Gastroenterology

ISSN 1007-9327 CN 14-1219/R

Gastroenterology

®

Volume 13 Number 32 August 28, 2007

World Journal of Gastroenterology

World Journal of

www.wjgnet.com Aug 28

Editorial Department of World Journal of Gastroenterology 77 Shuangta Xijie, Taiyuan 030001, Shanxi Province, China Telephone: +86-351-4078656 E-mail: [email protected] http://www.wjgnet.com

Number 32

Baishideng

Volume 13

National Journal Award 2005

2007

ISSN 1007-9327 CN 14-1219/R Local Post Offices Code No. 82-261

World Journal of

Gastroenterology Indexed and Abstracted in: Current Contents®/Clinical Medicine, Science Citation Index Expanded (also known as SciSearch®) and Journal Citation Reports/Science Edition, Index Medicus, MEDLINE and PubMed, Chemical Abstracts, EMBASE/Excerpta Medica, Abstracts Journals, Nature Clinical Practice Gastroenterology and Hepatology, CAB Abstracts and Global Health. ISI JCR 2003-2000 IF: 3.318, 2.532, 1.445 and 0.993.

Volume 13 Number 32 August 28, 2007

World J Gastroenterol 2007 August 28; 13(32): 4287-4412 Online Submissions wjg.wjgnet.com www.wjgnet.com Printed on Acid-free Paper

A Weekly Journal of Gastroenterology and Hepatology

World Journal of

Gastroenterology Editorial Board 2007-2009

Baishideng http://www.wjgnet.com E-mail: [email protected] HONORARY EDITORS-IN-CHIEF Ke-Ji Chen, Beijing Li-Fang Chou, Taipei Zhi-Qiang Huang, Beijing Shinn-Jang Hwang, Taipei Min-Liang Kuo, Taipei Nicholas F LaRusso, Rochester Jie-Shou Li, Nanjing Geng-Tao Liu, Beijing Lein-Ray Mo, Tainan Bo-Rong Pan, Xi'an Fa-Zu Qiu, Wuhan Eamonn M Quigley, Cork David S Rampton, London Rudi Schmid, Kentfield Nicholas J Talley, Rochester Guido NJ Tytgat, Amsterdam H-P Wang, Taipei Jaw-Ching Wu, Taipei Meng-Chao Wu, Shanghai Ming-Shiang Wu, Taipei Jia-Yu Xu, Shanghai Ta-Sen Yeh, Taoyuan EDITOR-IN-CHIEF Lian-Sheng Ma, Taiyuan ASSOCIATE EDITORS-IN-CHIEF Gianfranco D Alpini, Temple Bruno Annibale, Roma Roger William Chapman, Oxford Chi-Hin Cho, Hong Kong Alexander L Gerbes, Munich Shou-Dong Lee, Taipei Walter Edwin Longo, New Haven You-Yong Lu, Beijing Masao Omata, Tokyo Harry HX Xia, Hanover MEMBERS OF THE EDITORIAL BOARD Albania Bashkim Resuli, Tirana

Argentina Julio Horacio Carri, Córdoba Adriana M Torres, Rosario Australia Minoti Vivek Apte, Liverpool Richard B Banati, Lidcombe Michael R Beard, Adelaide Patrick Bertolino, Sydney Filip Braet, Sydney Andrew D Clouston, Sydney Darrell HG Crawford, Brisbane Guy D Eslick, Sydney Michael Anthony Fink, Melbourne Robert JL Fraser, Daw Park Mark D Gorrell, Sydney Yik-Hong Ho, Townsville Gerald J Holtmann, Adelaide Michael Horowitz, Adelaide John E Kellow, Sydney Daniel Markovich, Brisbane Phillip S Oates, Perth Stephen M Riordan, Sydney IC Roberts-Thomson, Adelaide Arthur Shulkes, Melbourne Ross C Smith, Sydney Kevin John Spring, Brisbane Nathan Subramaniam, Brisbane Herbert Tilg, Innsbruck Martin John Veysey, Gosford DL Worthley, Bedford Austria Valentin Fuhrmann, Vienna Alfred Gangl, Vienna Christoph Gasche, Vienna Kurt Lenz, Linz M Peck-Radosavljevic, Vienna RE Stauber, Auenbruggerplatz Michael Trauner, Graz Harald Vogelsang, Vienna Guenter Weiss, Innsbruck Belarus Yury K Marakhouski, Minsk

www.wjgnet.com

Belgium Rudi Beyaert, Gent Bart Rik De Geest, Leuven Inge Irma Depoortere, Leuven Olivier Detry, Liège BY De Winter, Antwerp Karel Geboes, Leuven Thierry Gustot, Brussels Yves J Horsmans, Brussels Geert G Leroux-Roels, Ghent Louis Libbrecht, Leuven Etienne M Sokal, Brussels Marc Peeters, De Pintelaan Gert A Van Assche, Leuven Yvan Vandenplas, Brussels Eddie Wisse, Keerbergen Brazil Heitor Rosa, Goiania Bulgaria Zahariy Krastev, Sofia Canada Fernando Alvarez, Québec David Armstrong, Ontario Olivier Barbier, Québec Nancy Baxter, Toronto Matthew Bjerknes, Toronto Frank J Burczynski, Winnipeg Michael F Byrne, Vancouver Wang-Xue Chen, Ottawa Hugh J Freeman, Vancouver Chantal Guillemette, Québec Samuel S Lee, Calgary Gary A Levy, Toronto Andrew Lawrence Mason, Alberta John K Marshall, Ontario Donna-Marie McCafferty, Calgary Thomas I Michalak, St. John's Gerald Y Minuk, Manitoba Paul Moayyedi, Hamilton Eldon Shaffer, Calgary Morris Sherman, Toronto Alan BR Thomson, Edmonton EF Verdu, Ontario

I

John L Wallace, Calgary Eric M Yoshida, Vancouver Chile Silvana Zanlungo, Santiago China Henry LY Chan, Hongkong Xiao-Ping Chen, Wuhan Zong-Jie Cui, Beijing Da-Jun Deng, Beijing Er-Dan Dong, Beijing Sheung-Tat Fan, Hong Kong Jin Gu, Beijing De-Wu Han, Taiyuan Ming-Liang He, Hong Kong Wayne HC Hu, Hong Kong Chee-Kin Hui, Hong Kong Ching Lung Lai, Hong Kong Kam Chuen Lai, Hong Kong James YW Lau, Hong Kong Yuk Tong Lee, Hong Kong Suet Yi Leung, Hong Kong Wai-Keung Leung, Hong Kong Chung-Mau Lo, Hong Kong Jing-Yun Ma, Beijing Lun-Xiu Qin, Shanghai Yu-Gang Song, Guangzhou Qin Su, Beijing Wai-Man Wong, Hong Kong Hong Xiao, Beijing Dong-Liang Yang, Wuhan Winnie Yeo, Hong Kong Yuan Yuan, Shenyang Man-Fung Yuen, Hong Kong Jian-Zhong Zhang, Beijing Xin-Xin Zhang, Shanghai Shu Zheng, Hangzhou Croatia Tamara Cacev, Zagreb Marko Duvnjak, Zagreb Cuba Damian Casadesus Rodriguez, Havana Czech Milan Jirsa, Praha Denmark Peter Bytzer, Copenhagen Hans Gregersen, Aalborg Jens H Henriksen, Hvidovre Claus Peter Hovendal, Odense Fin Stolze Larsen, Copenhagen SØren MØller, Hvidovre Egypt Abdel-Rahman El-Zayadi, Giza Amr Mohamed Helmy, Cairo Sanaa Moharram Kamal, Cairo Ayman Yosry, Cairo Finland Irma Elisabet Jarvela, Helsinki Katri Maria Kaukinen, Tampere Minna Nyström, Helsinki Pentti Sipponen, Espoo France Bettaieb Ali, Dijon Corlu Anne, Rennes Denis Ardid, Clermont-Ferrand Charles Paul Balabaud, Bordeaux Soumeya Bekri, Rouen Jacques Belghiti, Clichy

Ⅱ

Pierre Brissot, Rennes Patrice Philippe Cacoub, Paris Franck Carbonnel, Besancon Laurent Castera, Pessac Bruno Clément, Rennes Jacques Cosnes, Paris Thomas Decaens, Cedex Francoise Lunel Fabiani, Angers Gérard Feldmann, Paris Jean Fioramonti, Toulouse Catherine Guettier, Villejuif Chantal Housset, Paris Juan Lucio Iovanna, Marseille Rene Lambert, Lyon Philippe Mathurin, Lille Tamara Matysiak–Budnik, Paris Francis Mégraud, Bordeaux Richard Moreau, Clichy Thierry Piche, Nice Raoul Poupon, Paris Jean Rosenbaum, Bordeaux Jose Sahel, Marseille Jean-Philippe Salier, Rouen Jean-Yves Scoazec, Lyon Khalid Ahnini Tazi, Clichy Emmanuel Tiret, Paris Baumert F Thomas, Strasbourg MC Vozenin-brotons, Villejuif Jean-Pierre Henri Zarski, Grenoble Jessica Zucman-Rossi, Paris Germany HD Allescher, Garmisch-Partenkirchen Martin Anlauf, Kiel Rudolf Arnold, Marburg Max G Bachem, Ulm Thomas F Baumert, Freiburg Daniel C Baumgart, Berlin Hubert Blum, Freiburg Thomas Bock, Tuebingen Katja Breitkopf, Mannheim Dunja Bruder, Braunschweig Markus W Büchler, Heidelberg Christa Buechler, Regensburg Reinhard Buettner, Bonn Elke Cario, Essen CF Dietrich, Bad Mergentheim Rainer Josef Duchmann, Berlin Paul Enck, Tuebingen Fred Fändrich, Kiel Ulrich Robert Fölsch, Kiel Helmut Friess, Heidelberg Peter R Galle, Mainz Nikolaus Gassler, Aachen Andreas Geier, Aachen Dieter Glebe, Giessen Burkhard Göke, Munich Florian Graepler, Tuebingen Axel M Gressner, Aachen Veit Gülberg, Munich Rainer Haas, Munich Eckhart Georg Hahn, Erlangen Stephan Hellmig, Kiel Martin Hennenberg, Bonn Johannes Herkel, Hamburg Klaus Herrlinger, Stuttgart Eberhard Hildt, Berlin Joerg C Hoffmann, Berlin Ferdinand Hofstaedter, Regensburg Werner Hohenberger, Erlangen RG Jakobs, Ludwigshafen Jutta Keller, Hamburg Andrej Khandoga, Munich Sibylle Koletzko, München Stefan Kubicka, Hannover Joachim Labenz, Siegen Frank Lammert, Bonn Thomas Langmann, Regensburg Christian Liedtke, Aachen Matthias Löhr, Mannheim Christian Maaser, Muenster Ahmed Madisch, Dresden www.wjgnet.com

Michael Peter Manns, Hannover Stephan Miehlke, Dresden Sabine Mihm, Göttingen Silvio Nadalin, Essen Markus F Neurath, Mainz Johann Ockenga, Berlin Florian Obermeier, Regensburg Gustav Paumgartner, Munich Ulrich Ks Peitz, Magdeburg Markus Reiser, Bochum Steffen Rickes, Magdeburg Gerhard Rogler, Regensburg Tilman Sauerbruch, Bonn Dieter Saur, Munich Hans Scherubl, Berlin Joerg Schirra, Munich Roland M Schmid, München Volker Schmitz, Bonn AG Schreyer, Regensburg Tobias Schroeder, Essen Hans Seifert, Oldenburg Manfred V Singer, Mannheim Gisela Sparmann, Rostock Jurgen M Stein, Frankfurt Ulrike Susanne Stein, Berlin Manfred Stolte, Bayreuth Christian P Strassburg, Hannover WR Stremmel, Heidelberg Harald F Teutsch, Ulm Robert Thimme, Freiburg HL Tillmann, Leipzig Tung-Yu Tsui, Regensburg Axel Ulsenheimer, Munich Patrick Veit, Essen Claudia Veltkamp, Heidelberg Siegfried Wagner, Deggendorf Henning Walczak, Heidelberg Fritz von Weizsacker, Berlin Jens Werner, Heidelberg Bertram Wiedenmann, Berlin Reiner Wiest, Regensburg Stefan Wirth, Wuppertal Stefan JP Zeuzem, Homburg Greece Elias A Kouroumalis, Heraklion Ioannis E Koutroubakis, Heraklion Spiros Sgouros, Athens Hungary Peter Laszlo Lakatos, Budapest Zsuzsa Szondy, Debrecen Iceland H Gudjonsson, Reykjavik India KA Balasubramanian, Vellore Sujit K Bhattacharya, Kolkata Yogesh K Chawla, Chandigarh Radha K Dhiman, Chandigarh Sri Prakash Misra, Allahabad ND Reddy, Hyderabad Iran Seyed-Moayed Alavian, Tehran Reza Malekzadeh, Tehran Seyed Alireza Taghavi, Shiraz Ireland Billy Bourke, Dublin Ronan A Cahill, Cork Anthony P Moran, Galway Israel Simon Bar-Meir, Hashomer Abraham Rami Eliakim, Haifa

Yaron Ilan, Jerusalem Avidan U Neumann, Ramat-Gan Yaron Niv, Pardesia Ran Oren, Tel Aviv Italy Giovanni Addolorato, Roma Luigi E Adinolfi, Naples Domenico Alvaro, Rome V Annese, San Giovanni Rotond Adolfo Francesco Attili, Roma Giovanni Barbara, Bologna Gabrio Bassotti, Perugia Pier Maria Battezzati, Milan Stefano Bellentani, Carpi Antomio Benedetti, Ancona Mauro Bernardi, Bologna Livia Biancone, Rome Luigi Bonavina, Milano Flavia Bortolotti, Padova Giuseppe Brisinda, Rome Giovanni Cammarota, Roma Antonino Cavallari, Bologna Giuseppe Chiarioni, Valeggio Michele Cicala, Rome Amedeo Columbano, Cagliari Massimo Conio, Sanremo Dario Conte, Milano Gino Roberto Corazza, Pavia Francesco Costa, Pisa Antonio Craxi, Palermo Silvio Danese, Milan Roberto De Giorgio, Bologna Giovanni D De Palma, Naples Fabio Farinati, Padua Giammarco Fava, Ancona Francesco Feo, Sassari Stefano Fiorucci, Perugia Andrea Galli, Firenze Valeria Ghisett, Turin Gianluigi Giannelli, Bari Edoardo G Giannini, Genoa Paolo Gionchetti, Bologna Mario Guslandi, Milano Pietro Invernizzi, Milan Giacomo Laffi, Firenze Giovanni Maconi, Milan Lucia Malaguarnera, Catania ED Mangoni, Napoli Giulio Marchesini, Bologna Fabio Marra, Florence Marco Marzioni, Ancona Giuseppe Montalto, Palermo Giovanni Monteleone, Rome Giovanni Musso, Torino Gerardo Nardone, Napoli Valerio Nobili, Rome Luisi Pagliaro, Palermo Francesco Pallone, Rome Fabrizio R Parente, Milan F Perri, San Giovanni Rotondo Raffaele Pezzilli, Bologna A Pilotto, San Giovanni Rotondo Mario Pirisi, Novara Paolo Del Poggio, Treviglio Gabriele Bianchi Porro, Milano Piero Portincasa, Bari Bernardino Rampone, Siena Claudio Romano, Messina Marco Romano, Napoli Gerardo Rosati, Potenza Enrico Roda, Bologna Domenico Sansonno, Bari Vincenzo Savarino, Genova Mario Del Tacca, Pisa Giovanni Tarantino, Naples Roberto Testa, Genoa Pier Alberto Testoni, Milan

Dino Vaira, Bologna Japan Kyoichi Adachi, Izumo Yasushi Adachi, Sapporo Taiji Akamatsu, Matsumoto Sk Md Fazle Akbar, Ehime Takafumi Ando, Nagoya Akira Andoh, Otsu Taku Aoki, Tokyo Masahiro Arai, Tokyo Tetsuo Arakawa, Osaka Yasuji Arase, Tokyo Masahiro Asaka, Sapporo Hitoshi Asakura, Tokyo Takeshi Azuma, Fukui Yoichi Chida, Fukuoka Takahiro Fujimori, Tochigi Jiro Fujimoto, Hyogo Kazuma Fujimoto, Saga Mitsuhiro Fujishiro, Tokyo Yoshihide Fujiyama, Otsu Hirokazu Fukui, Tochigi Hiroyuki Hanai, Hamamatsu Kazuhiro Hanazaki, Kochi Naohiko Harada, Fukuoka Makoto Hashizume, Fukuoka Tetsuo Hayakawa, Nagoya Kazuhide Higuchi, Osaka Keisuke Hino, Ube Keiji Hirata, Kitakyushu Yuji Iimuro, Nishinomiya Kenji Ikeda, Tokyo Fumio Imazeki, Chiba Yutaka Inagaki, Kanagawa Yasuhiro Inokuchi, Yokohama Haruhiro Inoue, Yokohama Masayasu Inoue, Osaka Akio Inui, Kagoshima Hiromi Ishibashi, Nagasaki Shunji Ishihara, Izumo Toru Ishikawa, Niigata Kei Ito, Sendai Masayoshi Ito, Tokyo Hiroaki Itoh, Akita Ryuichi Iwakiri, Saga Yoshiaki Iwasaki, Okayama Terumi Kamisawa, Tokyo Hiroshi Kaneko, Aichi-Gun Shuichi Kaneko, Kanazawa Takashi Kanematsu, Nagasaki Mitsuo Katano, Fukuoka Junji Kato, Sapporo Mototsugu Kato, Sapporo Shinzo Kato, Tokyo Norifumi Kawada, Osaka Sunao Kawano, Osaka Mitsuhiro Kida, Kanagawa Yoshikazu Kinoshita, Izumo Tsuneo Kitamura, Chiba Seigo Kitano, Oita Kazuhiko Koike, Tokyo Norihiro Kokudo, Tokyo Satoshi Kondo, Sapporo Shoji Kubo, Osaka Masato Kusunoki, Tsu Mie Katsunori Iijima, Sendai Shin Maeda, Tokyo Masatoshi Makuuchi, Tokyo Osamu Matsui, Kanazawa Yasuhiro Matsumura, Chiba Yasushi Matsuzaki, Tsukuba Kiyoshi Migita, Omura Tetsuya Mine, Kanagawa Hiroto Miwa, Hyogo Masashi Mizokami, Nagoya Yoshiaki Mizuguchi, Tokyo Motowo Mizuno, Hiroshima www.wjgnet.com

Morito Monden, Suita Hisataka S Moriwaki, Gifu Yasuaki Motomura, Iizuka Yoshiharu Motoo, Kanazawa Kazunari Murakami, Oita Kunihiko Murase, Tusima Masahito Nagaki, Gifu Masaki Nagaya, Kawasaki Yuji Naito, Kyoto Hisato Nakajima, Tokyo Hiroki Nakamura, Yamaguchi Shotaro Nakamura, Fukuoka Mikio Nishioka, Niihama Shuji Nomoto, Nagoya Susumu Ohmada, Maebashi Masayuki Ohta, Oita Tetsuo Ohta, Kanazawa Kazuichi Okazaki, Osaka Katsuhisa Omagari, Nagasaki Saburo Onishi, Nankoku Morikazu Onji, Ehime Satoshi Osawa, Hamamatsu Masanobu Oshima, Kanazawa Hiromitsu Saisho, Chiba Hidetsugu Saito, Tokyo Yutaka Saito, Tokyo Isao Sakaida, Yamaguchi Michiie Sakamoto, Tokyo Yasushi Sano, Chiba Hiroki Sasaki, Tokyo Iwao Sasaki, Sendai Motoko Sasaki, Kanazawa Chifumi Sato, Tokyo Shuichi Seki, Osaka Hiroshi Shimada, Yokohama Mitsuo Shimada, Tokushima Tomohiko Shimatan, Hiroshima Hiroaki Shimizu, Chiba Ichiro Shimizu, Tokushima Yukihiro Shimizu, Kyoto Shinji Shimoda, Fukuoka Tooru Shimosegawa, Sendai Tadashi Shimoyama, Hirosaki Ken Shirabe, Iizuka Yoshio Shirai, Niigata Katsuya Shiraki, Mie Yasushi Shiratori, Okayama Masayuki Sho, Nara Yasuhiko Sugawara, Tokyo Hidekazu Suzuki, Tokyo Minoru Tada, Tokyo Tadatoshi Takayama, Tokyo Tadashi Takeda, Osaka Koji Takeuchi, Kyoto Kiichi Tamada, Tochigi Akira Tanaka, Kyoto Eiji Tanaka, Matsumoto Noriaki Tanaka, Okayama Shinji Tanaka, Hiroshima Wei Tang, Tokyo Hideki Taniguchi, Yokohama Kyuichi Tanikawa, Kurume Akira Terano, Shimotsugagun Hitoshi Togash, Yamagata Kazunari Tominaga, Osaka Takuji Torimura, Fukuoka Minoru Toyota, Sapporo Akihito Tsubota, Chiba Shingo Tsuji, Osaka Takato Ueno, Kurume Shinichi Wada, Tochigi Hiroyuki Watanabe, Kanazawa Toshio Watanabe, Osaka Yuji Watanabe, Ehime Chun-Yang Wen, Nagasaki Koji Yamaguchi, Fukuoka Takayuki Yamamoto, Yokkaichi Takashi Yao, Fukuoka

