscript final version. Hirano H and Catanozi S provided technical help with the operative procedure, were responsible for the data analysis and interpretation of results. Chaib E and D’Albuquerque LA provided advice and consultation, were responsible for the manuscript writing and approval of the manuscript final version.

REFERENCES 1. Wald A. Fecal incontinence in adults. New Engl J Med. 2007;356(16):164855, http://dx.doi.org/10.1056/NEJMcp067041. 2. Galvao FHF, Seid VE, dos Santos RMN, Kitamura M, Galvao RD, Pinto RA, et al. Anorectal transplantation. Tech Coloproctol. 2009;13(1):55-9, http://dx.doi.org/10.1007/s10151-009-0459-5. 3. Galvao FHF, Waisberg DR, Vianna RMD, Galvao RD, Seid VE, Andraus W, et al. Intestinal transplantation including anorectal segment in the rat. Microsurg. 2012;32(1):77-9, http://dx.doi.org/10.1002/micr.20958. 4. Araki J, Mihara M, Narushima M, Iida T, Sato T, Koshima I. Vascularized Anal Autotransplantation Model in Rats: Preliminary Report. Transplant P. 2011;43(9):3552-6, http://dx.doi.org/10.1016/j.transproceed.2011.08.042. 5. O’Bichere A, Shurey S, Sibbons P, Green C, Phillips RKS. Experimental model of anorectal transplantation. Brit J Surg. 2000;87(11):1534-9, http://dx.doi.org/10.1046/j.1365-2168.2000.01557.x.

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CLINICS 2012;67(8):973-975

DOI:10.6061/clinics/2012(08)21

CASE REPORT

Diagnosis of an ectopic adrenocorticotropic hormonesecreting bronchial carcinoid by somatostatin receptor scintigraphy ˙Inan Anaforog˘lu,I Kerem Ersoy,I Mehmet As¸ık,I Savas¸ Karyag˘ar,II Ekrem Algu¨nI I

Departments of Endocrinology and Metabolism, Trabzon Numune Education and Research Hospital, Trabzon, Turkey. Numune Education and Research Hospital, Trabzon, Turkey.

II

Nuclear Medicine, Trabzon

Email: ianaforoglu@hotmail.com Tel.: 90-462-230-56-49

(Figure 1). No involvement was present upon positron emission tomography (PET). SRS revealed a lesion at the left posterobasal segment consistent with the thorax CT, suggesting a bronchial carcinoid tumor (Figure 2). The patient underwent segmentectomy. An immunohistochemical evaluation revealed positive staining for chromogranin, synaptophysin, and ACTH (Figure 3). Two weeks after surgery, cortisol was suppressed upon lowdose DST (serum cortisol level, 0.2 mg/dL). After four months of follow up, she had lost 15 kg, her menstrual periods had become regular, and her acne and hirsutism had regressed significantly.

INTRODUCTION Ectopic adrenocorticotropic hormone (ACTH) syndrome accounts for 10%–20% of the cases of ACTH-dependent Cushing’s syndrome (1). Ectopic ACTH secretion typically results from an occult, slow-growing bronchial carcinoid tumor. The diagnosis of these relatively small tumors can be difficult with conventional imaging procedures, such as computerized tomography (CT) or magnetic resonance imaging (MRI) (2). Herein, we present a case of ectopic ACTH syndrome arising from a bronchial carcinoid tumor that was diagnosed with somatostatin receptor scintigraphy (SRS).

DISCUSSION

CASE DESCRIPTION

Ectopic ACTH syndrome represents a minority of all cases of Cushing’s syndrome (1,2). High-dose DST can be used to discriminate ectopic ACTH syndrome from classic Cushing’s disease; a suppression of more than 50% of the basal cortisol level can be observed in more than 80% of patients with Cushing’s disease (3). However, the diagnostic utility of highdose DST is limited, and this test is not recommended when IPSS is available (3). In our patient, although cortisol suppression could be compatible with a diagnosis of ectopic ACTH syndrome, she also had a microadenoma in the hypophysis. Because adenomas are incidentally discovered in up to 10% of the normal population, IPSS is advised for patients with ACTH-dependent Cushing’s syndrome and a hypophyseal adenoma smaller than 6 mm, particularly when test results conflict (4). IPSS can demonstrate the ratio of the ACTH level in the central sinus relative to the peripheral sinus. A central/ peripheral ratio of 2 before the administration of corticotropin-releasing hormone and a ratio of $3 after its administration strongly suggests Cushing’s disease (5). However, although a positive result is highly suggestive of Cushing’s disease, false-negative results might be more common than previously appreciated (2%–4%). In cases of a negative response, clinicians should perform a careful search for an ectopic source (6). In our patient, although she had a pituitary microadenoma (3 mm), the results of high-dose DST and IPSS were consistent with ectopic ACTH syndrome. The source of ectopic ACTH secretion should be established after diagnosis because the excision of an ACTHproducing tumor can be curative (2,7). The most likely site of ACTH-producing tumors is the thorax, and these tumors are

A 25-year-old Caucasian woman was admitted for oligomenorrhea and hirsutism. A physical examination revealed central obesity, a ‘‘buffalo hump’’, acne, and severe hirsutism (Ferriman-Gallwey score of 18). After overnight dexamethasone suppression testing (DST), her serum cortisol level was 14.4 mg/dL. She was hospitalized with a presumptive diagnosis of Cushing’s syndrome. Her diurnal cortisol secretion rhythm was found to be impaired; her serum cortisol level at midnight was 27.1 mg/ dL. Low-dose DST was also consistent with this diagnosis, with an unsuppressed serum cortisol level of 4.2 mg/dL. Her basal ACTH and cortisol levels were 67 pg/mL and 15.2 mg/dL, respectively. With a refined presumptive diagnosis of ACTH-dependent Cushing’s syndrome, we performed overnight highdose DST with 8 mg dexamethasone. Her serum cortisol level was 10 mg/dL, revealing a lack of suppression. An MRI revealed a 3-mm microadenoma of the hypophysis. However, inferior petrosal sinus sampling (IPSS) revealed no substantial gradient in ACTH levels between the center and the periphery (Table 1). Thus, a diagnosis of ectopic Cushing’s syndrome was proposed. Upon CT of the thorax and abdomen, we noted a contrastenhancing small lesion at the left posterobasal segment

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.