Ⅲ

Masashi Yoneda, Tochigi Hiroshi Yoshida, Tokyo Masashi Yoshida, Tokyo Norimasa Yoshida, Kyoto Kentaro Yoshika, Toyoake Masahide Yoshikawa, Kashihara Lebanon Bassam N Abboud, Beirut Ala I Sharara, Beirut Joseph Daoud Boujaoude, Beirut Lithuania Limas Kupcinskas, Kaunas Macedonia Vladimir Cirko Serafimoski, Skopje Malaysia Andrew Seng Boon Chua, Ipoh Khean-Lee Goh, Kuala Lumpur Jayaram Menon, Sabah Mexico Garcia-Compean Diego, Monterrey E R Marin-Lopez, Jesús García Saúl Villa-Treviño, México JK Yamamoto-Furusho, México Monaco Patrick Rampal, Monaco Netherlands Ulrich Beuers, Amsterdam Gerd Bouma, Amsterdam Lee Bouwman, Leiden J Bart A Crusius, Amsterdam Janine K Kruit, Groningen Ernst Johan Kuipers, Rotterdam Ton Lisman, Utrecht Yi Liu, Amsterdam Servaas Morré, Amsterdam Chris JJ Mulder, Amsterdam Michael Müller, Wageningen Amado Salvador Peña, Amsterdam Robert J Porte, Groningen Ingrid B Renes, Rotterdam Andreas Smout, Utrecht RW Stockbrugger, Maastricht Luc JW van der Laan, Rotterdam Karel van Erpecum, Utrecht GP VanBerge-Henegouwen,Utrecht New Zealand Ian David Wallace, Auckland Nigeria Samuel Babafemi Olaleye, Ibadan Norway Trond Berg, Oslo Tom Hemming Karlsen, Oslo Helge Lyder Waldum, Trondheim Pakistan Muhammad S Khokhar, Lahore Poland Tomasz Brzozowski, Cracow Robert Flisiak, Bialystok Hanna Gregorek, Warsaw DM Lebensztejn, Bialystok Wojciech G Polak, Wroclaw Marek Hartleb, Katowice

IV

Portugal MP Cecília, Lisbon Miguel Carneiro De Moura, Lisbon Russia Vladimir T Ivashkin, Moscow Leonid Lazebnik, Moscow Vasiliy I Reshetnyak, Moscow Saudi Arabia Ibrahim Abdulkarim Al Mofleh, Riyadh Serbia DM Jovanovic, Sremska Kamenica Singapore Bow Ho, Kent Ridge Khek-Yu Ho, Singapore Francis Seow-Choen, Singapore Slovakia Anton Vavrecka, Bratislava Slovenia Sasa Markovic, Ljubljana South Africa Michael C Kew, Parktown South Korea Byung Ihn Choi, Seoul Ho Soon Choi, Seoul M Yeo, Suwon Sun Pyo Hong, Gyeonggi-do Jae J Kim, Seoul Jin-Hong Kim, Suwon Myung-Hwan Kim, Seoul Chang Hong Lee, Seoul Jong Kyun Lee, Seoul Eun-Yi Moon, Seoul Jae-Gahb Park, Seoul Dong Wan Seo, Seoul Dong jin Suh, Seoul Spain Juan G Abraldes, Barcelona Agustin Albillos, Madrid Raul J Andrade, Málaga Luis Aparisi, Valencia Fernando Azpiroz, Barcelona Ramon Bataller, Barcelona Josep M Bordas, Barcelona Xavier Calvet, Sabadell Andres Cardenas, Barcelona Vicente Carreño, Madrid Jose Castellote, Barcelona Antoni Castells, Barcelona Vicente Felipo, Valencia Juan C Garcia-Pagán, Barcelona Jaime Bosch Genover, Barcelona Jaime Guardia, Barcelona Angel Lanas, Zaragoza María Isabel Torres López, Jaén José M Mato, Derio Juan F Medina, Pamplona MA Muñoz-Navas, Pamplona Julian Panes, Barcelona Miguel Minguez Perez, Valencia Miguel Perez-Mateo, Alicante Josep M Pique, Barcelona Jesús M Prieto, Pamplona Sabino Riestra, Pola De Siero Luis Rodrigo, Oviedo Manuel Romero-Gómez, Sevilla Sweden Einar Stefan Björnsson, Gothenburg Curt Einarsson, Huddinge www.wjgnet.com

Ulf Hindorf, Lund Hanns-Ulrich Marschall, Stockholm Lars Christer Olbe, Molndal Matti Sallberg, Stockholm Magnus Simrén, Göteborg Xiao-Feng Sun, Linköping Ervin Tóth, Malmö Weimin Ye, Stockholm Switzerland Chrish Beglinger, Basel Pierre A Clavien, Zurich Jean-Francois Dufour, Bern Franco Fortunato, Zürich Jean Louis Frossard, Geneva Gerd A Kullak-Ublick, Zurich Pierre Michetti, Lausanne Francesco Negro, Genève Bruno Stieger, Zurich Radu Tutuian, Zurich Stephan Robert Vavricka, Zurich Arthur Zimmermann, Berne Turkey Yusuf Bayraktar, Ankara Figen Gurakan, Ankara Aydin Karabacakoglu, Konya Serdar Karakose, Konya Hizir Kurtel, Istanbu Osman Cavit Ozdogan, Istanbul Özlem Yilmaz, Izmir Cihan Yurdaydin, Ankara United Arab Emirates Sherif M Karam, Al-Ain United Kingdom David Adams, Birmingham NK Ahluwalia, Stockport CG Antoniades, London Anthony TR Axon, Leeds Qasim Aziz, Manchester Nicholas M Barnes, Birmingham Jim D Bell, London Mairi Brittan, London Alastair David Burt, Newcastle Simon Scott Campbell, Manchester Simon R Carding, Leeds Paul Jonathan Ciclitira, London Eithne Costello, Liverpool Tatjana Crnogorac-Jurcevic, London Amar Paul Dhillon, London Emad M El-Omar, Aberdeen Annette Fristscher-Ravens, London Elizabeth Furrie, Dundee Daniel Richard Gaya, Edinburgh Subrata Ghosh, London William Greenhalf, Liverpool Indra Neil Guha, Southampton Peter Clive Hayes, Edinburgh Gwo-Tzer Ho, Edinburgh Anthony R Hobson, Salford Stefan G Hübscher, Birmingham Robin Hughes, London Pali Hungin, Stockton David Paul Hurlstone, Sheffield Rajiv Jalan, London Janusz AZ Jankowski, Oxford Brian T Johnston, Belfast David EJ Jones, Newcastle Michael A Kamm, Harrow Peter Karayiannis, London Laurens Kruidenier, Harlow Patricia F Lalor, Birmingham Hong-Xiang Liu, Cambridge K E L McColl, Glasgow Stuart AC McDonald, London

Dermot Patrick Mcgovern, Oxford Giorgina Mieli-Vergani, London Nikolai V Naoumov, London John P Neoptolemos, Liverpool James Neuberger, Birmingham Mark S Pearce, Newcastle Upon Tyne Stephen P Pereira, London D Mark Pritchard, Liverpool Stephen E Roberts, Swansea Marco Senzolo, Padova Soraya Shirazi-Beechey, Liverpool Robert Sutton, Liverpool Simon D Taylor-Robinson, London Paris P Tekkis, London Ulrich Thalheimer, London Nick Paul Thompson, Newcastle David Tosh, Bath Frank Ivor Tovey, London Chris Tselepis, Birmingham Diego Vergani, London Geoffrey Warhurst, Salford Peter James Whorwell, Manchester Roger Williams, London Karen Leslie Wright, Bath Min Zhao, Foresterhill United States Gary A Abrams, Birmingham Golo Ahlenstiel, Bethesda BS Anand, Houston Frank A Anania, Atlanta M Ananthanarayanan, New York Gavin Edward Arteel, Louisville Jasmohan Singh Bajaj, Milwaukee Jamie S Barkin, Miami Beach Kim Elaine Barrett, San Diego Marc Basson, Detroit Wallace F Berman, Durham Timothy R Billiar, Pittsburgh Edmund J Bini, New York Jennifer D Black, Buffalo Herbert L Bonkovsky, Farmington Andrea D Branch, New York Robert S Bresalier, Houston Alan L Buchman, Chicago Alan Cahill, Philadelphia John M Carethers, San Diego David L Carr-Locke, Boston Ravi S Chari, Nashville Jiande Chen, Galveston Xian-Ming Chen, Omaha Ramsey Chi-man Cheung, Palo Alto William D Chey, Ann Arbor John Y Chiang, Rootstown Parimal Chowdhury, Arkansas Raymond T Chung, Boston James M Church, Cleveland Mark G Clemens, Charlotte Vincent Coghlan, Beaverton David Cronin II, New Haven John Cuppoletti, Cincinnati Mark James Czaja, New York Peter V Danenberg, Los Angeles Kiron Moy Das, New Brunswick Sharon DeMorrow, Temple Deborah L Diamond, Seattle Peter Draganov, Florida Bijan Eghtesad, Cleveland Hala El-Zimaity, Houston Michelle Embree-Ku, Providence Ronnie Fass, Tucson Mark A Feitelson, Philadelphia Ariel E Feldstein, Cleveland Alessandro Fichera, Chicago Chris E Forsmark, Gainesville Chandrashekhar R Gandhi, Pittsburgh Susan L Gearhart, Baltimore Xupeng Ge, Boston

John P Geibel, New Haven Xin Geng, New Brunswick Jean-Francois Geschwind, Baltimore Ignacio Gil-Bazo, New York Shannon S Glaser, Temple Ajay Goel, Dallas Julia Butler Greer, Pittsburgh James Henry Grendell, New York David R Gretch, Seattle Stefano Guandalini, Chicago Anna S Gukovskaya, Los Angeles Sanjeev Gupta, Bronx David J Hackam, Pittsburgh Stephen B Hanauer, Chicago Gavin Harewood, Rochester Margaret McLean Heitkemper, Seattle Alan W Hemming, Gainesville Samuel B Ho, San Diego Colin William Howden, Chicago Hongjin Huang, Alameda Jamal A Ibdah, Columbia Atif Iqbal, Omaha Hajime Isomoto, Rochester Hartmut Jaeschke, Tucson Dennis M Jensen, Los Angeles Leonard R Johnson, Memphis Michael P Jones, Chicago Peter James Kahrilas, Chicago AN Kalloo, Baltimore Neil Kaplowitz, Los Angeles Rashmi Kaul, Tulsa Jonathan D Kaunitz, Los Angeles Ali Keshavarzian, Chicago Miran Kim, Providence Joseph B Kirsner, Chicago Leonidas G Koniaris, Miami Burton I Korelitz, New York Robert J Korst, New York Richard A Kozarek, Seattle Michael Kremer, Chapel Hill Shiu-Ming Kuo, Buffalo Daryl Tan Yeung Lau, Galvesto Joel E Lavine, San Diego Dirk J van Leeuwen, Lebanon Glen A Lehman, Indianapolis Alex B Lentsch, Cincinnati Andreas Leodolter, La Jolla Gene LeSage, Houston Ming Li, New Orleans Zhiping Li, Baltimore LM Lichtenberger, Houston GR Lichtenstein, Philadelphia Otto Schiueh-Tzang Lin, Seattle Martin Lipkin, New York Edward V Loftus, Rocheste Robin G Lorenz, Birmingham Michael Ronan Lucey, Madison JD Luketich, Pittsburgh Henry Thomson Lynch, Omaha Patrick M Lynch, Houston Peter J Mannon, Bethesda Charles Milton Mansbach, Memphis John Frank Di Mari, Texas John M Mariadason, Bronx WM Mars, Pittsburgh Laura E Matarese, Pittsburgh Lynne V McFarland, Washington Kevin McGrath, Pittsburgh Harihara Mehendale, Monroe Stephan Menne, New York Howard Mertz, Nashville George W Meyer, Sacramento G Michalopoulos, Pittsburgh James Michael Millis, Chicago Albert D Min, New York Pramod Kumar Mistry, New Haven www.wjgnet.com

Smruti Ranjan Mohanty, Chicago Satdarshan Singh Monga, Pittsburgh Timothy H Moran, Baltimore Steven F Moss, Providence Masaki Nagaya, Boston Laura Eleanor Nagy, Cleveland Hiroshi Nakagawa, Philadelphia Douglas B Nelson, Minneaplis Brant K Oelschlager, Washington Curtis T Okamoto, Los Angeles Stephen JD O’Keefe, Pittsburgh Dimitry Oleynikov, Omaha Natalia A Osna, Omaha Stephen J Pandol, Los Angeles Pankaj Jay Pasricha, Gaveston Zhiheng Pei, New York Michael A Pezzone, Pittsburgh CS Pitchumoni, New Brunswiuc Jay Pravda, Gainesville M Raimondo, Jacksonville GS Raju, Galveston Murray B Resnick, Providence Adrian Reuben, Charleston Douglas K Rex, Indianapolis Victor E Reyes, Galveston Richard A Rippe, Chapel Hill Marcos Rojkind, Washington Philip Rosenthal, San Francisco Hemant Kumar Roy, Evanston Shawn David Safford, Norfolk Bruce E Sands, Boston NJ Shaheen, Chapel Hill Harvey L Sharp, Minneapolis Stuart Sherman, Indianapolis Shivendra Shukla, Columbia Alphonse E Sirica, Virginia Shanthi V Sitaraman, Atlanta Shanthi Srinivasan, Atlanta Michael Steer, Boston Gary D Stoner, Columbus Liping Su, Chicago Christina Surawicz, Seattle Gyongyi Szabo, Worcester Yvette Taché, Los Angeles Seng-Lai Tan, Seattle Andrzej Tarnawski, Long Beach Andrzej S Tarnawski, Orange K-M Tchou-Wong, New York Neil D Theise, New York PJ Thuluvath, Baltimore Swan Nio Thung, New York Natalie J Torok, Sacramento RA Travagli, Baton Rouge G Triadafilopoulos, Stanford James F Trotter, Denver Chung-Jyi Tsai, Lexington Andrew Ukleja, Florida Hugo E Vargas, Scottsdale Scott A Waldman, Philadelphia Jian-Ying Wang, Baltimore Steven David Wexner, Weston Keith Tucker Wilson, Baltimore Jacqueline L Wolf, Boston Jackie Wood, Ohio George Y Wu, Farmington Jian Wu, Sacramento Samuel Wyllie, Houston Wen Xie, Pittsburgh Yoshio Yamaoka, Houston Vincent W Yang, Atlanta Francis Y Yao, San Francisco Min You, Tampa Zobair M Younossi, Virginia Liqing Yu, Winston-Salem David Yule, Rochester Ruben Zamora, Pittsburgh Michael E Zenilman, New York Zhi Zhong, Chapel Hill Uruguay Henry Cohen, Montevideo

Ⅴ

World Journal of

Gastroenterology



Weekly Established in October 1995 National Journal Award 2005

Volume 13 Number 32 August 28, 2007

Baishideng

Contents EDITORIAL

4287   Noninvasive diagnosis of hepatic fibrosis in chronic hepatitis C Stauber RE, Lackner C

4295

Female hepatology: Favorable role of estrogen in chronic liver disease with hepatitis B virus infection Shimizu I, Kohno N, Tamaki K, Shono M, Huang HW, He JH, Yao DF

4306   Is it relevant that intra-arterial chemotherapy may be effective for advanced  pancreatic cancer? Ishikawa T

REVIEW

4310   Duration of treatment with 5-aminosalicylic acid compounds

GASTRIC CANCER

4316

Moshkovska T, Mayberry JF

 Results of mass endoscopic examination for gastric cancer in Kamigoto  Hospital, Nagasaki Prefecture Matsumoto S, Yamasaki K, Tsuji K, Shirahama S

4321

  Syndrome differentiation in traditional Chinese medicine and E-cadherin/  ICAM-1 gene protein expression in gastric carcinoma  Sun DZ, Xu L, Wei PK, Liu L, He J

BASIC RESEARCH

4328

 Cystamine ameliorates liver fibrosis induced by carbon tetrachloride via  inhibition of tissue transglutaminase   Qiu JF, Zhang ZQ, Chen W, Wu ZY

RAPID COMMUNICATION 4333    Clinical presentation and endoscopic management of Dieulafoy’s lesions in an

 urban community hospital Nagri S, Anand S, Arya Y

4336

   Effects of long term hydrophilic bile acid therapy on in vitro contraction of gallbladder muscle strips in patients with cholesterol gallstones Mas MR, Comert B, Mas N, Yamanel L, Ozotuk H, Tasci I, Jazrawi RP

4340

  Effect of vitamin E on oxidative stress status in small intestine of  diabetic rat Shirpoor A, Ansari MHK, Salami S, Pakdel FG, Rasmi Y

4345

 Role of serum interleukin-18 as a prognostic factor in patients with  hepatocellular carcinoma   Tangkijvanich P, Thong-ngam D, Mahachai V, Theamboonlers A, Poovorawan Y

4350      Genetic polymorphism of UL144 open reading frame of human  cytomegalovirus DNA detected in colon samples from infants with  Hirschsprung’s disease Mao ZQ, Huang Y, Sun M, Ruan Q, Qi Y, He R, Huang YJ, Ma YP, Ji YH, Sun ZR, Gao H

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World Journal of Gastroenterology

Contents

Volume 13 Number 32 August 28, 2007 4355   Association of high expression in rat gastric mucosal heat shock protein 70  induced by moxibustion pretreatment with protection against stress injury Chang XR, Peng L, Yi SX, Peng Y, Yan J

4360    Effects of body-resistance strengthening and tumor-suppressing granules on  immune adhesion function of red blood cells and expression of metastasis  protein CD44 in tumor cells of patients with esophageal carcinoma Zhao JX, Li XF, Wang XX

4365    Change in expression of apoptosis genes after hyperthermia, chemotherapy  and radiotherapy in human colon cancer transplanted into nude mice Liang H, Zhan HJ, Wang BG, Pan Y, Hao XS

4372

 A meta-analysis of the yield of capsule endoscopy compared to  double-balloon enteroscopy in patients with small bowel diseases Chen X, Ran ZH, Tong JL

4379  Safety evaluation of donors for living-donor liver transplantation in Chinese  mainland: A single-center report Li GQ, Zhang F, Li XC, Sun BC, Cheng F, Ge WG, Wang XH

4385   New precut sphincterotomy for endoscopic retrograde  cholangiopancreatography in difficult biliary duct cannulation

Deng DH, Zuo HM, Wang JF, Gu ZE, Chen H, Luo Y, Chen M, Huang WN, Wang L, Lu W

CASE REPORTS

4391   L  isteria monocytogenes following orthotopic liver transplantation: Central  nervous system involvement and review of the literature Mizuno S, Zendejas IR, Reed AI, Kim RD, Howard RJ, Hemming AW, Schain DC, Soldevila-Pico C, Firpi RJ, Fujita S

4394

 Fulminant hepatic failure in a case of autoimmune hepatitis in hepatitis C  during peg-interferon-alpha 2b plus ribavirin treatment Kogure T, Ueno Y, Fukushima K, Nagasaki F, Inoue J, Kakazu E, Matsuda Y, Kido O, Nakagome Y, Kimura O, Obara N, Wakui Y, Iwasaki T, Shimosegawa T

4398  Successful treatment of hypovascular advanced hepatocellular carcinoma  with lipiodol-targetting intervention radiology

Kurokohchi K, Deguchi A, Masaki T, Himoto T, Yoneyama H, Kobayashi M, Maeta T, Kiuchi T, Kohi F, Miyoshi H, Taminato T, Kuriyama S

4401   Hepatocellular carcinoma with chronic B-type hepatitis complicated by  autoimmune hemolytic anemia: A case report Okada T, Kubota K, Kita J, Kato M, Sawada T

4405  Spontaneous rupture of the liver in a patient with chronic hepatitis B and D   Liu CJ, Chien RN, Yen CL, Chang JJ

ACKNOWLEDGMENTS

4408   Acknowledgments to Reviewers of World Journal of Gastroenterology

APPENDIX

4409    M eetings

FLYLEAF

4410

Instructions to authors

I-V

Editorial Board

INSIDE FRONT COVER

Online Submissions

INSIDE BACK COVER

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World Journal of Gastroenterology

Contents

Volume 13 Number 32 August 28, 2007

Responsible E-Editor for this issue: Wen-Hua Ma C-Editor for this issue: Dr. Richard A Rippe Responsible S-Editor for this issue: Ye Liu

World Journal of Gastroenterology ( World J Gastroenterol , WJG ), a leading international journal in gastroenterology and hepatology, has an established reputation for publishing first class research on esophageal cancer, gastric cancer, liver cancer, viral hepatitis, colorectal cancer, and H pylori infection, providing a forum for both clinicians and scientists, and has been indexed and abstracted in Current Contents®/Clinical Medicine, Science Citation Index Expanded (also known as SciSearch®) and Journal Citation Reports/Science Edition, Index Medicus, MEDLINE and PubMed, Chemical Abstracts, EMBASE/Excerpta Medica, Abstracts Journals, Nature Clinical Practice Gastroenterology and Hepatology, CAB Abstracts and Global Health. ISI JCR 2003-2000 IF: 3.318, 2.532, 1.445 and 0.993. WJG is a weekly journal published by WJG. The publication date is on 7th, 14th, 21st, and 28th every month. The WJG is supported by The National Natural Science Foundation of China, No. 30224801 and No.30424812, which was founded with a name of China National Journal of New Gastroenterology on October 1, 1995, and renamed as WJG on January 25, 1998. NAME OF JOURNAL World Journal of Gastroenterology

EDITOR-IN -CHIEF Lian-Sheng Ma, Taiyuan

RESPONSIBLE INSTITUTION Department of Science and Technology of Shanxi Province

SUBSCRIPTION RMB 50 Yuan for each issue, RMB 2400 Yuan for one year

SPONSOR Taiyuan Research and Treatment Center for Digestive Diseases, Taiyuan 77, Shuangta Xijie, Taiyuan 030001, Shanxi Province, China

CSSN ISSN 1007-9327 CN 14-1219/R

EDITING Editorial Board of World Journal of Gastroenterology, 77 Shuangta Xijie, Taiyuan 030001, Shanxi Province, China Telephone: +86-351-4078656 E-mail: [email protected] PUBLISHING Editorial Department of World Journal of Gastroenterology, 77 Shuangta Xijie, Taiyuan 030001, Shanxi Province, China Telephone: +86-351-4078656 E-mail: [email protected] http://www.wjgnet.com PRINTING Beijing Kexin Printing House OVERSEAS DISTRIBUTOR Beijing Bureau for Distribution of Newspapers and Journals (Code No. 82-261) China International Book Trading Corporation PO Box 399, Beijing, China (Code No. M4481) PUBLICATION DATE August 28, 2007

You-Yong Lu, Beijing Masao Omata, Tokyo Harry HX Xia, Hanover SCIENCE EDITORS Deputy Director: Ye Liu, Beijing Jian-Zhong Zhang, Beijing LANGUAGE EDITORS Director: Jing-Yun Ma, Beijing Deputy Director: Xian-Lin Wang, Beijing

HONORARY EDITORS-IN-CHIEF Ke-Ji Chen, Beijing Li-Fang Chou, Taipei Zhi-Qiang Huang, Beijing Shinn-Jang Hwang, Taipei Min-Liang Kuo, Taipei Nicholas F LaRusso, Rochester Jie-Shou Li, Nanjing Geng-Tao Liu, Beijing Lein-Ray Mo, Tainan Bo-Rong Pan, Xi'an Fa-Zu Qiu, Wuhan Eamonn M Quigley, Cork David S Rampton, London Rudi Schmid, kentfield Nicholas J Talley, Rochester Guido NJ Tytgat, Amsterdam H-P Wang, Taipei Jaw-Ching Wu, Taipei Meng-Chao Wu, Shanghai Ming-Shiang Wu, Taipei Jia-Yu Xu, Shanghai Ta-Sen Yeh, Taoyuan ASSOCIATE EDITORS-IN-CHIEF Gianfranco D Alpini, Temple Bruno Annibale, Roma Roger William Chapman, Oxford Chi-Hin Cho, Hong Kong Alexander L Gerbes, Munich Shou-Dong Lee, Taipei Walter Edwin Longo, New Haven

MEMBERS Gianfranco D Alpini, Temple BS Anand, Houston Richard B Banati, Lidcombe Giuseppe Chiarioni, Valeggio John Frank Di Mari, Texas Shannon S Glaser, Temple Mario Guslandi, Milano Martin Hennenberg, Bonn Atif Iqbal, Omaha Manoj Kumar, Nepal Patricia F Lalor, Birmingham Ming Li, New Orleans Margaret Lutze, Chicago Jing-Yun Ma, Beijing Daniel Markovich, Brisbane Sabine Mihm, Göttingen Francesco Negro, Genève Bernardino Rampone, Siena Richard A Rippe, Chapel Hill Stephen E Roberts, Swansea  Ross C Smith, Sydney Seng-Lai Tan, Seattle Xian-Lin Wang, Beijing Eddie Wisse, Keerbergen Daniel Lindsay Worthley, Bedford COPY EDITORS Gianfranco D Alpini, Temple Sujit Kumar Bhattacharya, Kolkata Filip Braet, Sydney Kirsteen N Browning, Baton Rouge

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Radha K Dhiman, Chandigarh John Frank Di Mari, Texas Shannon S Glaser, Temple Martin Hennenberg, Bonn Eberhard Hildt, Berlin Patricia F Lalor, Birmingham Ming Li, New Orleans Margaret Lutze, Chicago MI Torrs, Jaén Sri Prakash Misra, Allahabad Giovanni Monteleone, Rome Giovanni Musso, Torino Valerio Nobili, Rome Osman Cavit Ozdogan, Istanbul Francesco Perri, San Giovanni Rotondo Thierry Piche, Nice Bernardino Rampone, Siena Richard A Rippe, Chapel Hill Ross C Smith, Sydney Daniel Lindsay Worthley, Bedford George Y Wu, Farmington Jian Wu, Sacramento COPYRIGHT © 2007 Published by WJG. All rights reserved; no part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise without the prior permission of WJG. Authors are required to grant WJG an exclusive licence to publish. SPECIAL STATEMENT All articles published in this journal represent the viewpoints of the authors except where indicated otherwise. INSTRUCTIONS TO AUTHORS Full instructions are available online at http://www.wjgnet.com/wjg/help/ instructions.jsp. If you do not have web access please contact the editorial office.

Online Submissions: wjg.wjgnet.com www.wjgnet.com [email protected]

World J Gastroenterol 2007 August 28; 13(32): 4287-4294 World Journal of Gastroenterology ISSN 1007-9327 © 2007 WJG. All rights reserved.

EDITORIAL

Noninvasive diagnosis of hepatic fibrosis in chronic hepatitis C Rudolf E Stauber, Carolin Lackner Rudolf E Stauber, Department of Internal Medicine, Medical University of Graz, Graz, Austria Carolin Lackner, Institute of Pathology, Medical University of Graz, Graz, Austria Correspondence to: Rudolf E Stauber, MD, Department of Internal Medicine, Medical University of Graz, Auenbruggerplatz 15, A-8036 Graz, Austria. [email protected] Telephone: +43-316-3854388 Fax: +43-316-3852648 Received: 2007-04-18 Accepted: 2007-04-20

Abstract A s s e s s m e n t o f h e p a t i c f i b r o s i s i s i m p o r t a n t fo r determining prognosis, guiding management decisions, and monitoring disease. Histological evaluation of liver biopsy specimens is currently considered the reference test for staging hepatic fibrosis. Since liver biopsy carries a small but significant risk, noninvasive tests to assess hepatic fibrosis are desirable. This editorial gives an overview on noninvasive methods currently available to determine hepatic fibrosis and their diagnostic accuracy for predicting significant fibrosis and cirrhosis in chronic hepatitis C. Based on available data, the performance of simple tests derived from routine laboratory parameters appears to be similar to that of more complex and expensive fibrosis panels. Transient elastography seems more accurate than blood tests for diagnosing cirrhosis. © 2007 WJG . All rights reserved.

Key words: Noninvasive fibrosis tests; Significant fibrosis; Cirrhosis; Biomarkers; Transient elastography Stauber RE, Lackner C. Noninvasive diagnosis of hepatic fibrosis in chronic hepatitis C. World J Gastroenterol 2007;

13(32): 4287-4294

http://www.wjgnet.com/1007-9327/13/4287.asp

INTRODUCTION Hepatic fibrosis is the basis for the development of portal hypertension, complications of chronic liver disease including esophageal varices and/or ascites, and liver failure. Assessment of the degree of hepatic fibrosis (i.e. staging) is important for several reasons: (1) to determine

the prognosis of chronic liver disease, (2) to select patients for specific (antifibrotic) treatment, and (3) to monitor the success of treatment. Assessment of hepatic fibrosis is especially relevant in the context of chronic hepatitis C. In developed countries, chronic hepatitis C is one of the leading causes of cirrhosis, hepatocellular carcinoma, and liver transplantation. The stage of fibrosis carries important prognostic information as it is closely related to the risk for development of cirrhosis[1]. Antiviral treatment of chronic hepatitis C aims at viral eradication and/or prevention of fibrosis progression. However, current standard treatment with peginterferon/ribavirin has limited efficacy and is associated with severe side effects. Especially in difficultto-treat patients with hepatitis C virus (HCV) genotype 1 infection and average cure rates of only 50%, the indication for antiviral treatment is selective and based on several factors such as age, concomitant diseases, and fibrosis stage. Hence, in HCV genotype 1 patients antiviral treatment has been primarily recommended for patients with at least significant fibrosis[2]. Currently, histological scoring is the reference test for staging of hepatic fibrosis. However, since liver biopsy is associated with complications, noninvasive methods for assessment of hepatic fibrosis are desired by both clinicians and patients. Different approaches to estimate liver fibrosis noninvasively have been pursued, including indirect fibrosis tests based on routine liver function parameters, direct fibrosis tests based on extracellular matrix proteins, and physical methods that estimate fibrosis by measuring hepatic stiffness. These noninvasive fibrosis tests are being intensely investigated in liver disease of various etiologies and several comprehensive reviews have been published recently[3-6]. However, the clinical impact of the numerous proposed methods remains unclear at present. This Editorial is intended to give an overview on currently available blood tests and physical methods for assessment of hepatic fibrosis and focuses on comparison of their diagnostic accuracies for predicting clinically relevant stages (significant fibrosis, cirrhosis) of chronic HCV infection.

IS LIVER BIOPSY AN ADEQUATE REFERENCE TEST? Histological evaluation of percutaneous liver biopsy

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specimens is currently used as a gold standard for assessment of hepatic fibrosis in chronic HCV infection. Fibrosis is usually staged semi-quantitatively by the pathologist using one of several published scoring systems. However, liver biopsy carries a significant risk and histological staging has several shortcomings as outlined below that limit its diagnostic accuracy. Risk of percutaneous liver biopsy Among the complications of percutaneous liver biopsy are pain (10%-30%), bleeding (which may be severe and necessitate blood transfusion or emergency surgery), biliary peritonitis, and pneumothorax. In large series, mortality has been reported to range from 0.1%-0.01%[7,8]. Percutaneous liver biopsy is contraindicated in the presence of coagulopathy, thrombocytopenia, and ascites. These contraindications may be in part obviated by transjugular liver biopsy; however, this method is not widely available. Sampling error An average needle biopsy specimen represents only 1/50 000 of a human liver. Sampling error thus may play an important role in diseases that exhibit a patchy rather than homogenous distribution within the liver. In a study investigating simultaneous biopsies from the left and right liver lobes during laparoscopy, fibrosis scores obtained in both biopsy sites differed by at least one stage in 33% of the patients[9]. Furthermore, Bedossa et al[10] demonstrated that the length of the biopsy core is positively related to the precision of fibrosis scoring. Likewise, Colloredo et al[11] reported that in liver biopsy specimens of inadequate size stage is likely to be underscored in chronic viral hepatitis. Based on these studies, liver biopsy cylinders with a length of ≥ 20 mm (at a width of 1.4 mm) and/or at least 11 complete portal tracts have been identified as minimal requirements for optimal histological evaluation of fibrosis in chronic hepatitis C. However, with respect to the evaluation of noninvasive fibrosis tests, most studies did not accurately report whether these requirements for adequate liver biopsy specimens were met[12]. Interobserver variation As scoring is subjective, observer error also plays an important role. Interobserver variation is largely dependent on the experience of the pathologist. In a study evaluating interobserver variation between 10 different experienced pathologists, substantial agreement was found for staging fibrosis (kappa 0.78) while variation was considerably higher for grading inflammatory activity[13,14]. Lack of a universal scoring system of fibrosis The interpretation of studies assessing hepatic fibrosis is further hampered by the lack of standardization of hepatic fibrosis scores. Several scoring systems have been developed that classify the degree of hepatic fibrosis either on a 5-step scale (F0-F4) including the Knodell fibrosis score[15], the Scheuer fibrosis score[16], and the METAVIR fibrosis score [13], or on a 7-step scale (F0-F6) such as the Ishak fibrosis score[17]. Significant fibrosis has been defined as F2-F4 or F3-F6 and cirrhosis as F4 or F5-F6, www.wjgnet.com

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respectively. To date, liver pathologists have not reached a universal consensus on the standardization of scoring systems. However, histopathological scoring of fibrosis by different systems appears to be quite robust as comparison of the Ishak and METAVIR fibrosis scores yielded excellent agreement (weighted kappa 0.998)[14].

INDIRECT FIBROSIS TESTS Several indirect fibrosis tests (indices composed of routine laboratory parameters that reflect changes in liver function) have been suggested as surrogate marker of hepatic fibrosis. Most of them are readily available at no additional cost, albeit they may require the use of a pocket calculator or access to the internet. Aspartate aminotransferase/Alanine aminotransferase ratio Almost three decades ag o, the ratio of aspar tate aminotransferase to alanine aminotransferase (AST/ALT ratio, AAR) has been proposed as a surrogate marker of hepatic fibrosis, with values > 1 being suggestive of cirrhosis[18]. This finding is related to an increased release of mitochondrial AST, decreased AST clearance and/or impaired synthesis of ALT in advanced liver disease. However, discrepant results have been published on the diagnostic accuracy of the AAR. Giannini et al reported high diagnostic accuracy of the AAR for prediction of cirrhosis[19] and significant fibrosis[20]. In contrast, Lackner et al[21] found the diagnostic accuracy of AAR to be clearly inferior to that of other indirect fibrosis tests based on routine laboratory parameters. Platelet count Hepatic fibrosis may lead to thrombocytopenia as a consequence of impaired synthesis of thrombopoietin and/or sequestering of platelets in an enlarged spleen. Surprisingly, few data exist on the diagnostic value of platelet count per se although the platelet count has been included in several composite fibrosis scores. Ono et al[22] reported the use of platelet count could discriminate F4 from F1-F3 in 75%-80% of patients with chronic hepatitis C. In our own study, a platelet count of < 150 × 109/L had a positive predictive value (PPV) > 90% for significant fibrosis, whereas at a cut-off of ≥ 150 × 109/L it had a negative predictive value (NPV) > 90% for cirrhosis[21]. Platelet count has been combined with age in the ageplatelet index[23] or with AAR and prothrombin time in the cirrhosis discriminant score (CDS)[24] but the diagnostic accuracy of these composite scores was not superior to platelet count per se[21]. In addition, platelet count is a component of AST to platelet ratio index (APRI), model 3, Forns index, Fibrometer, and FibroIndex. AST to platelet ratio index (APRI) The APRI was described by Wai et al from Anna Lok’s group at Ann Arbor University[25]. It is calculated as APRI = [(AST/Upper limit of normal)/platelet count (109/ L)] × 100 This test is derived from readily available laboratory

Stauber RE et al . Noninvasive fibrosis tests

parameters and usually requires a pocket calculator. Its diagnostic accuracy for both significant fibrosis and cirrhosis has been confirmed by several external studies[21,26-30]. Using the cut-offs proposed by Wai et al, approximately 50% of the patients can be correctly classified without a liver biopsy. Model 3 Lok et al [31] proposed another prognostic model for prediction/exclusion of cirrhosis in patients with chronic hepatitis C which is based on platelet count, AST, and prothrombin time expressed as international normalized ratio (INR). This index may be derived from the regression formula: log odds = -5.56 - 0.0089 × platelet (× 109/L) + 1.26 × AST/ALT ratio + 5.27 × INR or by a calculator available at the web site of the HALT-C trial (www.haltctrial.org). Using model 3 at a cut-off of < 0.20, cirrhosis could be excluded with an NPV of 99%. Forns index Forns et al[32] developed an index derived from age, gammaglutamyl transferase (GGT), cholesterol, and platelet count in a study of 476 untreated HCV patients, which is calculated as follows: 7.811 - 3.131 × ln (platelet count) + 0.781 × ln (GGT) + 3.467 × ln (age) - 0.014 × (cholesterol [mg/dL]) In their study, the area under the receiver operating characteristic curve (AUROC) for prediction of significant fibrosis (F2-F4 according to the Scheuer classification) was 0.86 in the test set and 0.81 in the validation set. The diagnostic accuracy of this index has been confirmed in patients with HIV-HCV coinfection[33]. Fibrotest/Fibrosure French investigators analyzed an extensive array of biochemical tests in 339 patients with chronic hepatitis C and identified a panel of 5 markers which could best predict the stage of fibrosis: α 2-macroglobulin, haptoglobin, apolipoprotein A1, GGT, and total bilirubin[34]. This test has been marketed as FibrotestTM (Biopredictive, Paris, France) in Europe and as FibrosureTM (LabCorp, Burlington, NC) in the United States. In contrast to the above mentioned indirect fibrosis tests, calculation of the Fibrotest by a patented algorithm is subject to payment of a fee to the manufacturer. The Fibrotest has been validated internally and externally in several studies in patients with chronic hepatitis C[35-38]. Interestingly, a recent study suggested that Fibrotest was a better predictor than histologic staging for complications of chronic hepatitis C[39]. Fibrotest was also found to predict fibrosis in alcoholic[40] and non-alcoholic[41] fatty liver disease. The diagnostic accuracy of this test is limited by hemolysis (leading to a reduction in haptoglobin), Gilbert’s syndrome (increasing the bilirubin level), and recent or ongoing infection (leading to elevations of α2macroglobulin and haptoglobin). FibroIndex Recently, Japanese investigators proposed another index composed of platelet count, AST, and gamma globulin:

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1.738 - 0.064 (platelets [× 104/mm3]) + 0.005 (AST [IU/L]) + 0.463 (gamma globulin [g/dL]) The AUROC for prediction of significant fibrosis was 0.83 (0.82 in the validation set) which was slightly superior to APRI or the Forns index assessed in the same study[29].