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Table 1 - Adrenocorticotropic hormone (ACTH) levels in the inferior petrosal sinus before and after the infusion of corticotropin-releasing hormone. ACTH Level (pg/mL)

Time before infusion 5 minutes 2 minutes Time after infusion 2 minutes 5 minutes 10 minutes

Periphery

Right

Left

141 144

163 159

151 164

148 149 160

160 166 175

151 181 172

Figure 1 - Computerized tomography of the bronchial carcinoid.

Figure 2 - Somatostatin receptor scintigraphy of the bronchial carcinoid.

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Figure 3 - A) ACTH staining of bronchial cells. B) Vascular invasion by tumor cells. C) Lymphoid invasion by tumor cells.

frequently bronchial carcinoid tumors. Locating these tumors can be challenging; they are relatively small and slowgrowing, and conventional imaging studies, such as CT and MRI, identify the tumor in only 50% of cases (2,4,7). The tumor can remain occult long after the diagnosis of Cushing’s syndrome (4). Functional imaging studies, such as PET and SRS, are complementary imaging tools for revealing the presence of carcinoids (6). SRS might be superior to PET in detecting bronchial carcinoids. Although PET can detect highly active proliferative tumors, bronchial carcinoids usually have a low proliferation index and are slow growing, quite small lesions (7,8). However, the success of SRS depends only on the presence of somatostatin receptors, which have been identified on many cells of neuroendocrine origin (8). The radiolabelled somatostatin analog octreotide can bind to somatostatin receptors 2 and 5 with high affinity (8). Bronchial carcinoids express both receptors. Nevertheless, these tumors may show heterogeneity in the degree of somatostatin receptor expression (8). The diagnostic sensitivity of SRS is approximately 25%– 73% for the detection of an ectopic source of ACTH secretion (9). Ilias and colleagues reported that the sensitivity of SRS is only approximately 49%, and it is insufficient for lesions that are not present on CT or MRI (1). Zemskova and colleagues reported a 57% sensitivity and a 79% positive predictive value for SRS for detecting ectopic ACTH-producing tumors (7). The use of a single imaging tool may not be sufficient to diagnose ectopic ACTH-secreting tumors; conventional and functional imaging studies should be used in combination when needed (2,6-9). In our case, the ectopic lesion was shown on CT but not on PET. SRS confirmed the lesion shown on CT, and the tumor was extracted. Suppression by DST revealed biochemical recruitment, and ACTH staining of the excised tissue confirmed the ectopic tumor postoperatively. In conclusion, in cases of ectopic ACTH syndrome, the exact location of the lesion is needed for a curative approach. Conventional imaging studies may not always show the lesion. The use of somatostatin receptor scintigraphy in addition to conventional imaging may help localize lesions.

ACKNOWLEDGMENTS English language editing of this manuscript was performed by Patricia French from Left Lane Communications (Chapel Hill, NC, USA) and funded by Sanofi-Aventis (I˙stanbul, Turkey).

AUTHOR CONTRIBUTIONS Anaforoglu I drafted and approved the final version of the manuscript. Ersoy K drafted the manuscript. Asik M and Karyagar S substantially contributed to the acquisition and interpretation of data. Algun E critically reviewed the manuscript. Each author participated sufficiently to take public responsibility for appropriate portions of the content. All authors read and approved the final version of the manuscript.

REFERENCES 1. Ilias I, Torpy DJ, Pacak K, Mullen N, Wesley RA, Nieman LK. Cushing’s syndrome due to ectopic corticotropin secretion: twenty years’ experience at the National Institutes of Health. J Clin Endocrinol Metab. 2005;90(8):4955-62, http://dx.doi.org/10.1210/jc.2004-2527. 2. Tani Y, Sugiyama T, Hirooka S, Izumiyama H, Hirata Y. Ectopic ACTH syndrome caused by bronchial carcinoid tumor indistinguishable from Cushing’s disease. Endocr J. 2010;57(8):679-86, http://dx.doi.org/ 10.1507/endocrj.K10E-129. 3. Newell-Price J, Grossman AB. Differential diagnosis of Cushing’s syndrome. Arq Bras Endocrinol Metabol. 2007;51(8):1199-206. 4. Boscaro M, Arnaldi G. Approach to the patient with possible Cushing’s syndrome. J Clin Endocrinol Metab. 2009;94(9):3121-31, http:// dx.doi.org/10.1210/jc.2009-0612. 5. Lad SP, Patil CG, Laws ER Jr, Katznelson L. The role of inferior petrosal sinus sampling in the diagnostic localization of Cushing’s disease. Neurosurg Focus. 2007;23:E2. 6. Isidori AM, Lenzi A. Ectopic ACTH syndrome. Arq Bras Endocrinol Metabol. 2007;51(8):1217-25, http://dx.doi.org/10.1590/S0004-2730 2007000800007. 7. Zemskova MS, Gundabolu B, Sinaii N, Chen CC, Carrasquillo JA, Whatley M, Chowdhury I, Gharib AM, Nieman LK. Utility of various functional and anatomic imaging modalities for detection of ectopic adrenocorticotropin-secreting tumors. J Clin Endocrinol Metab. 2010;95(3):1207-19, http://dx.doi.org/10.1210/jc.2009-2282. 8. Pacak K, Ilias I, Chen CC, Carrasquillo JA, Whatley M, Nieman LK. The role of [(18)F]fluorodeoxyglucose positron emission tomography and [(111)In]-diethylenetriaminepentaacetate-D-Phe-pentetreotide scintigraphy in the localization of ectopic adrenocorticotropin-secreting tumors causing Cushing’s syndrome. J Clin Endocrinol Metab. 2004;89(5):221421, http://dx.doi.org/10.1210/jc.2003-031812. 9. Doi M, Sugiyama T, Izumiyama H, Yoshimoto T, Hirata Y. Clinical features and management of ectopic ACTH syndrome at a single institute in Japan. Endocr J. 2010;57(12):1061-9, http://dx.doi.org/10.1507/ endocrj.K10E-265.