DIRECT FIBROSIS TESTS Serum levels of extracellular matrix (ECM) proteins reflect the balance between hepatic fibrogenesis and fibrolysis and have been proposed as direct markers (biomarkers) of hepatic fibrosis. In addition, several fibrosis panels (i.e. combinations of such biomarkers) have been developed and some of them are now available for commercial use. While these biomarkers or panels thereof are directly related to the deposition of fibrotic material, their diagnostic performance in hepatic fibrosis may be limited by extrahepatic confounding factors such as systemic inflammation or renal failure. Hyaluronic acid Hyaluronic acid (HA) is a polysaccharide present in ECM and elevated in serum in patients with hepatic fibrosis. Commercial test kits are available from Corgenix (Westminster, Colorado). The diagnostic accuracy of HA was found to be superior to that of procollagen type Ⅲ N-terminal peptide (PⅢNP)[42] and its diagnostic accuracy was confirmed in a large study of 486 HCV patients[43]. HA was also found to be an accurate marker of severe hepatic fibrosis in nonalcoholic fatty liver disease[44]. Extracellular matrix proteins Procollagen peptides: PⅢNP is a product of cleavage of procollagen and has been proposed as a ser um marker of hepatic fibrosis more than two decades ago[45]. However, in several studies in patients with chronic HCV infection, this biomarker showed only moderate diagnostic accuracy[42,46,47]. Matrix metalloproteinases and inhibitors: Excess ECM proteins are degraded by matrix metalloproteinases (MMPs) which are in turn inhibited by tissue inhibitors of metalloproteinases (TIMPs). Since both MMPs and TIMPs are related to matrix protein turnover, their serum levels have been used as fibrosis markers. However, studies on the correlation of MMP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 with hepatic fibrosis have produced conflicting results[48-50]. TIMP-1 is a component of several composite fibrosis panels (see below). YKL-40: YKL-40 is a glycoprotein believed to play a role in ECM degradation. In a study of 109 patients with chronic HCV infection, this biomarker showed similar diagnostic accuracy to HA for significant fibrosis, while its ability to diagnose cirrhosis was inferior to that of HA[51].

COMPOSITE FIBROSIS PANELS Combinations of one or more biomarkers with indirect fibrosis tests have been suggested as noninvasive fibrosis tests.

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Fibrospect Ⅱ The Fibrospect Ⅱ assay (Prometheus Laboratories Inc., San Diego, CA) uses HA, TIMP-1 and α2-macroglobulin. This test was found to accurately predict significant fibrosis in a study of 294 HCV patients and validated in an external cohort of 402 HCV patients [52]. This test was further validated in a study comparing the diagnostic accuracies of APRI, Forns index, Fibrotest, and Fibrometer[26]. Enhanced liver fibrosis (ELF) In a European multicenter study of 1021 patients with chronic liver disease of different etiologies, an algorithm c o n s i s t i n g o f a g e, H A , P Ⅲ N P, a n d T I M P - 1 wa s developed[53]. Using the Scheuer fibrosis score as a reference test, its overall diagnostic accuracy was similar to that of other noninvasive fibrosis tests (AUROC 0.78 for significant fibrosis, 0.89 for cirrhosis). Performance of the algorithm was slightly lower in a subgroup of patients with chronic hepatitis C (n = 325, AUROC 0.77 for prediction of F3-F4). This test is being marketed as Enhanced Liver Fibrosis (ELFTM) test by Siemens Medical Solutions Diagnostics (Tarrytown, NY). Hepascore This model was developed by Australian investigators and is derived from bilirubin, GGT, HA, α2-macroglobulin, age and sex. High diagnostic performance was reported for both significant fibrosis and cirrhosis[54], but external validation yielded somewhat lower diagnostic accuracies[30]. Fibrometer The Fibrometer test incorporates α2-macroglobulin, HA, AST, platelet count, prothrombin index, urea, and age. Cales et al[26] reported superior diagnostic accuracy of Fibrometer to Forns index, Fibrotest, and APRI. However, this finding was not confirmed in an external validation study[30]. Several Fibrometers are now commercially available at BioLiveScale (Angers, France) for assessment of fibrosis in chronic viral hepatitis (Fibrometer V), alcoholic liver disease (Fibrometer A), and metabolic steatopathy (Fibrometer S).

PHYSICAL METHODS/IMAGING Radiological techniques such as ultrasound, CT scan or MRI can accurately detect cirrhosis at advanced stages with hepatic nodularity or signs of portal hypertension such as splenomegaly, presence of portosystemic collaterals or ascites. However, these techniques fail to detect earlier stages of hepatic fibrosis or cirrhosis. Recently, special techniques for assessment of liver stiffness (elastography) have been developed. Transient elastography Transient elastography by FibroscanTM (Echosens, Paris, France) is a promising technique that estimates the degree of hepatic fibrosis by measuring liver stiffness[55]. The Fibroscan transmits a vibration of low frequency into the liver from a probe that includes an ultrasonic transducer. The vibration waves induce an elastic shear wave whose velocity is proportional to the stiffness of the tissue. Shear www.wjgnet.com

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wave velocity is measured by pulse-echo ultrasound and results are expressed in kPa. In cirrhotic patients, liver stiffness measurements (LSM) show a wide range from approximately 12 to 75 kPa. The Fibroscan samples a volume of approximately 4 cm3 which is considered more representative of the entire liver than a needle biopsy specimen. Measurements can be quickly performed in 5 min and are highly reproducible. However, steatosis may confound its value to estimate fibrosis. Furthermore, measurement is heavily limited or impossible in obesity, ascites or in patients with narrow intercostal spaces. The Fibroscan has been studied in various liver diseases including chronic hepatitis C [56] and primary biliary cirrhosis[57] demonstrating good diagnostic accuracy. A small study in patients with chronic HCV infection and persistently normal transaminases even reported values of 100% for sensitivity, specificity, PPV and NPV, respectively[58]. In a study of 711 patients with chronic liver disease of various etiologies, LSM was closely related to fibrosis stage (r = 0.73) and high diagnostic accuracy was found for the diagnosis of cir rhosis (AUROC 0.96) [59]. Ganne-Carrie et al [60] further elaborated LSM for diagnosing cirrhosis and confirmed high diagnostic accuracy (AUROC 0.95) at a cut-off value of 14.6 kPa with some false-negative results due to inactive or macronodular cirrhosis. Another large study evaluated the success rate and performance of LSM in 935 patients with chronic HCV infection and reported successful measurements in 97% of the patients, while fatty thoracic belt was the major limiting factor[61]. Interestingly, recent data indicate a close correlation between LSM and portal pressure as estimated by the hepatic venous pressure gradient (HVPG) in patients with HCV-related cirrhosis[62] as well as recurring hepatitis C following liver transplantation[63]. Vizzutti et al[62] reported a good correlation between LSM and HVPG (r = 0.82) at lower portal pressures (< 12 mmHg) and suggested that, at a cut-off of 13.6 kPa, LSM could reliably predict or exclude clinically significant portal hypertension (CSPH, i.e. a portal pressure of ≥ 10 mmHg) which is the prerequisite for the formation of esophageal varices. Thus, LSM may be the method of choice to characterize the severity of cirrhosis in patients without CSPH while at the same time it could be used to identify patients with CSPH that may benefit from portal pressure measurement by hepatic vein catheterization. This approach should be prospectively tested in future studies. Real-time elastography Another attractive approach is the development of real-time ultrasound systems equipped with special elastography modules that allow estimation of liver fibrosis with conventional ultrasound probes during a routine sonography examination. First results obtained with the Hitachi EUB-8500 system were recently presented[64]. In 79 patients with chronic viral hepatitis, the AUROC for diagnosis of significant fibrosis was 0.75 (elasticity score) or 0.93 (combined elasticity-laboratory score). Magnetic resonance imaging Aguirre et al from the University of California at San

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Diego proposed noninvasive diagnosis of liver fibrosis using double contrast material-enhanced MR imaging. In a retrospective study in 101 patients, this method could detect advanced fibrosis (METAVIR fibrosis score ≥ 3) with a sensitivity and specificity of > 90%[65].

Table 1 Overview of studies on noninvasive fibrosis tests in chronic hepatitis C n

Indirect fibrosis tests AST/ALT ratio

COMBINED METHODS

Platelet count

The highest diagnostic accuracies have to date been reported with the combined use of different methods with synergistic performance.

APRI

Fibroscan/Fibrotest Castera et al investigated APRI, Fibrotest, Fibroscan and combinations thereof in 183 patients with chronic hepatitis C. For significant fibrosis (but not cirrhosis), the combination of Fibroscan and Fibrotest had superior diagnostic accuracy (AUROC 0.88) to that of respective individual tests[66].

Model 3 Forns index

Fibrotest/Fibrosure

Sequential algorithms The sequential use of several markers may overcome some of the limitations of individual markers. Sebastiani et al developed three different sequential algorithms (including APRI, Fibrotest and/or Forns index) for the diagnosis of significant fibrosis with elevated ALT, significant fibrosis with persistently normal ALT, and cirrhosis, respectively, which allowed to classify 100% of the patients for each entity[67].

FibroIndex Direct fibrosis tests Hyaluronic acid PIIINP TIMP-1 YKL-40 Composite fibrosis panels Fibrospect Ⅱ

COMPARISON OF DIFFERENT NONINVASIVE FIBROSIS TESTS Table 1 gives an overview of the diagnostic accuracies of various noninvasive tests for assessment of clinically relevant stages of hepatic fibrosis. All tests showed a better performance for diagnosing cirrhosis (AUROC 0.90-0.95) than for significant fibrosis (AUROC clustering around 0.80). With respect to significant fibrosis, the diagnostic accuracy of complex biomarker panels or newer physical methods was not superior to that of simple tests based on routine laboratory parameters. However, transient elastography appears more accurate than blood tests for diagnosing cirrhosis and may be especially useful for detection of clinically significant portal hypertension. It should be noted that many of the reported tests have not yet undergone adequate external validation. Besides, direct comparison of different studies is hampered by variation in the reference test (sampling error, observer variation, use of different scoring systems) and different distribution of fibrosis stages within the study populations. In a recent study, Poynard et al[68] reported an important influence of biopsy size and fragmentation on the diagnostic accuracy of the Fibrotest. Such variation of the ‘gold standard’ may lead to underestimation of the diagnostic performance of noninvasive tests. Few trials have directly compared different tests in the same populations of patients with chronic hepatitis C. Lackner et al[21] evaluated several noninvasive fibrosis tests based on routine laboratory parameters and found

ELF Hepascore Fibrometer

194 409 194 409 270 194 503 235 206 360 356 1141 476 503 235 360 339 352 323 125 503 519 235 356 360 326 206 326 78 109

Significant Cirrhosis Author fibrosis (AUROC) (AUROC) 0.57 0.73 0.75 0.71 0.89 0.73 0.80 (0.881) 0.89 (0.941) 0.8 0.9 0.79 0.71 0.81 0.82 0.84 0.79 (0.821) 0.76 0.92 0.78 (0.811) 0.86 (0.811) 0.82 0.76 0.79 (0.841) 0.84 (0.871) 0.92 0.73 0.84 0.74 0.81 0.79 0.81 0.82 0.79 0.86 0.83 (0.861) 0.86 0.88 0.69 0.71 0.81

Guechot[42] Parise[28] Guechot[42] Boeker[49] Saitou[51]

0.92 0.91 0.73 0.90 0.80

696 0.83 (0.821) 503 0.87 10212 0.78 0.89 503 0.83 221 0.85 (0.821) 0.94 (0.891) 356 0.76 0.89 503 0.88 356 0.78 0.94

Elastography Transient elastography 327 (Fibroscan) 7112 403 7752 935 Real-time elastography 79 Combined tests Fibroscan + Fibrotest 183

Lackner[21] Giannini[20] Lackner[21] Giannini[20] Wai[25] Lackner[21] Cales[26] Bourliere[27] Parise[28] Koda[29] Halfon[30] Lok[31] Forns[32] Cales[26] Bourliere[27] Koda[29] Imbert-Bismut[34] Poynard[36] Myers[35] Rossi[37] Cales[26] Halfon[38] Bourliere[27] Halfon[30] Koda[29]

Patel[52] Cales[26] Rosenberg[53] Cales[26] Adams[54] Halfon[30] Cales[26] Halfon[30]

0.79 0.80 1.00 0.79 0.75

0.97 0.96 0.95 0.91 0.69

Ziol[56] Foucher[59] Colletta[58] Ganne-Carrie[60] Kettaneh[61] Friedrich-Rust[64]

0.88

0.95

Castera[66]

1

Internal validation set; 2Chronic liver disease of various etiologies; 3Chronic HCV with persistently normal ALT.

superior diagnostic accuracy of APRI to AST/ALT ratio. Bourliere et al[27] reported similar performance of Fibrotest, APRI, and Forns index while Cales et al[26] found superior diagnostic accuracy of Fibrometer to APRI, Forns index, and Fibrotest. In contrast, in 356 patients with chronic hepatitis C, Halfon et al[30] reported similar diagnostic accuracies of Fibrotest, Fibrometer, APRI, and Hepascore for significant fibrosis (AUROC 0.79, 0.78, 0.76, 0.76, respectively). Based on these comparative studies, www.wjgnet.com

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the diagnostic accuracy of simple noninvasive fibrosis tests such as APRI appears to be similar to that of more complex and expensive fibrosis panels.

14 15

CONCLUSION The main advantages of noninvasive fibrosis tests are the absence of risks and the potential to reflect the status of the entire liver. However, while optimal results are obtained at both ends of the spectr um of liver fibrosis, their most important limitation is the lack of discrimination at intermediate stages of fibrosis. Parkes et al[5] evaluated the diagnostic accuracy of several direct and indirect noninvasive fibrosis tests at cut-off levels giving high predictive values (PPV ≥ 90%, NPV ≥ 95%) and concluded that serum markers can reliably predict fibrosis only in a minority (about 35%) of patients with chronic hepatitis C. Besides, liver biopsy is still needed to rule out concomitant pathologies known to influence response to antiviral treatment, such as steatosis, steatohepatitis and/or iron overload. Future practice guidelines should address the role of noninvasive tests in assessing the stage of fibrosis in chronic hepatitis C[69].

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of cirrhosis in patients with chronic liver diseases. Hepatology 2006; 44: 1511-1517 61 Kettaneh A, Marcellin P, Douvin C, Poupon R, Ziol M, Beaugrand M, de Ledinghen V. Features associated with success rate and performance of FibroScan measurements for the diagnosis of cirrhosis in HCV patients: a prospective study of 935 patients. J Hepatol 2007; 46: 628-634 62 Vizzutti F, Arena U, Romanelli RG, Rega L, Foschi M, Colagrande S, Petrarca A, Moscarella S, Belli G, Zignego AL, Marra F, Laffi G, Pinzani M. Liver stiffness measurement predicts severe portal hypertension in patients with HCVrelated cirrhosis. Hepatology 2007; 45: 1290-1297 63 Carrion JA, Navasa M, Bosch J, Bruguera M, Gilabert R, Forns X. Transient elastography for diagnosis of advanced fibrosis and portal hypertension in patients with hepatitis C recurrence after liver transplantation. Liver Transpl 2006; 12: 1791-1798 64 Friedrich-Rust M, Ong MF, Herrmann E, Dries V, Samaras P, Zeuzem S, Sarrazin C. Real-time elastography for noninvasive assessment of liver fibrosis in chronic viral hepatitis. AJR Am J Roentgenol 2007; 188: 758-764 65 Aguirre DA, Behling CA, Alpert E, Hassanein TI, Sirlin CB.

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Liver fibrosis: noninvasive diagnosis with double contrast material-enhanced MR imaging. Radiology 2006; 239: 425-437 Castera L, Vergniol J, Foucher J, Le Bail B, Chanteloup E, Haaser M, Darriet M, Couzigou P, De Ledinghen V. Prospective comparison of transient elastography, Fibrotest, APRI, and liver biopsy for the assessment of fibrosis in chronic hepatitis C. Gastroenterology 2005; 128: 343-350 Sebastiani G, Vario A, Guido M, Noventa F, Plebani M, Pistis R, Ferrari A, Alberti A. Stepwise combination algorithms of non-invasive markers to diagnose significant fibrosis in chronic hepatitis C. J Hepatol 2006; 44: 686-693 Poynard T, Halfon P, Castera L, Charlotte F, Le Bail B, Munteanu M, Messous D, Ratziu V, Benhamou Y, Bourliere M, De Ledinghen V. Variability of the area under the receiver operating characteristic curves in the diagnostic evaluation of liver fibrosis markers: impact of biopsy length and fragmentation. Aliment Pharmacol Ther 2007; 25: 733-739 Castera L, Denis J, Babany G, Roudot-Thoraval F. Evolving practices of non-invasive markers of liver fibrosis in patients with chronic hepatitis C in France: time for new guidelines? J Hepatol 2007; 46: 528-529; author reply 529-530 S- Editor Zhu LH L- Editor Zhu LH E- Editor Liu Y

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World J Gastroenterol 2007 August 28; 13(32): 4295-4305 World Journal of Gastroenterology ISSN 1007-9327 © 2007 WJG. All rights reserved.

EDITORIAL

Female hepatology: Favorable role of estrogen in chronic liver disease with hepatitis B virus infection Ichiro Shimizu, Nao Kohno, Katsuyoshi Tamaki, Masayuki Shono, Hui-Wei Huang, Jiang-Hong He, Deng-Fu Yao Ichiro Shimizu, Nao Kohno, Katsuyoshi Tamaki, Department of Digestive and Cardiovascular Medicine, Institute of Health Biosciences, University of Tokushima Graduate School, Kuramoto-cho, Tokushima 770-8503, Japan Masayuki Shono, Support Center for Advanced Medical Sciences, University of Tokushima Graduate School, Kuramotocho, Tokushima 770-8503, Japan Hui-Wei Huang, Department of Physiology, Faculty of Medicine, Nantong University, Nantong 226001, Jiangsu Province, China Jiang-Hong He, Key Laboratory of Neuroregeneration of Jiangsu Province, Nantong University, Nantong 226001, Jiangsu Province, China Deng-Fu Yao, Research Center of Clinical Molecular Biology, Affiliated Hospital, Nantong University, Nantong 226001, Jiangsu Province, China Correspondence to: Ichiro Shimizu, MD, Department of Digestive and Cardiovascular Medicine, Institute of Health Biosciences, University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan. [email protected] Telephone: +81-88-6337124 Fax: +81-88-6339235 Received: 2007-05-29 Accepted: 2007-06-04

with chronic liver disease than in females. These lines of evidence suggest that the greater progression of hepatic fibrosis and HCC in men and postmenopausal women may be due, at least in part, to lower production of estradiol and a reduced response to the action of estradiol. A better understanding of the basic mechanisms underlying the sex-associated differences in hepatic fibrogenesis and carciogenesis may open up new avenues for the prevention and treatment of chronic liver disease. © 2007 WJG. All rights reserved.

Key words: Hepatic fibrosis; Hepatocellular carcinoma; Hepatic stellate cell; Estradiol; Estrogen receptor; Oxidative stress; Menopause; Hepatic iron Shimizu I, Kohno N, Tamaki K, Shono M, Huang HW, He JH, Yao DF. Female hepatology: Favorable role of estrogen in chronic liver disease with hepatitis B virus infection. World J Gastroenterol 2007; 13(32): 4295-4305

http://www.wjgnet.com/1007-9327/13/4295.asp

Abstract Chronic hepatitis B virus (HBV) infection is the most common cause of hepatic fibrosis and hepatocellular carcinoma (HCC), mainly as a result of chronic necroinflammatory liver disease. A characteristic feature of chronic hepatitis B infection, alcoholic liver disease and nonalcoholic fatty liver disease (NAFLD) is hepatic steatosis. Hepatic steatosis leads to an increase in lipid peroxidation in hepatocytes, which, in turn, activates hepatic stellate cells (HSCs). HSCs are the primary target cells for inflammatory and oxidative stimuli, and these cells produce extracellular matrix components. Chronic hepatitis B appears to progress more rapidly in males than in females, and NAFLD, cirrhosis and HCC are predominately diseases that tend to occur in men and postmenopausal women. Premenopausal women have lower hepatic iron stores and a decreased production of proinflammatory cytokines. Hepatic steatosis has been observed in aromatase-deficient mice, and has been shown to decrease in animals after estradiol treatment. Estradiol is a potent endogenous antioxidant which suppresses hepatic fibrosis in animal models, and attenuates induction of redox sensitive transcription factors, hepatocyte apoptosis and HSC activation by inhibiting a generation of reactive oxygen species in primary cultures. Variant estrogen receptors are expressed to a greater extent in male patients

INTRODUCTION Worldwide chronic infection with hepatitis B virus (HBV)[1] and hepatitis C virus (HCV)[2] is seen in 350 million and 170 million people respectively. Chronic HBV infection is currently the most common cause of cirrhosis and hepatocellular carcinoma (HCC) in the world. HBV is transmitted by the prenatal, parenteral, and sexual routes. If an HBV infection is acquired at birth, there is a strong chance that the child will become chronically infected. However, if the infection is acquired during adulthood, the risk of chronic infection is relatively low (about 10%-20%) [3,4]. Fifteen to 40% of chronically infected people may develop cirrhosis and HCC[5]. The remaining individuals become inactive carriers, otherwise defined as asymptomatic or healthy carriers. Among individuals infected with HBV, those who express the HBV surface antigen (i.e., HBsAg carriers) are approximately 20 times more likely to develop HCC than those who do not [6]. Clinical observations and death statistics support the view that chronic hepatitis B and C appear to progress more rapidly in males than in females[7,8], and that cirrhosis is largely a disease of men and postmenopausal women, with the exception of autoimmune liver diseases, such www.wjgnet.com

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China Middle Africa Japan Eastern Africa South-Eastern Asia Melanesia Western Africa Southern Europe Micro/Polynesia Caribbean Southern Africa Western Europe Eastern Europe Northern America Central America Western Asia Northern Africa Australia/New Zealand South America Norhtern Europe South Central Asia

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0 30 20 10 10 Age standardized incidence per 100 000

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Figure 1 Gender-specific incidence of liver cancer in the world. Data on age standardized incidence of liver cancer was obtained from Cancer Incidence in Five Continents Vol. Ⅷ[14].

as primary biliary cirrhosis and chronic autoimmune hepatitis[9,10]. The most clearly established risk factors for HCC are chronic infection with HBV and HCV, cirrhosis, male sex, older age, alcohol abuse, and exposure to dietary aflatoxin[11]. Liver injury in chronic hepatitis B is predominantly caused by the cellular immune response to the virus, and over time the balance between HBV and the alterations in the host immune response[12]. By contrast, the high frequency of chronicity (54%-86%)[13] in HCV infection and evidence of high rates of HCV mutations are perhaps the result of an ineffective immune response or immunological escape by HCV. According to the International Agency for Research on Cancer[14], the male: female ratio of the age-standardized incidence per 100 000 of liver cancer worldwide is 2.9:1. In Asia (particularly in China, Japan, and Taiwan), the incidence of liver cancer is high and accounts for one-half of all liver cancer cases in the world (Figure 1). In the Asian-Pacific region including China and Taiwan, and sub-Saharan Africa, HBV is hyperendemic, whereas in Japan, Western Europe and the USA, HBV infection is much less common, whereas HCV infection is more prevalent and is recognized as a major causative factor for HCC. The inshore region of the Yangtze River, including Qidong, in eastern China has the highest incidence of HCC, due to high levels of aflatoxin, which is associated with HCC because of its carcinogenic potential in HBsAg carriers[15]. Moreover, there is growing concern in clinical practice with regard to the development of nonalcoholic fatty liver disease (NAFLD). Although in most cases, fatty liver does not progress to more severe liver disease, approximately 15%-20% of patients have histological signs of fibrosis and necroinflammation, indicating the presence of nonalcoholic steatohepatitis (NASH). These patients are at a higher risk of developing cirrhosis, terminal liver failure, and HCC [16]. Most studies show a female predilection for NASH[16]; however, two recent reports indicate that NAFLD is more prevalent in men in each age group, and that there is a male predominance for NASH as well[17,18]. Differences in the social environment and the lifestyles of men and women may be involved in the basic mechanisms underlying the sex-associated differences in chronic liver diseases. In general, men have a greater www.wjgnet.com

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risk of exposure to hepatitis viruses as well as greater opportunities for drinking and smoking. Environmental factors may result in a higher preponderance of nutritional and exercise-associated problems in men. However, it should be noted that some mechanisms related to sexlinked differences may be based on biological factors, including estrogen-related female sex hormones, such as estradiol, rather than simply gender differences in social environment and lifestyle. Hepatic estrogen receptors (ERs) mediate estrogen action in the liver. The present review summarizes the current knowledge of the biological functions of estrogens and the ER status as it relates to hepatic fibrogenesis and carcinogenesis.

HBV SEROCONVERSION IN FEMALES During the early phase of chronic HBV infection, patients are positive for HBV e antigen (HBeAg), a surrogate marker of active HBV replication, and have frequent acute flares characterized by substantial increases in the serum aminotransferase levels as the result of specific, T-lymphocyte-mediated cellular responses to viral antigens and apoptosis of the hepatocytes. Some of the acute flares may be followed by seroconversion from HBeAg to its antibody (anti-HBe) and clinical remission [19,20], while other patients progress to cirrhosis and HCC, particularly elderly men[21,22]. A positive test for HBeAg is associated with higher inflammatory activity in the liver and an increased risk of HCC[23]. Long term cohort studies show that a higher percentage of females clear their HBeAg and become HBsAg negative compared to males[24]. Seroconversion from HBeAg to anti-HBe and from HBsAg to anti-HBs occurs more frequently in female subjects than in males [25]. Generally, females produce more vigorous cellular and humoral immune reactions, and have a higher incidence of autoimmune diseases than males [26,27]. The underlying mechanism by which females seem more likely to achieve seroconversion of HBeAg and HBsAg remains unclear. However, estradiol has been reported to induce the production of interferon (INF)- γ in lymphocytes [28], and augments an antigenspecific primary antibody response in human peripheral blood mononuclear cells[29]. IFN-γ is a potent cytokine with immunomodulatory and antiproliferative properties. Therefore, female subjects, particularly before menopause, may produce antibodies against HBeAg and HBsAg at a higher frequency than males with chronic HBV infection. Immunization is the most effective method of preventing transmission of HBV. After immunization with HBV vaccine, approximately 90% of healthy adults and 95% of infants, children, and adolescents develop a protective serum level of anti-HBs. Predictors associated with a nonresponse to HBV vaccination, include male sex, older age, obesity and immunocompromising chronic diseases[30]. After an initial vaccination in newborns of Alaska Natives who were seronegative for HBsAg and anti-HBs at birth, the mean concentrations of anti-HBs were higher in females (975 mIU/mL) than males (722 mIU/mL), but after 15 years these values decreased to 23 mIU/mL in females compared to 32 mIU/mL in males[31]. In Taiwan, the chronic HBsAg carrier rate was lower in

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Figure 2 Schematic representation of the hepatic sinusoidal wall, based on the studies by Wake[35]. HSCs cause contraction of both endothelial cells and hepatocytes. Collagen fibrils extend through the space of Disse.

females than males (4.4% vs 10.7%) who were born to HBsAg carrier mothers, vaccinated against HBV at birth, and followed for over 18 years[32].

OXIDATIVE STRESS IN LIVER INJURY AND HEPATIC STELLATE CELL ACTIVATION Damage to the parenchymal cell membranes produces reactive oxygen species (ROS) derived from the lipid peroxidative processes, which are a feature of sustained inf lammator y response and liver injur y, once the antioxidant mechanisms have been depleted. Cells are well equipped to neutralize the effects of ROS, by virtue of a series of antioxidant protective systems, including superoxide dismutase (SOD), glutathione peroxidase and glutathione. Although mild liver injury usually results in an almost complete resolution, persistence of the original insult causes prolonged activation of tissue repair mechanisms, leading to hepatic fibrosis rather than to effective tissue repair. Hepatic fibrosis, or collagen deposition, is associated with inflammation and cell death, which are a consequence of severe liver damage that occurs in many patients with chronic liver disease, regardless of the etiology such as HBV/HCV infection, alcohol abuse, and iron overload state[33]. In other words, collagen production predominates over hepatocellular regeneration. Collagens are mainly produced by cells known as hepatic stellate cells (HSCs). HSCs are located in the space of Disse in close contact with hepatocytes and sinusoidal endothelial cells[34] (Figure 2). In the injured liver, HSCs are regarded as the primary target cells for inflammatory and peroxidative stimuli and are transformed into myofibroblast-like cells. These HSCs are referred to as activated cells and their activation is accompanied with a loss of cellular retinoid, and the synthesis of α-smooth muscle actin (SMA), and large quantities of the major components of the extracellular matrix, including collagen typesⅠand Ⅲ. Transgenic mice expressing HBsAg exhibit oxidative

Hypercarcinogenic state

Collagen deposition

Hepatic fibrosis

Figure 3 Hepatic fibrosis and hypercarcinogenic state associated with oxidative stress in hepatocytes and HSCs. HSCs are activated by MDA and HNE as well as by ROS. HSCs produce ROS and TGF-β in response to ROS and TGF-β (autocrine) with increased collagen expression in the injured liver.

stress and DNA damage, leading to the development of HCC[35,36]. In addition, HBV X (HBx) protein alters the mitochondrial transmembrane potential and enhances ROS production in the liver[37]. A primary source of ROS production in hepatocytes and HSCs is mitochondrial NADH/NADPH oxidase. Hydrogen peroxide is more stable and membrane permeable compared to other ROS, which has lead to the hypothesis that it acts as a second messenger causing the following reactions: (1) induction of gene expression of redox sensitive transcription factors, such as activator protein (AP)-1 and nuclear factor (NF)[38] [39] κB , (2) stimulation of apoptosis , and (3) modulation [40] of cell proliferation . AP-1 and NF- κ B induce the expression of multiple genes involved in inflammation, cell death and fibrogenesis, including cytokines and growth factors such as platelet-derived growth factor and transforming growth factor (TGF)-β. TGF-β is a major fibrogenic cytokine, acting as a paracrine and autocrine (from HSCs) mediator, which triggers and induces the activation of HSCs in vivo. Hydrogen peroxide is converted into the hydroxyl radical, a harmful and highly reactive ROS, in the presence of transition metals such as iron. The hydroxyl radical is able to induce DNA cleavage and lipid peroxidation in the structure of membrane phospholipids, leading to cell death. Malondialdehyde (MDA) and hydroxynonenal (HNE), end products of lipid peroxidation, are discharged from injured hepatocytes into the space of Disse. Paracrine stimuli derived from hepatocytes undergoing oxidative stress induce HSC proliferation and collagen synthesis, and these HSCs are activated by ROS as well as by MDA and HNE[41,42]. HSCs are also able to produce ROS through the activation of NADH/NADPH oxidase by stimuli from outside the cell [43]. Exogenous TGF- β increases ROS production by HSCs, whereas the addition of ROS induces TGF-β production and secretion by these cells[44]. This so-called autocrine loop of ROS by HSCs is regarded as the mechanism that corresponds to the autocrine loop of www.wjgnet.com

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Figure 4 Distribution of serum ferritin levels by decade of age for females and males. The serum ferritin levels (ng/mL) were examined by gender and age in 305 healthy Japanese individuals (mean age 50.6 years, 47.2% males).

TGF-β which HSCs produce in response to this cytokine with an increased collagen expression in the injured liver[45] (Figure 3).

ESTROGENS AND OXIDATIVE STRESS IN HEPATOCYTES It should be noted that estradiol and its derivatives are strong endogenous antioxidants that reduce lipid peroxide levels in the liver and serum[46,47]. Recent studies show that estradiol suppresses iron (ferric nitrilotriacetate)-induced ROS generation, lipid peroxidation, activation of AP-1 and NF-κB, and the loss of SOD and glutathione peroxidase activities in cultured rat hepatocytes[48,49]. Estradiol also inhibits iron (FeSO4)-induced lipid peroxidation in isolated rat liver mitochondria [48]. These findings suggest that the inhibitory effect of estradiol on AP-1 and NF- κ B activation may be produced by scavenging ROS and/or by reducing the intracellular production of ROS via antioxidant enzyme induction. Many of the actions of estradiol are mediated through the ER subtypes, ERα and ERβ. In addition to the action of ER as a classical estrogen response element, ER α and ERβ also mediate gene transcription from an AP-1 enhancer element. Paech et al reported that ER α and ERβ from an AP-1 site signaled in opposite ways when combined with estradiol: estradiol activated transcription with ER α , whereas estradiol inhibited transcription with ER β [50] . A high level of ER β expression and a low level of ERα expression is seen in human and rat hepatocytes[49,51,52]. In addition, estradiol up-regulates the Bcl-2 expression in cultured rat hepatocytes undergoing oxidative stress[49]. The overexpression of Bcl-2 is known to suppress lipid peroxidation and to prevent apoptosis, leading to an increase in cellular longevity. These findings suggest that estradiol may protect hepatocytes from oxidative damage, inflammatory cell injury and cell death by the suppression of AP-1 and NF-κB activation and induction of Bcl-2 expression.

FEMALES AND HEPATIC IRON STORES Iron is known to be a potent in vivo factor in the www.wjgnet.com

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production of hydroxy radicals, leading to DNA mutagenesis [53]. In humans with iron overload due to genetic or acquired causes, fibrosis and cirrhosis are the predominant features of liver disease. The clinical expression of genetic hemochromatosis is 5 to 10 times more frequent in men than in women, possibly because of iron loss during menstruation and increased requirements during pregnancy. In the absence of hepatic inflammation, serum ferritin is a reliable marker of hepatic iron stores. In a study from Japan, serum ferritin levels were examined by gender and age in 305 healthy individuals. In men, the serum ferritin values reached the maximum level in the age group of 40 to 49 years and declined thereafter. By contrast, in women the serum ferritin remained relatively low until menopause, at a level which was one-fifth of that for men of comparable age, and reached the maximum level after menopause to approximately one-half of that for men of comparable age. These findings correlate with the data obtained from the third National Health and Nutrition Examination Survey in the USA[54] and indicate that women, especially before menopause, have lower iron stores in the liver (Figure 4). Alizadeh et al analyzed the liver histology of age- and sex-matched untreated patients with chronic hepatitis B (n = 20) and chronic hepatitis C (n = 43) and showed that the hepatocyte iron-staining score was similar in chronic hepatitis B and C[55]. The frequency of iron-staining in chronic hepatitis B and C was significantly greater than that seen in alcoholic liver disease[56]. It should be noted that hepatic iron overload is an independent risk factor associated with hepatic fibrosis and cell death [10,57]. In Chinese patients with HBV-associated HCC, stainable iron in non-cancerous and cancerous liver specimens was preferentially enriched in HBV replicating hepatocytes, and was lower in 25% (1/4) of female patients compared to 71% (25/35) of males [58] . Deugnier et al reported that HBsAg seropositivity in male patients with genetic hemochromatosis complicated with HCC was significantly higher (6.2%) than in sex- and age-matched male blood donors (0.075%) and female controls (0.04%), whereas female patients with and without HCC showed no serum HBV marker[59]. These findings suggest that hepatic iron overload may facilitate viral replication in hepatocytes, or that the virus infected hepatocytes tend to accumulate iron. In addition, the lower hepatic iron stores in females may, in part, explain their greater cytoprotection against liver injury. Chronic alcohol consumption in moderate to heavy quantities results in increased serum ferritin, which may result in increased hepatic iron stores[60]. Hepcidin is a circulatory peptide synthesized in the liver that appears to regulate iron absorption in the duodenum. HarrisonFindik et al treated mice with ethanol added to drinking water for 7 d[61]. Ethanol-treated mice showed significantly lower hepatic hepcidin mRNA expression compared to controls, and also demonstrated greater suppression of hepcidin gene expression in males than females. This effect was abrogated by an antioxidant, vitamin E, while ROS production by ethanol via the alcohol dehydrogenaseand CYP2E1-metabolizing system appeared to result in decreased expression of hepcidin. These findings indicate

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that alcohol-induced oxidative stress downregulates hepcidin transcription, leading to increased duodenal iron transport, and chronic alcohol consumption leads to increased hepatic iron stores, which is more likely to occur in males than in females. However, females are more vulnerable to alcohol-related damage because they have smaller volume of distribution and lower gastric alcohol dehydrogenase activity[62]. In addition, it has been reported that after alcohol consumption, estrogen stimulation of Kupffer cells increases their sensitivity to endotoxins and leads to higher levels of chemical mediators [63]. These findings suggest that chronic alcohol use may induce more rapid and more severe liver injury in females than in males[64].

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ESTROGENS AND HEPATIC FIBROSIS During ongoing HBV replication irrespective of the HBeAg seroconversion status, hepatic fibrosis eventually progresses to the stage of cirrhosis. The incidence of cirrhosis in chronic HBV infection, based on data mostly from tertiary care centers, ranges from 2% to 7% annually[20,65-68], although, in a study on 1506 asymptomatic HBV carriers, the cirrhosis incidence rate was 0.7% annually[69]. Activated HSCs are responsible for much of the collagen synthesis observed during hepatofibrogenesis. Male gender and older age are associated with a more rapid progression of hepatic fibrosis [64]. Using multivariate analysis, Iloeje et al in Taiwan, China[70] and Zarski et al in France[71] observed that the independent predictors for cirrhosis in patients with chronic HBV infection were male sex and age > 50 years. McMahon also demonstrated that male sex and old age were the most important factors associated with disease progression in chronic hepatitis B[72]. Patients infected with HBV genotype C have delayed HBeAg seroconversion, a longer duration of viremia, higher risk of progressive disease, and a correspondingly higher rate of cirrhosis and HCC[73]. Genotypes C and B are prevalent in Asia, whereas genotypes A and D are common in Western countries. In addition, the significance of older age as a risk factor may reflect a more prolonged duration of the liver disease and accumulated exposure to environmental risk factors such as aflatoxin, in highly endemic areas[74]. An increased prevalence of HBeAg negative chronic hepatitis B has recently been observed in several countries[75,76]. Patients with HBeAg negative chronic hepatitis B are usually males who are older than those with HBeAg positive chronic hepatitis B [76]. Moreover, the age at the time of acute infection plays an important role in the development of chronicity. Neonatal infection, common in areas of high or intermittent HBV prevalence, is associated with high rates of chronicity, while exposure at an adult age, only occasionally results in chronic infection. An ideal method of assessment of disease progression would be to prospectively follow a large cohort of untreated patients with all the major HBV genotypes (A to D) from infection until death, with repeated liver biopsy and measurement of HBV serological markers. However, such a study would be ethically and pragmatically impossible to perform in an unselected population.

Figure 5 Estrogens and hepatic fibrosis. A: Immunohistochemical findings of collagen typeⅠin fibrotic liver sections of male rats treated with a neutralizing antibody against rat estradiol (+ Antibody) or estradiol (+ Estradiol). The animals were given a single injection of dimethylnitrosamine and were killed on d 14; B: Immunocytochemical findings for α-SMA of a male rat HSCs cultured with estradiol (+ Estradiol).

In the hepatic fibrosis model in male rats, estradiol results in suppression of early apoptosis and hepatic fibrosis. These changes are accompanied with reduced collagen content and lower levels of procollagen type Ⅰand Ⅲ mRNA and α -SMA expression as well as induced Bcl-2 expression [77-79]. In addition, the use of neutralizing antibody against estradiol in male rats (Figure 5A) and ovarectomy in female rats leads to enhanced fibrogenesis[77]. Rat HSCs possess functional ERβ, but not ERα, which respond directly to estradiol exposure; estradiol attenuates the production of collagen type Ⅰ, α-SMA expression and cell proliferation in cultured rat HSCs [51,77,78] (Figure 5B). A recent report indicates that estradiol inhibits ROS generation and antioxidant enzyme loss via the suppression of NADH/NADPH oxidase activity, and blocks hydrogen peroxide-induced TGF-β expression, activation of AP-1 and NF-κB, and proliferation and transformation of cultured rat HSCs[45]. These findings suggest that, by suppressing NADH/ NADPH oxidase activity, estradiol prevents the autocrine loop of ROS and TGF-β by HSCs (Figure 3) as well as HSC activation, and has a cytoprotective effect against hepatocyte injury.