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DOI:10.6061/clinics/2012(08)22

CASE REPORT

A possible case of Churg-Strauss syndrome in a 9year-old child Jinling Liu,I Yingchun Xu,I Zhimin Chen,I Xuefeng Xu,I Meiping Lu,I Yingshuo Wang,I Yunlian Zhou,I Weizhong GuII I

The Children’s Hospital of Zhejiang University School of Medicine Department of Pulmonology, Zhejiang Province, China. II The Children’s Hospital of Zhejiang University School of Medicine, Department of Pathology, Zhejiang Province, China. Email: drchenzm@163.com Tel.: 8613958096916

Additionally, he began to experience lower back pain, whole body fatigue, pyknocardia, hepatomegaly, mild splenomegaly, hyperpyrexia, bilateral swelling of the parotid gland, submaxillary gland enlargement, discrete cervical lymphadenectasis, acute abdominal pain, chest pain, polyarthralgia, myalgia, and muscle weakness. His chest X-ray demonstrated worsening of the pulmonary infiltration (Figure 1B). The initial laboratory examination at the time of admission showed a peripheral white blood cell (WBC) count of 14.96109/L (normal: 4-106109/L) with 30.7% eosinophils (normal: 0%–5%), 41.4% neutrophils, 21.6% lymphocytes, and a hemoglobin level of 138 g/L. Notably, 11 days later, his eosinophil count increased to 72.2% with an absolute eosinophil count of 27.96109/ L and a WBC count of 38.66109/L. The patient’s erythrocyte sedimentation rate was 77 mm/hour, and immunoglobulin E (IgE) was elevated to 1258 KU/L (reference range: 0–100 KU/L). Tests for cytoplasmic (classical) antineutrophil cytoplasmic antibodies (c-ANCA) and peri-nuclear (protoplasmic-staining) antineutrophil cytoplasmic antibodies (p-ANCA) were negative. The levels of proteinase 3 (PR3) and myeloperoxidase (MPO) were normal. Computed tomography of the chest demonstrated interstitial infiltration. Cultures and/or stains of blood and sputum for viruses, fungi, acid-fast bacilli and bacteria were all negative. His parents declined bone marrow aspiration. A peripheral blood smear analysis produced normal results. His lactate dehydrogenase level was 235 U/L (normal, 120-300 U/L). Peripheral blood lymphocyte immunophenotyping indicated that his T-cell subsets were normal. All tumor markers and antinuclear antibodies were negative. Stool cultures for parasites and the results of serum analyses for anti-Schistosoma and antiSparganum species antibodies were all negative, and serum samples were negative for HIV and hepatitis B and C. No other significant laboratory abnormalities were found, including blood gas levels, hepatic, renal and myocardial function, coagulation function, antistreptolysin o, complement levels, 1-3-b-D glucosan (G test) and galactomannan (GM test). Cardiac evaluations, including electrocardiography and echocardiography, revealed no abnormalities. A skin biopsy specimen obtained from a rash on the patient’s left lower extremity revealed a dermal infiltration with a considerable quantity of eosinophils (Figure 2A). Flexible bronchoscopy revealed moderate secretions. Bronchoalveolar lavage cytology revealed a predominance of in flammatory cells with 93% eosinophils, 2% macrophages (Figure 2B), and no microorganisms. A diagnosis of

INTRODUCTION Churg-Strauss syndrome (CSS), also known as allergic granulomatous angiitis, is a systemic necrotizing vasculitis of the small and medium vessels. It typically occurs in conjunction with a history of asthma, eosinophilia, pulmonary infiltrates, and vasculitis, but cases without asthma have also been reported (1-2). CSS has rarely been reported in pediatric patients. Here, we report a nine-year-old boy with a possible diagnosis of CSS who presented with prominent pulmonary involvement accompanied by eosinophilia, rash, abdominal pain, and hepatomegaly but without anti-neutrophil cytoplasmic antibody (ANCA). The present case indicates that physicians should consider CSS when eosinophilia is noted in bronchoalveolar lavage, serum, or a tissue specimen. The clinical features and treatment for childhood CSS are reviewed.

CASE DESCRIPTION A nine-year-old boy presented to our clinic with a oneweek history of a productive cough, blood-streaked sputum and increased wheezing and a five-day history of fever. He complained of a one-year history of asthma, which had previously been treated with inhaled corticosteroids with the addition of montelukast. A BCG scar confirmed previous immunization against tuberculosis. He had no history of travel to helminth endemic areas and showed no weight loss. The boy was admitted because of the exacerbation of his symptoms and a chest radiograph revealing infiltration in both lungs in April 2011 (Figure 1A). The possibility of pneumonia was considered. After taking a five-day course of antibiotics, which included ceftriaxone for five days and azithromycin for three days, his respiratory symptoms and physical condition improved. However, several days later, his condition worsened, with cough, severe dyspnea, perioral cyanosis, urticaria-like lesions and vesicular rashes on the skin of both lower extremities, and purpura on the hands.

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.