FEMALES AND HEPATIC STEATOSIS Hepatic steatosis is a characteristic feature of chronic HBV and HCV infections. The frequency of hepatic steatosis in chronic hepatitis B varies from 27% to 51%, and in chronic hepatitis C from 31% and 72%[80-83]. It has been suggested that hepatic steatosis may reflect a direct cytopathic effect of HCV and plays a role in disease progression. In support of this hypothesis is a transgenic mouse model that expresses HCV core gene and develops progressive hepatic steatosis and HCC [84,85]. It is conceivable that www.wjgnet.com

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Figure 6 Prevalence of NAFLD by age and gender. NAFLD prevalence was determined ultrasonographically in 3229 Japanese adults (mean age 52.4 years, 45.9% males) in the Tokushima health screening center. The subjects had no history of alcohol abuse, defined as an alcohol intake of > 20 g per day, and were seronegative for HBsAg and HCV antibody. The overall prevalence of NAFLD was 25.6%.

following hepatocyte injury, hepatic steatosis leads to increased lipid peroxidation, which contributes to HSC activation by releasing soluble mediators[86], and thereby induces hepatic fibrosis. In contrast to HCV, there is little information on the correlation between HBV-associated hepatic steatosis and hepatic fibrosis. Furthermore, the molecular mechanism by which HBV mediates hepatic steatosis has not been clearly characterized. Although a cross-sectional study from Australia failed to confirm the impact of steatosis on hepatic fibrosis in chronic hepatitis B (but not in hepatitis C) [87] , another cross-sectional analysis in Taiwanese adults revealed that liver damage, evaluated by serum alanine aminotransferase (ALT) levels, was independently associated with HBV carrier status, ultrasonographic fatty liver and male sex[88]. Fatty liver, also termed hepatic steatosis, is the result of the deposition of triglycerides via the accumulation of free fatty acids in hepatocytes. Fatty liver is detected by ultrasonography based on the comparative assessment of image brightness relative to the kidneys [89]. Recently Kim et al reported that HBx protein induces hepatic lipid accumulation mediated by sterol regulatory element binding protein 1 and peroxisome proliferator-activated receptor γ, leading to hepatic steatosis[90]. There is mounting evidence that hepatic lipid accumulation is related to hepatic fibrosis, inflammation, apoptosis, and cancer[91,92]. Increased lipid peroxidation and accumulation are also commonly observed in alcoholic liver disease and NAFLD based on studies in human alcohol-related liver injury and animal models of diet-induced hepatic steatosis and druginduced steatohepatitis[93-95]. In the progression of fatty liver disease, lipid peroxidation products are generated because of impaired oxidation of the accumulated fatty acids. The key mediators of impaired β-oxidation include increased activity of cytochrome P450 and reduced electron transport in hepatocyte mitochondria. The secretion of inflammatory mediators including TNF-α and ROS by Kupffer cells resulting in HSC activation with disordered collagen production, are believed to play an important role in NASH-associated cryptogenic cirrhosis. www.wjgnet.com

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In Japan, Australia, Western Europe and the USA, ultrasonographic sur veys of the general population indicate that nearly one-quarter of the adult population has hepatic steatosis[96,97]. NAFLD is more common in males than females, particularly in Asians[98]. In a study on the gender differences in NAFLD among Asians, the prevalence of ultrasonographic NAFLD was examined in 3229 Japanese adults in a health screening center in Tokushima. Prevalence of NAFLD was 2.5-fold higher in males than females (31.5% vs 12.4%). The biggest difference in NAFLD prevalence between females and males was observed in individuals < 50 years (Figure 6). Recent studies have shown that visceral fat is an independent predictor of fatty liver, even in patients with a normal body mass index, and is much more harmful than subcutaneous accumulation of adipose tissue[99,100]. Human adipose tissue contains ER α and ER β . Low estrogen levels in menopausal women are associated with a loss in subcutaneous fat and gain in visceral fat[101]. Estrogen treatment of male-to-female transsexuals increases the amount of subcutaneous adipose tissue. Thus, estrogen alters the male type of visceral fat distribution into the female type[102]. It has been reported that women treated with estrogen have a lower visceral accumulation of adipose tissue[103]. An animal study showed that hepatic steatosis becomes evident spontaneously in aromatase-deficient mouse, which lack the ability to produce estrogen and are impaired with respect to hepatocellular fatty acid β -oxidation. Estradiol replacement reduces hepatic steatosis and restores the impairment in mitochondrial and peroxisomal fatty acid β -oxidation to the wild-type level [104] . In addition, tamoxifen an antiestrogen compound used in the treatment of ER positive breast cancer, is associated with an increased risk of fatty liver and NASH[105,106]. Therefore, the greater progression of liver injury with steatosis in males, regardless of the etiology, may be due at least in part to the decreased production of estradiol and/or the lower response to the actions of estradiol.

MENOPAUSE AND PROINFLAMMATORY CYTOKINES I n i n f l a m m a t o r y a n d ox i d a t ive l ive r i n j u r y, t h e accumulation of leukocytes and macrophages including Kupffer cells at sites of inflammation and injury is thought to be mediated by chemokines, such as macrophage chemotactic protein (MCP)-1 and interleukin (IL)-8. The mononuclear cells and macrophages are in turn able to release proinflammatory cytokines such as tumor necrosis factor (TNF)-α, IL-1β and IL-6, leading to persistent liver injury. There is a large body of evidence indicating that the decline in ovarian function with menopause is associated with spontaneous increase in TNF-α, IL-1β and IL-6[107]. Estradiol, at physiological concentrations, inhibits the spontaneous secretion of proinflammatory cytokines in whole blood cultures[108] or peripheral blood mononuclear cells (PBMCs) [109] . The spontaneous production of TNF-α and IL-1β by PBMCs is higher in patients with chronic hepatitis C than in healthy subjects[110]. Endotoxin-

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60 40 % of HCC cases

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Figure 7 Comparison of male-to-female ratio between two age groups of HBVrelated HCC patients without HCV infection. The age-specific male-to-female ratio in Japanese patients with HBV-associated HCC was examined from 1994 to 2006 in Tokushima. Nine hundred and one patients (mean age 49.3 yr, 76.4% males) were divided into two age groups, based on the menopausal age of 50 yr. HCC was detected at the indicated age. All subjects were seropositive for HBsAg and seronegative for HCV antibody.

stimulated TNF-α production by PBMCs is also higher in HBsAg carriers with elevated ALT levels than in HBsAg carriers with normal ALT levels [111]. Moreover, TNF-α production by hepatocytes in patients with chronic HBV infection is reported to be transcriptionally up-regulated by HBx protein[112,113]. Estradiol is able to attenuate IL-1β in ER expressing HepG2 cells[114], and to ameliorate burninduced increase in serum TNF-α levels in rats[115]. In vivo studies show that transdermal administration of estradiol in postmenopausal women decreases spontaneous IL-6 production by PBMCs after 12 mo of therapy [109] . A preliminary study has also shown that hydrogen peroxideinduced TNF-α and MCP-1 expressions are attenuated by estradiol in the peritoneal macrophages of female mice[116]. These findings suggest that estradiol has a hepatoprotective effect against inflammation and oxidative stress, at least in part, by preventing macrophage accumulation and inhibiting proinflammatory cytokine production. However, it appears that macrophages respond differently to endotoxins compared to Kupffer cells as far as the signaling pathways are concerned[117]. Estrogen increases the sensitivity of Kupffer cells to endotoxin[63], while estradiol augments the increase in the serum levels of TNF-α after endotoxin treatment in animals[118].

FEMALES AND HCC Both direct and indirect carcinogenic mechanisms are involved in the pathogenesis of HCC induced by chronic HBV infection. HBV may induce HCC indirectly by causing chronic necroinflammatory liver disease [23] . When HBV replication is sustained, hepatocytes undergo a process of continuous damage and regeneration. Chronic necroinflammation may induce a malignant transformation by producing mutagenic ROS during the inflammatory process along with hepatic fibrosis, leading to the development of cirrhosis and HCC (Figure 3). The active replication of HBV may also initiate malignant transformation through a direct carcinogenic mechanism

Figure 8 Estrogens and preneoplastic liver. Preneoplastic liver lesions were evaluated by means of an immunohistochemical analysis of glutathione-Stransferase placental form (GST-P) expression. GST-P-positive liver foci were induced using the DEN-AAF-PH model in male rats with estradiol (+ Estradiol).

by increasing the probability of insertion of viral DNA in or near proto-oncogenes, tumor-suppressor genes, or their regulatory elements in the cellular DNA [119]. The integration of viral DNA may increase the production of transactivator protein HBx antigen, which may induce the malignant transformation of hepatocytes, and bind to the p53 tumor-suppressor gene and disrupt its activity[23,120]. As with the risk factors for hepatic fibrosis, male sex and age > 50 years are important risk factors for HCC[121], although it is unclear whether the susceptibility to the integration of viral DNA inducing malignant transfor mation of hepatocytes is different between males and females. Conversely, premenopausal women, without the risk factors of male sex and older age, are least vulnerable to HCC. The age-specific male-to-female ratio was examined in 901 Japanese patients with HBVassociated HCC in Tokushima. When the subjects were divided into two age groups, based on whether they were younger or older than the menopausal age of 50 years, the younger group had a significantly lower proportion of females (10.5%) than the older group (32.8%, Figure 7). Moreover, variant ERs are expressed in HCC patients and, to a greater extent, in male patients than in female patients, even at an early stage of chronic liver disease[122,123]. The occurrence of variant ERs leads to the loss of estrogen responsiveness. Experimentally induced carcinomas using carcinogens, as well as spontaneous neoplasms, occur at a higher rate in male rats and mice. Another study demonstrated the suppressive effect of estradiol on chemical hepatocarcinogenesis in rats induced by dimethylnitrosamine (DEN)-2-acetylaminofluorene (AAF)partial hepatectomy (PH)[124] (Figure 8). Taken together, these lines of evidence suggest that both estradiol and the ER status play a critical role in the biological defense against hepatocarcinogenesis.

CONCLUSION Gender-associated differences are not limited to chronic www.wjgnet.com

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liver disease and are of potential interest in other chronic fibrogenic disorders as well. The predominance of atherosclerosis and the higher rate of renal fibrosis progression in men are excellent lines of evidence that point to the role of estrogen in the wound healing/ fibrogenic process[125]. It has been reported that estradiol inhibits the proliferation of vascular smooth muscle cells (VSMCs)[126]. VSMCs are anatomically analogous to HSCs, and are reported to express ERβ at a higher level after vascular injury, without any accompanying changes in ERα expression[127]. Several studies have documented the antifibrogenic effect of estrogen on VSMCs [128,129]. Moreover, renal mesangial cells which are analogous to HSC, have similar properties including playing a prominent role in fibrogenesis. The cell proliferation and collagen synthesis by mesangial cells have also been shown to be modulated by estradiol[130]. The present review indicates that estradiol may have a beneficial effect in the progression of chronic liver disease. However, it should be noted that administration of estradiol in women has potential problems, including an increased risk of breast cancer and endometrial abnormalities[131,132]. In addition, estradiol and ER subtypes have been reported to play a role in the modulation of cholangiocyte proliferation[133], which is a hallmark for the progression of cholestatic liver disease. Being a male or a female is an important basic human variable that affects health and liver disease throughout ones life span. A better understanding of the biological mechanisms underlying the gender-associated differences observed in chronic HBV infection may provide valuable information on more effective treatment modalities in liver disease in both males and females.

REFERENCES Lee WM. Hepatitis B virus infection. N Engl J Med 1997; 337: 1733-1745 WHO. Hepatitis C: global prevalence. Wkly Epidemiol Rec 1997; 2 72: 341-344 Seeff LB, Beebe GW, Hoofnagle JH, Norman JE, Buskell-Bales 3 Z, Waggoner JG, Kaplowitz N, Koff RS, Petrini JL Jr, Schiff ER. A serologic follow-up of the 1942 epidemic of post-vaccination hepatitis in the United States Army. N Engl J Med 1987; 316: 965-970 Lok AS, McMahon BJ. Chronic hepatitis B. Hepatology 2001; 34: 4 1225-1241 Lok AS. Chronic hepatitis B. N Engl J Med 2002; 346: 1682-1683 5 6 Yu MW, Chen CJ. Hepatitis B and C viruses in the development of hepatocellular carcinoma. Crit Rev Oncol Hematol 1994; 17: 71-91 McMahon BJ, Alberts SR, Wainwright RB, Bulkow L, Lanier 7 AP. Hepatitis B-related sequelae. Prospective study in 1400 hepatitis B surface antigen-positive Alaska native carriers. Arch Intern Med 1990; 150: 1051-1054 8 Poynard T, Ratziu V, Charlotte F, Goodman Z, McHutchison J, Albrecht J. Rates and risk factors of liver fibrosis progression in patients with chronic hepatitis c. J Hepatol 2001; 34: 730-739 Shimizu I. Impact of oestrogens on the progression of liver 9 disease. Liver Int 2003; 23: 63-69 10 Shimizu I, Ito S. Protection of estrogens against the progression of chronic liver disease. Hepatol Res 2007; 37: 239-247 11 Kao JH, Chen DS. Recent research progress in hepatocellular carcinoma. J Formos Med Assoc 2002; 101: 239-248 12 Chisari FV, Ferrari C. Hepatitis B virus immunopathogenesis. 1

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Annu Rev Immunol 1995; 13: 29-60 13 Seeff LB. Natural history of chronic hepatitis C. Hepatology 2002; 36: S35-S46 14 Cancer Incidence in Five Continents Vol. VIII. IARC Scientific Publications No. 155. Lyon, France: International Agency for Research on Cancer, 2002: 566-568 15 Kensler TW, Qian GS, Chen JG, Groopman JD. Translational strategies for cancer prevention in liver. Nat Rev Cancer 2003; 3: 321-329 16 Harrison SA, Kadakia S, Lang KA, Schenker S. Nonalcoholic steatohepatitis: what we know in the new millennium. Am J Gastroenterol 2002; 97: 2714-2724 17 Weltman MD, Farrell GC, Hall P, Ingelman-Sundberg M, Liddle C. Hepatic cytochrome P450 2E1 is increased in patients with nonalcoholic steatohepatitis. Hepatology 1998; 27: 128-133 18 American Gastroenterological Association. American Gastroenterological Association medical position statement: nonalcoholic fatty liver disease. Gastroenterology 2002; 123: 1702-1704 19 Brunetto MR, Oliveri F, Coco B, Leandro G, Colombatto P, Gorin JM, Bonino F. Outcome of anti-HBe positive chronic hepatitis B in alpha-interferon treated and untreated patients: a long term cohort study. J Hepatol 2002; 36: 263-270 20 Hsu YS, Chien RN, Yeh CT, Sheen IS, Chiou HY, Chu CM, Liaw YF. Long-term outcome after spontaneous HBeAg seroconversion in patients with chronic hepatitis B. Hepatology 2002; 35: 1522-1327 21 Bortolotti F, Jara P, Crivellaro C, Hierro L, Cadrobbi P, Frauca E, Camarena C, De La Vega A, Diaz C, De Moliner L, Noventa F. Outcome of chronic hepatitis B in Caucasian children during a 20-year observation period. J Hepatol 1998; 29: 184-190 22 Chan HL, Hui AY, Wong ML, Tse AM, Hung LC, Wong VW, Sung JJ. Genotype C hepatitis B virus infection is associated with an increased risk of hepatocellular carcinoma. Gut 2004; 53: 1494-1498 23 Yang HI, Lu SN, Liaw YF, You SL, Sun CA, Wang LY, Hsiao CK, Chen PJ, Chen DS, Chen CJ. Hepatitis B e antigen and the risk of hepatocellular carcinoma. N Engl J Med 2002; 347: 168-174 24 Alward WL, McMahon BJ, Hall DB, Heyward WL, Francis DP, Bender TR. The long-term serological course of asymptomatic hepatitis B virus carriers and the development of primary hepatocellular carcinoma. J Infect Dis 1985; 151: 604-609 25 Zacharakis GH, Koskinas J, Kotsiou S, Papoutselis M, Tzara F, Vafeiadis N, Archimandritis AJ, Papoutselis K. Natural history of chronic HBV infection: a cohort study with up to 12 years follow-up in North Greece (part of the Interreg I-II/ECproject). J Med Virol 2005; 77: 173-179 26 Ansar Ahmed S, Penhale WJ, Talal N. Sex hormones, immune responses, and autoimmune diseases. Mechanisms of sex hormone action. Am J Pathol 1985; 121: 531-551 27 Grossman CJ. Interactions between the gonadal steroids and the immune system. Science 1985; 227: 257-261 28 Fox HS, Bond BL, Parslow TG. Estrogen regulates the IFNgamma promoter. J Immunol 1991; 146: 4362-4367 29 Clerici E, Bergamasco E, Ferrario E, Villa ML. Influence of sex steroids on the antigen-specific primary antibody response in vitro. J Clin Lab Immunol 1991; 34: 71-78 30 Sjogren MH. Prevention of hepatitis B in nonresponders to initial hepatitis B virus vaccination. Am J Med 2005; 118 Suppl 10A: 34S-39S 31 McMahon BJ, Bruden DL, Petersen KM, Bulkow LR, Parkinson AJ, Nainan O, Khristova M, Zanis C, Peters H, Margolis HS. Antibody levels and protection after hepatitis B vaccination: results of a 15-year follow-up. Ann Intern Med 2005; 142: 333-341 32 Su FH, Chen JD, Cheng SH, Lin CH, Liu YH, Chu FY. Seroprevalence of Hepatitis-B infection amongst Taiwanese university students 18 years following the commencement of a national Hepatitis-B vaccination program. J Med Virol 2007; 79: 138-143 33 Poli G. Pathogenesis of liver fibrosis: role of oxidative stress. Mol Aspects Med 2000; 21: 49-98 34 Wake K. Cell-cell organization and functions of 'sinusoids' in