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DISCUSSION Churg-Strauss syndrome was first described by Churg and Strauss in 1951 (3). The annual incidence of CSS in adults ranges between 0.5 and 3.1 cases per million. In children, the incidence of CSS is unknown but is thought to be very low. To our knowledge, only 44 pediatric patients with CSS have been reported, including our present case. The reported patients’ ages ranged from 2 to 18 years (20 males and 24 females), with clinical manifestations rarely appearing before one year of age. The rarity of CSS in childhood makes the diagnosis much more difficult. In 1990, the American College of Rheumatology (ACR) developed specific clinical criteria for the classification of CSS, which comprised a history of asthma, eosinophilia .10%, mononeuropathy/polyneuropathy, nonfixed pulmonary infiltrates, paranasal sinus abnormality, and extravascular eosinophils revealed by biopsy. The presence of at least four criteria yields a sensitivity of 85% and a specificity of 99.7% (4). Our patient met four of the six ACR criteria, namely, a history of asthma, eosinophilia .10%, pulmonary infiltrates, and a skin biopsy revealing extravascular eosinophils. However, although commonly used to diagnose CSS, the ACR criteria were not meant as diagnostic criteria and were instead meant to distinguish one form of vasculitis from another. Unfortunately, there have been no new diagnostic criteria for CSS. The differential diagnosis of CSS depends on the predominant clinical manifestations of each patient. For example, hypereosinophilic syndrome (HES) is difficult to distinguish from CSS because HES patients may have hypereosinophilia and end-organ damage, but characteristics such as asthma, atopic history and lung infiltrates support a diagnosis of CSS. Concomitant eosinophilia, asthma, and lung infiltrates may resemble certain parasitic infections, but stool cultures for parasites and serum analyses for antibodies were negative, and our patient had no history of travel to helminth endemic areas. Moreover, neoplastic disorders, particularly lymphomas, typically progress by the 11-month follow-up examination, but our patient’s prompt response to treatment and persistent remission did not support the diagnosis of neoplastic disease. Nevertheless, a definite diagnosis was a challenge, and we still follow up with the patient in the event that evidence of a malignant disorder is discovered. The accurate diagnosis of CSS requires the presence of a constellation of findings that may have presented and evolved over a period of years rather than any individual pathological finding at one time point (5-7). After reviewing the 43 cases of pediatric CSS in the literature, we outlined the clinical features of CSS in children and compared them with those in adults. The mortality rate of CSS is approximately 5% in adults (8) and is significantly worse in children, at 15.9% (7/44). In the past four years, 2 of the 11 pediatric CSS patients died due to pulmonary abscess and sepsis despite treatment with immunosuppressive drugs and intravenous immunoglobulin (1,9). The other predominant causes of death include heart failure, renal failure, cerebral failure, and gastrointestinal perforation/hemorrhage. In our review, a history of asthma was found in 39 (88.6%) of the 44 children with CSS. Almost all CSS patients show a marked peripheral blood eosinophilia. The highest absolute eosinophilic count reported in all ages was 51.66109/L and leukocytosis in leukemic ranges (65.86109/L) (10). In our

Figure 1 - Radiological imaging. A) A computed tomography scan showing pulmonary infiltrates in both lungs. B) A chest x-ray showing aggravation. C) A computed tomography scan showing improvement. D) A chest x-ray showing improvement.

Churg-Strauss syndrome (CSS) was suggested. Corticosteroid therapy (intravenous methylprednisolone) was initiated at a dose of 80 mg daily (2.7 mg/kg/day), which improved the symptoms. Five days later corticosteroid (methylprednisolone tablets) was tapered at 48 mg daily (1.7 mg/kg/day). After two weeks of corticosteroids treatment, his condition stabilized and a peripheral eosinophil count decreased to 1.3%, WBC count decreased to 8.076109/L, and the sedimentation rate decreased from 77 to 16 mm/hour. Chest computed tomography (Figure 1C) and x-ray (Figure 1D) revealed an improvement in pulmonary infiltration. Ten months later, the last flexible fiberoptic examination showed a clear airway, and the bronchoalveolar lavage cytology assay was normal. The corticosteroids were tapered without any systemic vasculitis over the following 11 months. At the latest follow up in late February 2012, the child was started on methylprednisolone tablets at a dose of 8 mg daily (0.3 mg/Kg/day) for two months.

Figure 2 - A) A skin biopsy showing the accumulation of extravascular eosinophilic infiltrates (hematoxylin-eosin stain 6200). B) Bronchoalveolar lavage fluid (BALF) showing a considerable quantity of eosinophils (Giemsa staining 6200).

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patient, the highest level of leukocytosis was 38.66109/L with an absolute eosinophil count of 27.96109/L (72.2%). Of the 44 pediatric CSS patients, 23 had high IgE levels and only 2 had normal IgE levels; the 19 remaining cases lacked relevant data. Our patient was negative for ANCAs, which is consistent with the literature demonstrating that only 5 of 23 patients were positive for ANCAs. However, the frequency of ANCA positivity is uncertain because the ANCA status was not available for the other 21 children. Two recent independent studies on large cohorts have found that ANCAs are present in only approximately 40% of adult CSS patients (11,12). It has been postulated (11,12) that CSS might encompass two clinical subsets or phenotypes: one is an ANCA-associated process that primarily has the features of small-vessel vasculitis (such as glomerulonephritis, mononeuritis multiplex, and purpura), and the other is ANCA-negative and more related to eosinophilic tissue infiltration that leads predominantly to pulmonary involvement and cardiopulmonary manifestations. The clinical presentation of CSS is variable, and virtually any organ can be affected by the disease. In our review, pneumonia was the most common disorder (37/44 cases; 84.1%) followed by cutaneous manifestations (29/44, 65.9%). Skin manifestations were a common early presentation of CSS in children, which ranged from discrete to diffuse patchy, petechial or purpuric lesions and can be itchy, edematous, or tender. Paranasal sinus abnormalities were also common early presentations (24/37; 64.9%). Neurologic involvement, especially multiple mononeuropathy, was described in 40.5% (17/42) of children with CSS and has been reported in 36% of adult patients with CSS (8). Heart involvement was reported in 38.6% (17/44) of the children, gastrointestinal tract involvement in 34.1% (15/44) and kidney involvement in 13.6% (6/43). A number of reports reveal more subtle presentations of CSS, including swelling of the legs and feet, oral aphthae, hepatomegaly, hepatic venous outflow obstruction, respiratory distress, polyarthralgia, myalgia, polyneuropathies presenting as dysesthesia, muscle weakness, gait disturbances, venous thrombosis and pulmonary embolism, hemichorea, sudden painless loss of vision, retinal artery occlusions, discrete cervical lymphadenopathy, testicular pain, hypertension, seizures and necrosis of the toe tips. Although the diagnosis of CSS is established mainly based on clinical observations, a tissue biopsy should be performed whenever possible (5,13). In addition to skin biopsies, the diagnosis of CSS is increasingly aided by transbronchial lung biopsy, open or thoracoscopic lung biopsy and, less frequently, by sinus biopsy, nasal mucosa biopsy or liver biopsy. A controlled study for the optimal treatment of CSS is not yet available because of the limited number of patients. The cornerstone of treatment for CSS remains corticosteroid therapy, but the response is variable. In patients with severe organ involvement or uncontrolled vasculitis symptoms, additional therapy with cyclophosphamide and/or azathioprine chlorambucil and methotrexate is required. The use of these various immunosuppressive medications was effective in treating refractory CSS and in minimizing relapses. Plasma exchange was also used as an alternative to cytotoxic therapy, but prospective studies have not shown any difference in mortality or outcome (5). Moreover, intravenous immunoglobulin is a treatment option in patients with CSS with neuropathy (14). Rituximab, in fliximab, MMF (mycophenolate mofetil), and

a-interferon are other therapeutic options used to treat CSS (15). In conclusion, our present case emphasizes the importance of considering the diagnosis of CSS in children with uncontrolled pneumonia, hypereosinophilia, skin lesions, neuropathy, cardiomyopathy or hepatomegaly in conjunction with a previous history of asthma and sinusitis. Delayed diagnosis and severe organ involvement may worsen the prognosis and even lead to a fatal outcome. If started early, treatment with corticosteroids is often successful.

ACKNOWLEDGMENTS This study was partially supported by grants from the National Natural Science Foundation of China (Nos. 30872798, 81070004 and 81000 765).

AUTHOR CONTRIBUTIONS Jinling L wrote and revised the manuscript, collected data, performed the follow up, and served as an attending physician of the patient. Yingchun X served as an attending physician of the patient and participated in the data collection and writing. Zhimin C, the corresponding author, was responsible for the structure of the manuscript and the design of the treatment and follow up. Xuefeng X participated in writing the manuscript and constructed the figures. Meiping L co-designed the patient’s treatment scheme and served as an attending physician of the patient. Yingshuo W participated in data collection and writing. Yunlian Z participated in data collection and follow up. Weizhong G analyzed the skin biopsy specimens and bronchoalveolar lavage fluid.

REFERENCES 1. Hamdan MA, Al-Rumaithi S, Tolaymat N, Ionescu G, Al-Ashari M, ElTeraifi H. Churg–Strauss syndrome presenting as an abdominal mass in a non-asthmatic child. Ann Trop Paediatr. 2007;27(4):311-4, http:// dx.doi.org/10.1179/146532807X245724. 2. Sevinc A, Hasanoglu HC, Gokirmak M, Yildirim Z, Baysal T, Mizrak B. Allergic granulo-matosis and angiitis in the absence of asthma and blood eosinophilia: a rare presentation of limited Churg–Strauss syndrome. Rheumatol Int. 2004;24(5):301-4. 3. Churg J, Strauss L. Allergic granulomatosis, allergic angiitis, and periarteritis nodosa. Am J Pathol. 1951;27(2):277-301. 4. Masi AT, Hunder GG, Lie JT, Michel BA, Bloch DA, Arend WP, et al. The American College of Rheumatology 1990 criteria for the classification of Churg–Strauss syndrome (allergic granulomatosis and angiitis). Arthritis Rheum. 1990;33(8):1094-100. 5. Abril A, Calamia KT, Cohen MD. The Churg Strauss syndrome (allergic granulomatous angiitis): Review and update. Semin Arthritis Rheum. 2003;33(2):106-14, http://dx.doi.org/10.1016/S0049-0172(03)00083-0. 6. Noth I, Strek ME, Leff AR. Churg Strauss syndrome. Lancet. 2003;361(9357):587-94, http://dx.doi.org/10.1016/S0140-6736(03)125184. 7. Sinico RA, Bottero P. Churg-Strauss angiitis. Best Practice & Research Clinical Rheumatology. 2009;23(3):355-66, http://dx.doi.org/10.1016/ j.berh.2009.02.004. 8. Zwerina J, Eger G, Englbrecht M, Manger B, Schett G. Churg–Strauss Syndrome in Childhood: A Systematic Literature Review and Clinical Comparison with Adult Patients. Semin Arthritis Rheum. 2009;39(2):10815, http://dx.doi.org/10.1016/j.semarthrit.2008.05.004. 9. Twardowsky AO, Paz JA, Pastorino AC, Jacob CM, Marques-Dias MJ, Silva CA. Chorea in a child with Churg-Strauss syndrome. Acta Reumatol. 2010;35(1):72-5. 10. Mutsaers ER, Witteveen R, van den Bosch-Ruis W, Kuijpers TW, van Houten MA, van den Berg JM. A pseudoleukemic blood differentiation in a 13-year-old child: an extraordinary presentation of Churg-Strauss syndrome. Clin Rheumatol. 2009;Sep 9. [Epub ahead of print]. 11. Sinico RA, Di Toma L, Maggiore U, Bottero P, Radice A, Tosoni C, et al. Prevalence and clinical significance of antineutrophil cytoplasmic antibodies in Churg-Strauss Syndrome. Arthritis Rheum. 2005;52(9): 2926-35, http://dx.doi.org/10.1002/art.21250. 12. Sable-Fourtassou R, Cohen P, Mahr A, Pagnoux C, Mouthon L, Jayne D, et al. Antineutrophil cytoplasmic antibodies and the Churg-Strauss syndrome. Ann Intern Med 2005;143(9):632-8. 13. Mukhtyar C, Guillevin L, Cid MC, Dasgupta B, de Groot K, Gross W, et al. EULAR Recommendations for the management of primary small

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Neurosurg. 2009;111(8):683-7, http://dx.doi.org/10.1016/j.clineuro.2009. 07.004. 15. Demircin G, Baysun S, Bu¨lbu¨l M, Erdog˘an O, Oner A. Mycophenolate mofetil therapy in a child with Churg–Strauss syndrome. Pediatr Int. 2010;52(3):164-6.

and medium vessel vasculitis. Ann Rheum Dis. 2009;68(3):310-7, http:// dx.doi.org/10.1136/ard.2008.088096. 14. Nakamura M, Yabe I, Yaguchi H, Kishimoto R, Mito Y, Fujiki N, et al. Clinical characterization and successful treatment of 6 patients with Churg-Strauss syndrome-associated neuropathy. Clin Neurol

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DOI:10.6061/clinics/2012(08)23

CASE REPORT

MSX2 copy number increase and craniosynostosis: copy number variation detected by array comparative genomic hybridization Karla de Oliveira Pelegrino,I Sofia Sugayama,II Karina Lezirovitz,III Ana Lu´cia Catelani,I Fernando Kok,I Maria de Lourdes ChauffailleI I Instituto de Pesquisa e Desenvolvimento, Grupo Fleury, Sa˜o Paulo/SP, Brazil. II Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Departamento de Pediatria, Sa˜o Paulo/SP, Brazil. III Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Departamento de Otorrinolaringologia (LIM32), Sa˜o Paulo/SP, Brazil.