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Zarski JP, Marcellin P, Leroy V, Trepo C, Samuel D, Ganne-Carrie N, Barange K, Canva V, Doffoel M, Cales P. Characteristics of patients with chronic hepatitis B in France: predominant frequency of HBe antigen negative cases. J Hepatol 2006; 45: 355-360 McMahon BJ. The natural history of chronic hepatitis B virus infection. Semin Liver Dis 2004; 24 Suppl 1: 17-21 Fung SK, Lok AS. Hepatitis B virus genotypes: do they play a role in the outcome of HBV infection? Hepatology 2004; 40: 790-792 Kensler TW, Egner PA, Wang JB, Zhu YR, Zhang BC, Lu PX, Chen JG, Qian GS, Kuang SY, Jackson PE, Gange SJ, Jacobson LP, Munoz A, Groopman JD. Chemoprevention of hepatocellular carcinoma in aflatoxin endemic areas. Gastroenterology 2004; 127: S310-S318 Funk ML, Rosenberg DM, Lok AS. World-wide epidemiology of HBeAg-negative chronic hepatitis B and associated precore and core promoter variants. J Viral Hepat 2002; 9: 52-61 Hadziyannis SJ, Papatheodoridis GV. Hepatitis B e antigennegative chronic hepatitis B: natural history and treatment. Semin Liver Dis 2006; 26: 130-141 Yasuda M, Shimizu I, Shiba M, Ito S. Suppressive effects of estradiol on dimethylnitrosamine-induced fibrosis of the liver in rats. Hepatology 1999; 29: 719-727 Shimizu I, Mizobuchi Y, Yasuda M, Shiba M, Ma YR, Horie T, Liu F, Ito S. Inhibitory effect of oestradiol on activation of rat hepatic stellate cells in vivo and in vitro. Gut 1999; 44: 127-136 Lu G, Shimizu I, Cui X, Itonaga M, Tamaki K, Fukuno H, Inoue H, Honda H, Ito S. Antioxidant and antiapoptotic activities of idoxifene and estradiol in hepatic fibrosis in rats. Life Sci 2004; 74: 897-907 Scheuer PJ, Ashrafzadeh P, Sherlock S, Brown D, Dusheiko GM. The pathology of hepatitis C. Hepatology 1992; 15: 567-571 Bach N, Thung SN, Schaffner F. The histological features of chronic hepatitis C and autoimmune chronic hepatitis: a comparative analysis. Hepatology 1992; 15: 572-577 Lefkowitch JH, Schiff ER, Davis GL, Perrillo RP, Lindsay K, Bodenheimer HC Jr, Balart LA, Ortego TJ, Payne J, Dienstag JL. Pathological diagnosis of chronic hepatitis C: a multicenter comparative study with chronic hepatitis B. The Hepatitis Interventional Therapy Group. Gastroenterology 1993; 104: 595-603 Czaja AJ, Carpenter HA. Sensitivity, specificity, and predictability of biopsy interpretations in chronic hepatitis. Gastroenterology 1993; 105: 1824-1832 Moriya K, Yotsuyanagi H, Shintani Y, Fujie H, Ishibashi K, Matsuura Y, Miyamura T, Koike K. Hepatitis C virus core protein induces hepatic steatosis in transgenic mice. J Gen Virol 1997; 78 ( Pt 7): 1527-1531 Moriya K, Fujie H, Shintani Y, Yotsuyanagi H, Tsutsumi T, Ishibashi K, Matsuura Y, Kimura S, Miyamura T, Koike K. The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice. Nat Med 1998; 4: 1065-1067 Gressner AM, Lotfi S, Gressner G, Lahme B. Identification and partial characterization of a hepatocyte-derived factor promoting proliferation of cultured fat-storing cells (parasinusoidal lipocytes). Hepatology 1992; 16: 1250-1266 Gordon A, McLean CA, Pedersen JS, Bailey MJ, Roberts SK. Hepatic steatosis in chronic hepatitis B and C: predictors, distribution and effect on fibrosis. J Hepatol 2005; 43: 38-44 Lin YC, Hsiao ST, Chen JD. Sonographic fatty liver and hepatitis B virus carrier status: synergistic effect on liver damage in Taiwanese adults. World J Gastroenterol 2007; 13: 1805-1810 Saadeh S, Younossi ZM, Remer EM, Gramlich T, Ong JP, Hurley M, Mullen KD, Cooper JN, Sheridan MJ. The utility of radiological imaging in nonalcoholic fatty liver disease. Gastroenterology 2002; 123: 745-750 Kim KH, Shin HJ, Kim K, Choi HM, Rhee SH, Moon HB, Kim HH, Yang US, Yu DY, Cheong J. Hepatitis B virus X protein induces hepatic steatosis via transcriptional activation of SREBP1 and PPARgamma. Gastroenterology 2007; 132: 1955-1967

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Ohata K, Hamasaki K, Toriyama K, Matsumoto K, Saeki A, Yanagi K, Abiru S, Nakagawa Y, Shigeno M, Miyazoe S, Ichikawa T, Ishikawa H, Nakao K, Eguchi K. Hepatic steatosis is a risk factor for hepatocellular carcinoma in patients with chronic hepatitis C virus infection. Cancer 2003; 97: 3036-3043 Powell EE, Jonsson JR, Clouston AD. Steatosis: co-factor in other liver diseases. Hepatology 2005; 42: 5-13 Letteron P, Duchatelle V, Berson A, Fromenty B, Fisch C, Degott C, Benhamou JP, Pessayre D. Increased ethane exhalation, an in vivo index of lipid peroxidation, in alcoholabusers. Gut 1993; 34: 409-414 Letteron P, Fromenty B, Terris B, Degott C, Pessayre D. Acute and chronic hepatic steatosis lead to in vivo lipid peroxidation in mice. J Hepatol 1996; 24: 200-208 Berson A, De Beco V, Letteron P, Robin MA, Moreau C, El Kahwaji J, Verthier N, Feldmann G, Fromenty B, Pessayre D. Steatohepatitis-inducing drugs cause mitochondrial dysfunction and lipid peroxidation in rat hepatocytes. Gastroenterology 1998; 114: 764-774 Sanyal AJ. AGA technical review on nonalcoholic fatty liver disease. Gastroenterology 2002; 123: 1705-1725 Clark JM, Diehl AM. Defining nonalcoholic fatty liver disease: implications for epidemiologic studies. Gastroenterology 2003; 124: 248-250 Weston SR, Leyden W, Murphy R, Bass NM, Bell BP, Manos MM, Terrault NA. Racial and ethnic distribution of nonalcoholic fatty liver in persons with newly diagnosed chronic liver disease. Hepatology 2005; 41: 372-379 Bellentani S, Saccoccio G, Masutti F, Croce LS, Brandi G, Sasso F, Cristanini G, Tiribelli C. Prevalence of and risk factors for hepatic steatosis in Northern Italy. Ann Intern Med 2000; 132: 112-117 Omagari K, Kadokawa Y, Masuda J, Egawa I, Sawa T, Hazama H, Ohba K, Isomoto H, Mizuta Y, Hayashida K, Murase K, Kadota T, Murata I, Kohno S. Fatty liver in non-alcoholic non-overweight Japanese adults: incidence and clinical characteristics. J Gastroenterol Hepatol 2002; 17: 1098-1105 Toth MJ, Tchernof A, Sites CK, Poehlman ET. Effect of menopausal status on body composition and abdominal fat distribution. Int J Obes Relat Metab Disord 2000; 24: 226-231 Elbers JM, Asscheman H, Seidell JC, Gooren LJ. Effects of sex steroid hormones on regional fat depots as assessed by magnetic resonance imaging in transsexuals. Am J Physiol 1999; 276: E317-E325 Haarbo J, Marslew U, Gotfredsen A, Christiansen C. Postmenopausal hormone replacement therapy prevents central distribution of body fat after menopause. Metabolism 1991; 40: 1323-1326 Nemoto Y, Toda K, Ono M, Fujikawa-Adachi K, Saibara T, Onishi S, Enzan H, Okada T, Shizuta Y. Altered expression of fatty acid-metabolizing enzymes in aromatase-deficient mice. J Clin Invest 2000; 105: 1819-1825 Van Hoof M, Rahier J, Horsmans Y. Tamoxifen-induced steatohepatitis. Ann Intern Med 1996; 124: 855-856 Oien KA, Moffat D, Curry GW, Dickson J, Habeshaw T, Mills PR, MacSween RN. Cirrhosis with steatohepatitis after adjuvant tamoxifen. Lancet 1999; 353: 36-37 Pfeilschifter J, Koditz R, Pfohl M, Schatz H. Changes in proinflammatory cytokine activity after menopause. Endocr Rev 2002; 23: 90-119 Rogers A, Eastell R. The effect of 17beta-estradiol on production of cytokines in cultures of peripheral blood. Bone 2001; 29: 30-34 Rachon D, Mysliwska J, Suchecka-Rachon K, Wieckiewicz J, Mysliwski A. Effects of oestrogen deprivation on interleukin-6 production by peripheral blood mononuclear cells of postmenopausal women. J Endocrinol 2002; 172: 387-395 Kishihara Y, Hayashi J, Yoshimura E, Yamaji K, Nakashima K, Kashiwagi S. IL-1 beta and TNF-alpha produced by peripheral blood mononuclear cells before and during interferon therapy in patients with chronic hepatitis C. Dig Dis Sci 1996; 41: 315-321 Hsu HY, Chang MH, Ni YH, Lee PI. Cytokine release of peripheral blood mononuclear cells in children with chronic

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World J Gastroenterol 2007 August 28; 13(32): 4306-4309 World Journal of Gastroenterology ISSN 1007-9327 © 2007 WJG. All rights reserved.

EDITORIAL

Is it relevant that intra-arterial chemotherapy may be effective for advanced pancreatic cancer? Toru Ishikawa To r u I s h i k a w a , Departments of Gastroenterology and Hepatology, Saiseikai Niigata Second Hospital, Niigata, Japan Correspondence to: Toru Ishikawa, MD, Department of Gastroenterology, Saiseikai Niigata Second Hospital, Teraji 280-7, Niigata 950-1104, Japan. [email protected] Telephone: +81-25-2336161 Fax: +81-25-2338880 Received: 2007-04-04 Accepted: 2007-04-12

Abstract Unresectable pancreatic cancers have an extremely dismal prognosis and chemoresistant nature. The treatment of pancreatic cancer is still problematic. Gemcitabine is a promising new agent that has been studied recently for palliation of advanced pancreatic cancer. However, the response rates have been highly variable, and are often irreproducible. To improve this low response rate, various treatments are needed because no standard treatment exists. Intra-arterial chemotherapy is considered to take advantage of the first pass effect of the drug, generating higher local drug concentrations in tumor cells with lower toxicity. Regional intra-arterial chemotherapy may provide high levels of cytostatic concentrations within the tumor and, simultaneously, a low rate of systemic side effects compared with systemic administration of anti-neoplastic drugs. Intra-arterial chemotherapy has been introduced as an alternative treatment for advanced pancreatic cancer. Further clinical trials of this method should be subjected to a prospective randomized controlled study for advanced pancreatic cancer. © 2007 WJG . All rights reserved.

Key words: Pancreatic cancer; Intra-arterial chemotherapy; Systemic chemotherapy Ishikawa T. Is it relevant that intra-arterial chemotherapy may be effective for advanced pancreatic cancer? World J Gastroenterol 2007; 13(32): 4306-4309

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INTRODUCTION Pancreatic cancer is a malignancy with a poor prognosis[1]. Surgery is considered the only curative therapy. However, the 5-year survival rate after resection of pancreatic cancer is still very low, even when radical surgery is performed[2,3]. www.wjgnet.com

The cause of death in patients with advanced pancreatic cancer is primarily local progression of cancer and distant metastasis. At diagnosis pancreatic cancer is most commonly locally advanced and often accompanied by distant metastases to the liver and peritoneum[4]. Unresectable pancreatic cancers have an extremely dismal prognosis and chemoresistant nature because systemic chemotherapy is of limited effectiveness for this disease[5-9]. The treatment of pancreatic cancer is still problematic and a major challenge for oncologists because of its highly dismal outcomes. Many chemotherapeutic agents have been tested as single or combination therapies for pancreas cancer. Recently, it has been reported that gemcitabine (Gemzar: Eli Lilly Co Ltd., Tokyo, Japan) is an effective drug in the treatment of patients with metastatic pancreas cancer. Gemcitabine is a promising agent that improves the survival of patients with unresectable pancreas cancer, and now a standard first-line agent for these patients[10,11]. It is a nucleoside analogue with broad-spectrum antitumor activity. The major mechanism of action of gemcitabine is the inhibition of DNA synthesis. The primary endpoint of this trial was improvement in the clinical benefit response (CBR) score defined by performance status, weight gain, and pain control. However, the response rates have been highly variable, and are often irreproducible. To improve this low response rate, various treatments have been applied; however, no standard treatment exists. For these reasons, none of the reported treatment regimens can be considered to be standard treatment and in order to evaluate if intra-arterial regional chemotherapy is indeed superior to systemic chemotherapy, randomized trials must be conducted. In this paper, we review recent approaches to pancreas cancer in terms of regional chemotherapy.

SYSTEMIC CHEMOTHERAPY Pancreas cancer is considered an almost chemoresistant tumor; the average tumor response rate with 5-FU alone or in combination with other agents is in the range of 7%-28% [3,12]. Systemic adjuvant chemotherapy for pancreatic cancer has not increased the 5-year survival rate. Some researchers, therefore, have proposed that only symptomatic support should be performed for stage [13] Ⅳ advanced pancreatic cancer . Even though many chemotherapeutic agents have been evaluated in patients with pancreatic cancer, 5-fluorouracil (5-FU) has been the most important drug for 20 years[14].

Ishikawa T. Intra-arterial chemotherapy for advanced pancreatic cancer

Gemcitabine (Gemzar: Eli Lilly Co Ltd., Tokyo, Japan) is a promising new agent that has been recently studied for palliation of advanced (stage Ⅳ) unresectable pancreatic cancer and in pancreatic cancer it showed a response rate comparable to 5-FU, but with an improved clinical benefit [11]. Moreover, the action of gemcitabine seems to be synergetic with 5-FU. Both gemcitabine and 5-FU have shown well-recognized antitumor activity against pancreatic cancer and in in vitro assays showed synergistic activity[15]. It is clinically more beneficial in more patients than other chemotherapeutic agents such as 5-FU, but its efficacy is still insufficient even when combined with other agents. Systemic chemotherapy is of limited effectiveness. Approaches beyond systemic chemotherapy are needed for advanced pancreas cancer.

HEMODYNAMICS OF PANCREATIC CANCER Chemotherapy may not be effective because the sparse vascularity of pancreatic cancer may not allow for adequate drug accumulation in tumor tissues. The ineffectiveness of systemic chemotherapy is probably due to failure to reach a drug concentration within the tumor because of doselimited toxicity produced in bone marrow and epithelial tissue. Intratumoral blood vessels are immature, lacking both smooth muscle cells and immunoreactive nerves[16]. Therefore, tumor vessels are unable to react to vasoconstricting agents[17,18]. Angiotensin-Ⅱ (AT-Ⅱ) causes arteriolar constriction in normal blood vessels. It is a powerful vasoconstrictor which has been shown to alter the distribution of blood flow in favor of intrahepatic tumor perfusion during short (3-4 min) intra-arterial infusions of the compound[19]. We reported previously that chemotherapy with AT-Ⅱ was effective in some cancers[20-25]. So, we supposed that it is important for the strategy of pancreas cancer treatment to comprehend the hemodynamics of this cancer. We showed that administration of AT-Ⅱ enhanced the metastatic liver tumor as compared with normal tissue. Intratumoral blood flow increased in all patients with malignant tumors due to the pressure effect of AT-Ⅱ[26]. Furthermore, high detection rate of liver metastasis revealed by pharmacoangiographic CT administrating AT-Ⅱ sug gests the improvement of diagnosis on preoperative staging[26]. A relative increase in tumor blood flow may enable higher doses of regional chemotherapy to be given while avoiding hepatotoxicity. Thus, selective increase in tumor blood flow by AT-Ⅱ induces a marked increase in the tumor vascular area, which may improve not only the chemotherapeutic effects, but also the diagnostic effects in cancer patients. These results support that the delivery of anticancer drugs could be selectively enhanced in tumor tissue under AT-Ⅱ induced hypertension. This condition for drug delivery to tumor tissue may play a major role in enhancing therapeutic effects of chemotherapy.



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INTRA-ARTERIAL CHEMOTHERAPY Intra-arterial regional chemotherapy has improved the response rates and quality of life in patients with liver metastases from colorectal cancer[27]. Pancreas cancer is considered a chemoresistant tumor and up to now an individual drug with a high level of activity has been lacking. The most important reasons for this drug resistance are the presence of both a biological and a mechanical barrier. The first is the multidrug resistance gene (MDR1) product and the second a very dense, poorly vasculized, fibrotic envelope that is almost impenetrable by drugs[28]. Furthermore, the chemotherapy drugs are often quickly eliminated by a vigorous multidrug resistance mechanism in pancreas cancer[29]. In addition, pancreas cancer is highly resistant because it expresses moderate to high levels of P-170 glycoprotein. P-glycoprotein is part of a drug or toxin efflux enzyme system that rapidly clears chemotherapeutic agents from the tumor cell[30]. However, it is expected that the drug dose delivered by regional chemotherapy must be increased at least five-fold to overcome the tumor cell resistance derived from the P-170 drug efflux enzyme system. Intra-arterial chemotherapy for pancreatic cancer is still in its infancy and the ideal schedule does not yet exist. To improve the effect of chemotherapeutic agents against pancreatic cancers, effective methods for drug delivery into tumor tissues should be developed. Intra-arterial infusion allows higher drug concentrations to reach the tumor, overcoming the problem of poor blood flow to the tumor mass in comparison with healthy tissue. The dosedependent sensitivity of pancreatic cancer to locoregional chemotherapy has been shown in previous studies[31]. Although the purpose of intra-arterial chemotherapy is to deliver a high dose of anticancer agents into the cancer tissue and a low dose into the non-cancerous tissues, the conventional intra-arterial chemotherapy methods[32] for pancreatic cancer have not succeeded in this goal. Moreover, intra-arterial infusion is considered to take advantage of the first pass effect of the drug, generating higher local drug concentrations in tumor cells with lower toxicity. The chemotherapy is usually given through catheters placed in celiac/hepatic artery or portal vein. Early reports of intraarterial adjuvant therapy have been published[33,34]. In these studies, catheters were placed in the main tributaries of the celiac trunk and portal vein postoperatively to administer 5-FU. In the long-term follow-up, 1-, 3- and 5- year survivals were 92%, 54% and 39%, respectively[34]. Noteworthy in this study is the significantly lower rate of death from hepatic metastases, 8%, with intraarterial adjuvant therapy compared with controls, and the authors suggest that this effect improved survival. Further studies using intraarterial chemotherapy through a transcutaneous catheter via the femoral artery have been published by Beger et al[35,36]. In the long-term follow-up[35], this treatment resulted in a mean survival of 23 mo and 3-year survival of 48%. In the control group, the mean survival was a mere 10.5 mo. As adjuvant chemotherapy, a significantly better survival www.wjgnet.com

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Mambrini[37]

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Maurer[39] Homma[40] Cantone[41]

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Gausauge[42] Lorenz[43] Author's data[44]

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Cytostatic agents 5-FU, Leucovorin Epirubicin, Carboplatin 5-FU, MMC, Doxorubicin MXT, 5-FU 5-FU, CDDP 5-FU/Folic Acid, Carboplatin MXT GEM, MMC GEM, 5-FU, CDDP (Tegafur/Uracil) Angiotensin-Ⅱ

Response rate (%)

Survival time (mo)

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5.2

8.0 73.9 15.0

6.0 18.0 9.9

19.0 24.0 25.0

7.5 9.1 12.0

5-FU: 5-fluorouracil; MMC: mitomycin C; CDDP: cisplatin; CPA: cyclophosphamide; MTX: methotrexate; MXT: mitoxantrone; VCR: vincristine; GEM: gemcitabine.

was found with intraarterial treatment compared with historical controls. The calculated 1-, 2-, 3-year survivals were 95%, 59% and 43% in 19 patients with active treatment compared with 50%, 10% and 5% in controls. Hence, the therapy seems safe, but the intravascular catheters have caused some problems, including intimal damage in 2% and one case of pseudoaneurysm[35]. The results of intra-arterial chemotherapy for advanced pancreatic cancer are displayed in Table 1. Recently, Mambrini et al also reported that intra-arterial infusion of 5-fluorouracil, leucovorin, epirubicin and carboplatin (FLEC regimen) is well-tolerated and effective in patients with unresectable pancreatic cancer[37]. According to the definition of the World Health Organization (WHO), the objective response rate includes the reporting complete and partial remissions (Table 1). A complete remission is defined as the disappearance of any evidence of tumor (either primary tumor or metastases). A partial remission is defined as a minimum of a 50% reduction in tumor size without the reappearance of any new lesions[32,38-43]. Moreover, we reported that intra-arterial chemotherapy including changes in distribution of blood flow induced by AT-Ⅱ appears to be an effective palliative treatment for advanced pancreatic cancer not only for prolonging patient survival but also for improving the quality of life even for high aging patients (Figure 1)[44]. Intra-arterial chemotherapy is sug gested to take advantage of the first pass effect of chemotherapeutics, generating higher local drug concentrations at the tumor cell membrane and therefore enhancing cellular drug uptake as compared to intravenous infusion. Therefore, changing the route of drug delivery, which can increase the concentration of the drug in the regional area, thus reducing systemic side-effects, is an effective approach to solving these problems. In addition, this may lead to an increase of tumor response rate and prolongation of survival time. We suggest that, in the near future, regional chemotherapy method should be subjected to a prospective www.wjgnet.com

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Table 1 Overview of the important published results from trials in regional intra-arterial chemotherapy in the palliative treatment of pancreas cancer Author

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  0 0

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Figure 1 Survival of pancreatic cancer patients who were treated with arterial infusion chemotherapy. The differnt circles mean whether censored data or uncensored data.Censored data means black-lacquered circle.

randomized controlled study for advanced pancreatic cancer. Further clinical trials of this method for pancreatic cancer are needed.