Email: mlourdes.chauffaille@grupofleury.com.br Tel.: 55 11 5014-7621.

was obtained from the family. The propositus (a girl) was the first live born child of a healthy and non-consanguineous couple (the mother was 38 years old and the father was 40 years old at the time of conception). The mother is of Japanese descent, and the father is of African descent. The mother had experienced two previous miscarriages due to incontinence of the endocervical isthmus. The pregnancy was uneventful except for mild vaginal bleeding during the first trimester. The patient was born preterm (31 weeks) after vaginal delivery, with Apgar scores of 8 and 9, a weight of 1550 g, a length of 38.5 cm, and an OFC of 28.0 cm. At birth, the patient exhibited respiratory distress and underwent orotracheal intubation (3 days). The patient was hypotonic, had poor suction, and exhibited neonatal jaundice and metabolic disturbances (hyponatremia and hypocalcemia). She was discharged at 2 months of age at a weight of 1910 g. The patient also exhibited failure to thrive, recurrent otitis and global developmental delay. She was able to sit without support at 15 months and able to walk with no support and say simple words at 24 months. Physical examination at 19 months showed a weight of 6500 g, a length of 68.5 cm, and an OFC of 42.5 cm. The patient’s face was peculiar, with a narrowing of the frontal diameter, upslanting palpebral fissures, an enlarged nasal root, low and posteriorly rotated ears, bilateral convergent strabismus, a high arched palate and mild retrognathia. Neuroimaging studies (CT) revealed coronary craniosynostosis. No heart defects were observed. Abdominal ultrasonography showed a mild abnormality of the renal parenchyma, but the patient’s kidney function is normal. Phytohemagglutinin (PHA)-stimulated lymphocytes were subjected to G-banding karyotyping (500 bands) of the propositus and progenitors. The results were described according to the recommendations of the ISCN (2008). Gbanding karyotype analysis of the patient showed a normal 46,XX pattern with no structural or numeric variations. The same was observed for both progenitors. For the aCGH experiments, DNA from the patient and progenitors was extracted from blood samples using a QIAamp DNA Blood Midi kit (Qiagen, Germany). Labeling and hybridization reactions were performed as recommended by the manufacturer (Perkin Elmer, Norwalk). aCGH was performed with Constitutional Chip 4.0 (Perkin Elmer, Norwalk), which includes 5000 BAC clones spotted in

INTRODUCTION Craniosynostosis is a disorder characterized by the premature fusion of the calvarial sutures, causing an abnormal skull shape. Because this disorder occurs with a relatively high frequency, estimated at 1 in 2500 individuals, craniosynostosis represents a relevant medical problem (1). More than 100 syndromes have been shown to be associated with this disorder. It is believed that at least 20% of cases are due to single gene mutations or chromosome abnormalities (2). Although most cases can be considered both clinically and genetically heterogeneous, there is evidence that six genes are involved in many cases: MSX2, FGFR1, FGFR2, FGFR3, FBN1, and TWISTY (1). The Msh homeobox 2 (Msx2) gene encodes a homeodomain transcription factor protein and is expressed in migrating cranial neural crest cells during development (3). Msx2 has central roles in craniofacial development (4) and limb and tissue formation (6). Furthermore, Msx2 overexpression was demonstrated to be associated with craniosynostosis in mice (5,7), indicating that normal craniofacial formation is dependent on the Msx2 dosage. Corroborating these findings, increases in the copy number in the MSX2 region have been reported in craniosynostosis patients (8-11). In the present study, we describe the findings from a whole-genome array comparative genomic hybridization (aCGH) analysis of a four-year-old patient exhibiting craniosynostosis, microcephaly, psychomotor development delay, short stature, and cognitive impairment, among other abnormalities. We found a gain in a region of chromosome 5q35.2 that contains MSX2 and has been shown to be associated with craniosynostosis. Quantitative PCR confirmed the increase in the MSX2 copy number.

CASE DESCRIPTION This study was approved by the Hospital das Clı´nicas Institutional Ethics Committee, and written informed consent

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.

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Table 1 - Clones that exhibited copy number variation detected through aCGH screening of the patient’s genome. Clones RP11-203P12 RP11-603O17 RP11-606P24 RP11-10J21 RP11-153P4 RP11-311J21

Status of alteration

Cytogenetic location

Loss Gain Gain Loss Loss Loss

4q22.1 5q35.2 5q35.2 8q24.3 9q34.2 9q34.3

Genomic coordinates (GRCh36/ hg18) 4:88,282,3685:173,984,9265:174,718,4448:142,215,0929:135,531,5669:136,982,786-

88,336,595 174,145,340 174,907,513 142,412,727 135,710,693 137,166,802

Genes present in the region KLHL8 MSX2 DRD1, SFXN1 DENND3, SLC45A4 SARDH, VAV2 OLFM1

Figure 1 - A spectral image of chromosome 5 from the patient revealing a gain in 5q35.2, which was detected by aCGH.

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MSX2 COPY number increase and craniosynostosis Pelegrino KO et al.

duplicate with a resolution of at least 650 kb and human DNA segments of 100,300 kb distributed through the whole genome. The slides were scanned with InnoScan 710 (Innopsys, Carbonne, France), and MAPIX 4.5 software was used to generate GPR files. Data analysis was conducted with SpectralWareH v2.3.3 aCGH Analysis System software (12) from Perkin Elmer. The computational parameters used were pin linear normalization, a threshold between 0.7 and 1.3, a Loess alpha of 0.1 for normalization and a confidence level of 95%. Median values were used for the interpretation of the results. The Database of Genomics Variants was consulted to determine the size of chromosome regions and to identify CNVs (13). These analyses revealed gains in two neighboring BAC clones on 5q32.2 and losses in 4q22.1, 8q24.3 and 9q34.2-34.3 (Table 1). A spectral image of chromosome 5 from the patient demonstrating the gain in 5q35.2 is presented in Figure 1. The patients revised karyotype was 46,XX.arr 4q22.1(88,282,368-88,336,595)x1, 5q35.2(173,984,926-174,145,340)(174,718,444-174,907,513)x5, 8q24.3(142,215,092-142,412,727)x1, 9q34.2q34.3(136,982,786137,166,802)x1[hg18]. DNA samples from the patient and progenitors were used for MSX2 gene copy number assessment. A TaqMan copy number assay (Applied Biosystems, Foster City, CA) consisting of an MGB probe labeled with FAM and unlabeled PCR primers for MSX2 (assay Hs01821094_cn) was employed. Unlabeled primers and a VIC dye-labeled TAMRA probe for RNase P gene, which is known to be present in two copies in human diploid genome, were used for normalization.

Reactions were performed as recommended by the manufacturer in triplicate. Duplex real-time polymerase chain reactions were conducted in a 7500 Real-Time PCR System, and the data were analyzed with CopyCallerTM Software v1.0, both from Applied Biosystems. Cycle thresholds (CT) were calculated using the relative quantification method (Applied Biosystems). Copy numbers were calculated by determining the difference in the CT between the target and control probes (DCT). Cycle thresholds greater than 32 were excluded from analyses. A quantitative PCR assay to detect MSX2 CNVs revealed two copies for both progenitors and five copies for the patient (Figure 2). Phenotypically normal subjects unrelated to the studied family also had two copies. The mean delta Ct, calculated as the FAM Ct divided by the VIC Ct, and the standard deviation of the delta Ct were 0.2192¡0.13 for Subject 1, 0.5772¡0.29 for Subject 2, 0.5807¡0.1 for the patient’s mother, -0.8858¡0.12 for the patient, 0.3630¡0.16 for Subject 3 and 0.7518¡0.12 for the patient’s father.

DISCUSSION A search for similar abnormalities in DECIPHER (Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources) (14) returned three cases with chromosome 4 alterations related to that observed in the present study (cases 753, 994, and 249573). The phenotypes in these cases included mental retardation/developmental delay, short stature, atrial septum defects, hypotonia and eyelid defects, among other abnormalities. However, it is important to stress that no correlation between a loss in the

Figure 2 - Assessment of MSX2 copy number variation. The estimated copy number variations for the patient, the progenitors, and three unrelated subjects are presented. The bars represent the variation in the calculated copy number. The values were ¡0.41 for Subject 1, ¡0.63 for Subject 2, ¡0.23 for the patient’s mother, ¡0.79 for the patient, ¡0.45 for Subject 3 and ¡0.30 for the patient’s father.

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additional data on Decipher patient 994, Dr. Anne Philippe from Universite´ Rene´ Descartes for data on Decipher patient 753 and Dr. Koen Devriendt for data on patient 249573. This study was supported by Fleury Group.

4q22.1 region and craniosynostosis has been described. In addition, no patient with case data reported in DECIPHER who had alterations in 8q24.3 or 9q34.2-34.3 similar to those in our patient exhibited craniosynostosis. In a detailed study that investigated common CNVs in Asian population, it was found that the KLHL8 (4q22.1), VAV2 (9q34.2) and OLFM1 (9q34.3) genes exhibited copy number variations in phenotypically normal subjects (15), indicating that copy number losses in these genes most likely do not contribute to the craniosynostosis phenotype. Corroborating these findings, a search of the Online Mendelian Inheritance in Man (OMIM) database (16) returned no correlations between the genes KLHL8, VAV2, DRD1, SFXN1, DENND3, SLC45A4 and OLFM1 and clinical phenotypes. The SARDH gene, located in the 9q34.2 region, was shown to be associated with sarcosinemia, which is generally considered a benign condition unrelated to neurologic symptoms or significant clinical problems (17). Among the CNVs found in the present work, the gain in the 5q35.2 chromosome region where the MSX2 gene is located has already been reported to be associated with craniosynostosis (8); however, the MSX2 gain was confirmed using molecular techniques in only four cases (8-11). MSX2 is a member of the homeobox MSX family and is related to Drosophila muscle segment homeobox (msh). MSX2 has important roles in tissue and organ development (3) and is also expressed in several regions of the developing skull. In mice, Msx2 is strongly expressed in osteogenic cells from calvarial sutures, and Msx2 activation is thought to initiate the release of factors from the dura mater that affect osteoblastic suture cells (18,19). Consistent with this role of Msx2, transgenic mice that over expressed this gene exhibited premature suture closure and craniofacial abnormalities (5), whereas other evidence suggests that MSX2 haploinsufficiency leads to a delayed or incomplete closure of the opening between the frontal and parietal bones. MSX2 haploinsufficiency has been demonstrated to be responsible for the majority of foramina parietalia permagna (FPP) cases (20-22). There are 39 reported cases of 5q distal trisomy, but craniosynostosis was observed in only ten of these cases. Because most craniosynostosis patients exhibit a distal 5q duplication, it is hypothesized that in patients with larger duplications, MSX2 expression may be inhibited due to extra copies of other genes (8). Additional cases may help to elucidate the validity of this hypothesis. In conclusion, our analysis indicates that an increase in MSX2 copy number is correlated with this disorder, corroborating previous findings that subjects with gains in 5q35.2 in the MSX2 gene region exhibit craniosynostosis. In addition, the aCGH technique was shown to be useful for the detection of CNVs throughout the whole genome. Further studies should now be undertaken to determine which of these CNVs are normal individual variations and which are of clinical relevance, e.g., associated with specific syndromes.

AUTHOR CONTRIBUTIONS Pelegrino KO performed the aCGH experiments, the qPCR assays and the data analysis and interpretation and participated in the preparation and revision of the manuscript. Sugayama S was responsible for the patient examination and clinical description and participated in the preparation of the manuscript. Lezirovitz K performed the DNA extraction and participated in the aCGH experiments. Catelani AL performed the karyotype analysis. Kok F participated in the writing of the discussion section and in the revision of the manuscript. Chauffaille ML coordinated the study, designed the project, was responsible for fund obtaining, contributed to the data interpretation, manuscript writing and review.

REFERENCES 1. Wilkie AO. Craniosynostosis: genes and mechanisms. Hum Mol Genet. 1997;6(10):1647-56, http://dx.doi.org/10.1093/hmg/6.10.1647. 2. Johnson D and Wilkie AO. Craniosynostosis. Europ J Hum Genet. 2011;19(4):369-76, http://dx.doi.org/10.1038/ejhg.2010.235. 3. Finnerty JR, Mazza ME, Jezewski PA. Domain duplication, divergence, and loss events in Msx paralogs reveal phylogenomically diseases markers. BMC Evolutionary Biology. 2009;9-18. 4. Davidson D. The function and evolution of Msx genes: pointers and paradoxes. Trends Genet. 1995;11(10):405-11, http://dx.doi.org/ 10.1016/S0168-9525(00)89124-6. 5. Liu YH, Kundu R, Wu L, Luo W, Ignelzi MA Jr, Snead ML, et al. Premature suture closure and ectopic cranial bone in mice expressing Msx2 transgenes in the developing skull. Proc Natl Acad Sci USA. 1995;92(13):6137-41, http://dx.doi.org/10.1073/pnas.92.13.6137. 6. Satokata I, Ma L, Ohshima H, Bei M, Woo I, Nishizawa K, et al. Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation. Nat Genet. 2000;24(4):391-5. 7. Winograd J, Reilly MP, Roe R, Lutz J, Laughner E, Xu X, et al. Perinatal lethality and multiple craniofacial malformations in MSX2 transgenic mice. Hum Mol Genet. 1997;6(3):369-79, http://dx.doi.org/10.1093/hmg/6.3.369. 8. Kariminejad A, Kariminejad R, Tzschach A, Ullmann R, Ahmed A, Asghari-Roodsari A, et al. Craniosynostosis in a patient with 2q37.3 deletion 5q34 duplication: Association of extra copy of MSX2 with craniosynostosis. Am J Med Genet. 2009;149A(7):1544-9, http:// dx.doi.org/10.1002/ajmg.a.32949. 9. Shiihara T, Kato M, Kimura T, Hayasaka K, Yamamori S, Ogata T. Craniosynostosis with extra copy of MSX2 in a patient with partial 5qtrisomy. (Letter) Am J Med Genet. 2004;128A(2):214-6, http:// dx.doi.org/10.1002/ajmg.a.20552. 10. Bernardini L, Castori M, Capalbo A, Mokini V, Mingarelli R, Simi P, et al. Syndromic craniosynostosis due to complex chromosome 5 rearrangement and MSX2 gene triplication. Am J Med Genet Part A. 2007; 143A(24):2937-43. 11. Wang JC, Steinraths M, Dang L, Lomax B, Eydoux P, Stockley T, et al. Craniosynostosis associated with distal 5q-trisomy: Further evidence that extra copy of MSX2 gene leads to craniosynostosis. 2007;143A(24):2931-6. 12. SpectralWareH v2.3.3 aCGH Analysis System software, available at http://service.spectralgenomics.com. 13. Database of Genomics Variants, available at http://projects.tcag.ca/ variation/. 14. Park H, Kim JI, Ju YS, Gokcumen O, Mills RE, Kim S, et al. Discovery of common Asian copy number variants using integrated high-resolution array CGH and massively parallel DNA sequencing. Nat Genet. 2010;42(5):400-5, http://dx.doi.org/10.1038/ng.555. 15. Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources, available at http://decipher.sanger.ac.uk. 16. Online Mendelian Inheritance in Man (OMIM), available at http:// omim.org/. 17. Scott CR. Sarcosinemia. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds.): The Metabolic and Molecular Bases of Inherited Disease. Vol. II New York: McGraw-Hill (pub.) (8th ed.). 2001;2057-63. 18. Jabs EW, Muller U, Li X, Ma L, Luo W, Haworth IS, et al. A mutation in the homeodomain of the human MSX2 gene in a family affected with autosomal dominant craniosynostosis. Cell. 1993;75(3):443-50, http:// dx.doi.org/10.1016/0092-8674(93)90379-5. 19. Ma L, Golden S, Wu L, Maxson R. The molecular basis of Boston-type craniosynostosis: the Pro148-His mutation in the N-terminal arm of the MSX2 homeodomain stabilizes DNA binding without altering nucleotide sequence preferences. Hum Mol Genet. 1996;5(12):1915-20, http:// dx.doi.org/10.1093/hmg/5.12.1915.

ACKNOWLEDGMENTS We would like to thank the patient and her parents for their participation in our research. This study used data generated by the DECIPHER Consortium. A full list of the centers that contributed to the generation of the data is available from http://decipher.sanger.ac.uk and via e-mail from: decipher@sanger.ac.uk. We would specifically like to thank Dr. J. M. Friedman and Dr. McGillivray from the University of British Columbia for

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MSX2 COPY number increase and craniosynostosis Pelegrino KO et al. causes defects in skull ossification. Nat Genet. 2000;24(4):387-90, http:// dx.doi.org/10.1038/74224. 22. Mavrogiannis LA, Taylor IB, Davies SJ, Ramos FJ, Olivares JL, Wilkie AO. Enlarged parietal foramina caused by mutations in the homeobox genes ALX4 and MSX2: from genotype to phenotype. Europ J Hum Genet. 2006;14(2):151-8, http://dx.doi.org/10.1038/sj.ejhg.5201526.

20. Wuyts W, Reardon W, Preis S, Homfrey T, Rasore-Quartino A, Christians H, et al. Identification of mutations in the MSX2 homeobox gene in families affected with foramina parietalia permagna. Hum. Mol. Genet. 2000;9(8):1251-5, http://dx.doi.org/10.1093/hmg/9.8.1251. 21. Wilkie AO, Tang Z, Elanko N, Walsh S, Twigg SR, Hurst JA, et al. Functional haploinsufficiency of the human homeobox gene MSX2

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