CONCLUSION Regional intra-arterial chemotherapy may provide high levels of cytostatic concentrations within the tumor and, simultaneously, a low rate of systemic side effects compared with systemic administration of anti-neoplastic drugs. In order to achieve higher local drug concentrations in the tumor without causing side-effects of a comparable level of systemic treatment, this method has been introduced as an alternative treatment for advanced pancreatic cancer.

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Gold EB, Cameron JL. Chronic pancreatitis and pancreatic cancer. N Engl J Med 1993; 328: 1485-1486 Cameron JL, Crist DW, Sitzmann JV, Hruban RH, Boitnott JK, Seidler AJ, Coleman J. Factors influencing survival after pancreaticoduodenectomy for pancreatic cancer. Am J Surg 1991; 161: 120-124; discussion 124-125 Baumel H, Huguier M, Manderscheid JC, Fabre JM, Houry S, Fagot H. Results of resection for cancer of the exocrine pancreas: a study from the French Association of Surgery. Br J Surg 1994; 81: 102-107 Connolly MM, Dawson PJ, Michelassi F, Moossa AR, Lowenstein F. Survival in 1001 patients with carcinoma of the pancreas. Ann Surg 1987; 206: 366-373 Carter SK, Comis RL. The integration of chemotherapy into a combined modality approach for cancer treatment. VI. Pancreatic adenocarcinoma. Cancer Treat Rev 1975; 2: 193-214 Zimmerman SE, Smith FP, Schein PS. Chemotherapy of pancreatic carcinoma. Cancer 1981; 47: 1724-1728 Douglass HO. Current approaches to multimodality management of advanced pancreatic cancer. Hepatogastroenterology 1993; 40: 433-442 Nicolson M, Webb A, Cunningham D, Norman A, O'Brien M, Hill A, Hickish T. Cisplatin and protracted venous infusion 5-fluorouracil (CF)--good symptom relief with low toxicity in advanced pancreatic carcinoma. Ann Oncol 1995; 6: 801-804 Evans TR, Lofts FJ, Mansi JL, Glees JP, Dalgleish AG, Knight MJ. A phase II study of continuous-infusion 5-fluorouracil with cisplatin and epirubicin in inoperable pancreatic cancer. Br J Cancer 1996; 73: 1260-1264

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Moore M. Activity of gemcitabine in patients with advanced pancreatic carcinoma. A review. Cancer 1996; 78: 633-638 Rothenberg ML, Moore MJ, Cripps MC, Andersen JS, Portenoy RK, Burris HA 3rd, Green MR, Tarassoff PG, Brown TD, Casper ES, Storniolo AM, Von Hoff DD. A phase II trial of gemcitabine in patients with 5-FU-refractory pancreas cancer. Ann Oncol 1996; 7: 347-353 Fung MC, Sakata T. What's new in pancreatic cancer treatment? J Hepatobiliary Pancreat Surg 2002; 9: 61-75 Burris HA 3rd, Moore MJ, Andersen J, Green MR, Rothenberg ML, Modiano MR, Cripps MC, Portenoy RK, Storniolo AM, Tarassoff P, Nelson R, Dorr FA, Stephens CD, Von Hoff DD. Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial. J Clin Oncol 1997; 15: 2403-2413 Bosch X. Fluorouracil and mitomycin for pancreatic cancer. Lancet Oncol 2005; 6: 644 Whitehouse PA, Cooper AJ, Johnson CD. Synergistic activity of gamma-linolenic acid and cytotoxic drugs against pancreatic adenocarcinoma cell lines. Pancreatology 2003; 3: 367-373; discussion 373-374 Ashraf S, Loizidou M, Crowe R, Turmaine M, Taylor I, Burnstock G. Blood vessels in liver metastases from both sarcoma and carcinoma lack perivascular innervation and smooth muscle cells. Clin Exp Metastasis 1997; 15: 484-498 Peterson HI, Mattson J. Vasoactive drugs and tumor blood flow. Biorheology 1984; 21: 503-508 Mattson J, Appelgren L, Karlsson L, Peterson HI. Influence of vasoactive drugs and ischaemia on intra-tumour blood flow distribution. Eur J Cancer 1978; 14: 761-764 Sasaki Y, Imaoka S, Hasegawa Y, Nakano S, Ishikawa O, Ohigashi H, Taniguchi K, Koyama H, Iwanaga T, Terasawa T. Changes in distribution of hepatic blood flow induced by intra-arterial infusion of angiotensin II in human hepatic cancer. Cancer 1985; 55: 311-316 Ishikawa T, Mizuno K, Togashi T, Watanabe K, Seki K, Ohta H, Yoshida T, Kamimura T. A case of advanced gastric cancer with bone metastasis and severe DIC responding to hypertensive subselective chemotherapy with pharmacokinetic modulating chemotherapy. Gan To Kagaku Ryoho 2005; 32: 523-527 Ishikawa T, Mizuno K, Togashi T, Watanabe K, Seki K, Ohta H, Yoshida T, Kamimura T. Modified pharmacokinetic modulating chemotherapy for progressive gastric cancer accompanied by peritoneal dissemination. Gan To Kagaku Ryoho 2005; 32: 469-472 Ishikawa T, Mizuno K, Watanabe K, Baba Y, Oota H, Yoshida T, Kamimura T. A case of successful management of nonresectable pancreas cancer with liver metastasis by intra-arterial infusion chemotherapy with gemcitabine hydrochloride, 5-FU, CDDP and administration of tegafur/ uracil. Gan To Kagaku Ryoho 2004; 31: 1555-1558 Ishikawa T, Nomura K, Baba Y, Hayashi S, Oota H, Yoshida T, Kamimura T. A case of advanced gastric cancer with liver and intra-abdominal lymph node metastasis treated by hypertensive selective chemotherapy with pharmacokinetic modulating chemotherapy. Gan To Kagaku Ryoho 2003; 30: 1151-1155 Ishikawa T, Mita Y, Kobayashi M, Tashiro K, Tashiro S, Matsuki H. A case of nonresectable scirrhous type gastric cancer treated by hypertensive subselective chemotherapy with pharmacokinetic modulating chemotherapy. Gan To Kagaku Ryoho 2001; 28: 1137-1140 Ishikawa T, Sato S, Matsuzawa J, Mita Y, Matsui S, Tashiro K, Tashiro S, Matsuki H. A case of successful management of nonresectable pancreas cancer with liver metastasis by intra-arterial infusion chemotherapy with angiotensin-II and administration of tegafur/uracil. Gan To Kagaku Ryoho 2001; 28: 521-525 Ishikawa T, Ushiki T, Kamimura H, Togashi T, Tsuchiya A, Watanabe K, Seki K, Ohta H, Yoshida T, Takeda K, Kamimura T. Angiotensin-II administration is useful for the

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detection of liver metastasis from pancreatic cancer during pharmacoangiographic computed tomography. World J Gastroenterol 2007; 13: 3080-3083 Rougier P, Laplanche A, Huguier M, Hay JM, Ollivier JM, Escat J, Salmon R, Julien M, Roullet Audy JC, Gallot D. Hepatic arterial infusion of floxuridine in patients with liver metastases from colorectal carcinoma: long-term results of a prospective randomized trial. J Clin Oncol 1992; 10: 1112-1118 Goldstein LJ. MDR1 gene expression in solid tumours. Eur J Cancer 1996; 32A: 1039-1050 Muchmore JH, Preslan JE, George WJ. Regional chemotherapy for inoperable pancreatic carcinoma. Cancer 1996; 78: 664-673 Goldstein LJ, Galski H, Fojo A, Willingham M, Lai SL, Gazdar A, Pirker R, Green A, Crist W, Brodeur GM. Expression of a multidrug resistance gene in human cancers. J Natl Cancer Inst 1989; 81: 116-124 Muchmore JH, Preslan JE, George WJ. Regional chemotherapy for inoperable pancreatic carcinoma. Cancer 1996; 78: 664-673 Theodors A, Bukowski RM, Hewlett JS, Livingston RB, Weick JK. Intermittent regional infusion of chemotherapy for pancreatic adenocarcinoma. Phase I and II pilot study. Am J Clin Oncol 1982; 5: 555-558 Ishikawa O, Ohigashi H, Sasaki Y, Furukawa H, Kabuto T, Kameyama M, Nakamori S, Hiratsuka M, Imaoka S. Liver perfusion chemotherapy via both the hepatic artery and portal vein to prevent hepatic metastasis after extended pancreatectomy for adenocarcinoma of the pancreas. Am J Surg 1994; 168: 361-364 Ishikawa O, Ohigashi H, Sasaki Y, Masao K, Kabuto T, Furukawa H, Imaoka S. Adjuvant therapies in extended pancreatectomy for ductal adenocarcinoma of the pancreas. Hepatogastroenterology 1998; 45: 644-650 Beger HG, Link KH, Gansauge F. Adjuvant regional chemotherapy in advanced pancreatic cancer: results of a prospective study. Hepatogastroenterology 1998; 45: 638-643 Beger HG, Gansauge F, Buchler MW, Link KH. Intraarterial adjuvant chemotherapy after pancreaticoduodenectomy for pancreatic cancer: significant reduction in occurrence of liver metastasis. World J Surg 1999; 23: 946-949 Mambrini A, Sanguinetti F, Pacetti P, Caudana R, Iacono C, Guglielmi A, Guadagni S, Del Freo A, Fiorentini G, Cantore M. Intra-arterial infusion of 5-fluorouracil, leucovorin, epirubicin and carboplatin (FLEC regimen) in unresectable pancreatic cancer: results of a ten-year experience. In Vivo 2006; 20: 751-755 Miller AB, Hoogstraten B, Staquet M, Winkler A. Reporting results of cancer treatment. Cancer 1981; 47: 207-214 Maurer CA, Borner MM, Lauffer J, Friess H, Z'graggen K, Triller J, Buchler MW. Celiac axis infusion chemotherapy in advanced nonresectable pancreatic cancer. Int J Pancreatol 1998; 23: 181-186 Homma H, Doi T, Mezawa S, Takada K, Kukitsu T, Oku T, Akiyama T, Kusakabe T, Miyanishi K, Niitsu Y. A novel arterial infusion chemotherapy for the treatment of patients with advanced pancreatic carcinoma after vascular supply distribution via superselective embolization. Cancer 2000; 89: 303-313 Cantore M, Pederzoli P, Cornalba G, Fiorentini G, Guadagni S, Miserocchi L, Frassoldati A, Ceravolo C, Smerieri F, Muchmore JH. Intra-arterial chemotherapy for unresectable pancreatic cancer. Ann Oncol 2000; 11: 569-573 Gansauge F, Link KH, Rilinger N, Kunz R, Beger HG. Regional chemotherapy in advanced pancreatic carcinoma. Med Klin (Munich) 1995; 90: 501-505 Lorenz M, Heinrich S. Regional chemotherapy. Hematol Oncol Clin North Am 2002; 16: 199-215 Ishikawa T, Kamimura H, Tsuchiya A, Togashi T, Watanabe K, Seki K, Ohta H, Yoshida T, Takeda K, Kamimura T. Clinical Efficacy of Intra-arterial Pharmacokinetic Chemotherapy with 5-Fluorouracil, CDDP, Gemcitabine, and AngiotensinII in Patients with Advanced Pancreatic Cancer. HepatoGastroenterology 2007; in press S- Editor Zhu LH L- Editor Zhu LH E- Editor Lu W www.wjgnet.com

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World J Gastroenterol 2007 August 28; 13(32): 4310-4315 World Journal of Gastroenterology ISSN 1007-9327 © 2007 WJG . All rights reserved.

REVIEW

Duration of treatment with 5-aminosalicylic acid compounds T Moshkovska, JF Mayberry T Moshkovska, JF Mayberry, Leicester General Hospital, University Hospitals of Leicester NHS Trust, Gwendolen Road, Leicester LE5 4PW, United Kingdom Correspondence to: JF Mayberry, Leicester General Hospital, Gwendolen Road, Leicester LE5 4PW, United Kingdom. [email protected] Telephone: +44-116-2584786 Fax: +44-116-2588107 Received: 2006-09-15 Accepted: 2006-10-09

Abstract The development of 5-aminosalicylic acid (5-ASA) therapy as a life long treatment for ulcerative colitis is reviewed from its origins in the 1940s to the present day. The drug was designed to treat rheumatoid arthritis, but was found helpful in the management of nine patients with ulcerative colitis. This discovery preceded the emergence of the clinical trial as a tool for assessing a new drug’s efficacy; as a result it lacked scientific rigour and was selective in its presentation of results. Nevertheless it identified the future cornerstone of therapy in ulcerative colitis. In 1962, the first double blind controlled trial of sulphasalazine was conducted on 40 patients. Outcome measures were subjective and included symptoms and an assessment of the rectal mucosa. In 1973, the first two papers on the role of sulphasalazine in maintenance of remission were published. Both used placebo controls and had a stratified design. Outcomes were measured using “an intention to treat” approach. The British study of 64 patients used both subjective and objective criteria to assess outcomes. Patients on placebo had a relapse rate four times patients on active treatment and this founded the basis for a life long approach to therapy with 5-ASA compounds in ulcerative colitis. However, in 1985, a small “on demand” study of 32 patients suggested this approach might be as effective as continuous treatment. Some support for this view came from an Italian study which showed no benefit to continued treatment for those in remission for two years or more. The central problem these studies identify is that of adherence to treatment in the long-term. Few studies have considered patients’ attitudes to continuous therapy and it is an area that needs further investigation. © 2007 WJG . All rights reserved.

Key words: Ulcerative colitis; 5-aminosalicylic acid compounds; Sulphasalazine mesalazine adherance trials Moshkovska T, Mayberry JF. Duration of treatment with 5-aminosalicylic acid compounds. World J Gastroenterol

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INTRODUCTION Ulcerative colitis is a debilitating disease of young and middle aged people characterised by recurrent attacks of bloody diarrhea. Its cause is unknown and treatment has been aimed at control rather than cure. By the end of the twentieth century most clinicians advised patients to take 5-aminosalicylic (5-ASA) compounds, such as sulphasalazine or mesalazine, on a twice daily basis. This has become embedded in the guidelines put forward by the British Society of Gastroenterology in 2004: “Lifelong maintenance therapy is generally recommended for all patients, especially those with left sided or extensive disease, and those with distal disease who relapse more than once a year”[1]. The purpose of this review is to critically explore the basis for such treatment. Papers were identified through PubMed using the search words: ulcerative colitis, maintenance, trial, long-term, and non-adherence in different combinations. The references in these papers were then explored for further relevant studies. As a result this paper will look at initial case reports which identified a role for sulphasalazine, its subsequent investigation in clinical trials and more recent questioning of the need for regular treatment. During this review an attempt will be made to investigate the myths that developed around its clinical use and the question that will be addressed is: “Do 5-ASA compounds prevent relapse in ulcerative colitis and how frequently are they taken in practice?”

THE DISCOVERY AND EARLY ASSESSMENT OF SULPHASALAZINE Sulphasalazine was first made in 1940 in Sweden by the pharmaceutical company Pharmacia. It was probably the first designer drug in medical history. Professor Nana Svartz was interested in developing new therapies for the treatment of infective arthritis and speculated that the sulphur component of the molecule would act as an anti-infective agent while the salicylate constituent would have an antiinflammatory activity. During clinical work at the Karolinska Institute on the role of this new drug in rheumatoid arthritis, Professor Svartz and her colleagues found patients with ulcerative colitis reported a significant improvement in symptoms[2]. This paper preceded the emergence of the

Moshkovska T et al . Duration of treatment with 5-ASA compounds

clinical trial as a common method for assessing new drugs. The data she presented were based upon: “my material and represent patients who reacted rapidly or favourably to the treatment”. Svartz recognised that such an approach was selective and excluded patients in whom the drug had failed to be effective. In addition diagnosis depended upon history, radiological interpretation of barium enemas or a visual inspection of the rectal mucosa at proctoscopy. No evidence was presented that the diagnosis had been confirmed with a rectal biopsy or infection excluded through faecal culture. The paper reported nine cases of ulcerative colitis of varying degrees of severity and presented data on stool frequency and shape together with temperature charts. These end points largely reflected the patient’s subjective interpretation of progress on treatment. In two cases the rectal mucosa was examined after successful treatment but the interpretation of an improved appearance was subjective. The doctor was aware the patient had received treatment with a new drug and there had been an apparent clinical improvement. However, criticism needs to be tempered by the fact that the randomised controlled trial (RCT) had not been introduced into the assessment of drug treatment in 1942[3]. The study showed a beneficial effect from sulphasalazine in ulcerative colitis. However, it lacked rigor, was selective in the results presented, with poor case definition, a lack of clear and reproducible endpoints and no control group. It was an opportunity to record in some depth through structured interviews the views of patients on this new agent, its benefits and side effects. However, the methodology for recording and interpreting such qualitative data did not then exist within clinical practice.

EARLY CLINICAL TRIALS OF EFFICACY The concept of a “double-blind controlled trial of sulphasalazine against a placebo in mild cases of ulcerative colitis” was not investigated until 1962[4]. Forty patients were randomly allocated to a treatment or placebo group. There was no attempt to stratify the groups so that they contained patients of similar age and extent of disease. Two outcomes were measured-the patients’ symptoms as reported to an observer and a different observer’s assessment of the rectal mucosa at sigmoidoscopy. These again are subjective measures and where there was a discrepancy between symptoms and mucosal appearance “the final assessment was reached by discussion”. Sixteen of the twenty patients in the treatment group improved compared to eight on placebo. This result was statistically significant and launched the widespread use of sulphasalazine to control ulcerative colitis. Although the study was placebo controlled it was small and outcome measures were subjective. In 1964, a second placebo controlled trial of sulphasalazine was reported from a different centre[5]. It was based on 11 pairs of patients, one of whom received active treatment and the other placebo. It had the same limitations of being small, data were open to patient and observer subjectivity. Despite these limitations, such papers helped establish a central role for 5-ASA compounds, such as sulphasalazine, in the prevention of recurrent attacks of ulcerative colitis. They also generated a new research question, namely for

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how long should this medication be prescribed. In 1973, two papers were published on the role of sulphasalazine in maintenance of remission. The Danish study was of 50 patients in remission over a 6 mo period[6]. Half continued to receive sulphasalazine, while the rest were given placebo. Selection was random, but with a stratified design so that both groups had patients with disease of comparable extent and severity. Adherence to therapy was checked with a returned tablet count. However, poor adherence did not exclude a patient from the study. Rather an “intention to treat” approach was used. This added strength to the study and addressed any criticism that adherence was not an important factor in the assessment of long-term treatment programs. At the end of the study recurrence rates were comparable at 29% for those on placebo and 24% in the active group. The definition of recurrence chosen was again subjective and based on rectal bleeding for three days or more than three defecations on five successive days. In contrast, the British study of sixty four patients used both subjective and more objective endpoints, including a blinded assessment of sigmoidoscopic appearance and histology[7]. Patients who had been stable on active treatment for one year were entered into a placebo controlled study of the efficacy of sulphasalazine. Patients on placebo had a relapse rate four times greater than those receiving active treatment with sulphasalazine. The relapse rate amongst patients on active treatment was 12% compared to 55% for those on placebo. The study had a stratified design and the outcome for patients who had been on maintenance treatment for at least three years is of particular interest. The relapse rate for those who continued with active treatment was 13% compared with 58% for those on placebo. It was on the basis of these results that the authors considered that there was: “a good case for continuing maintenance treatment with sulphasalazine indefinitely provided there are no harmful side effects” and so the continuous use of sulphasalazine to maintain remission in ulcerative colitis became accepted practice. Riis et al’s study[6] was based on symptoms, while that of Dissanayake & Truelove[7] depended upon both symptoms and histological criteria. As the criteria used for remission in Dissanayake & Truelove’s study[7] were stricter and consistent with current views that histological remission is a more objective measure it is not surprising that life long 5-ASA therapy became the norm.

STUDIES ON DURATION OF TREATMENT In 1985, thirty two patients in remission and on continuous treatment with sulphasalazine were randomly allocated to one of two groups[8]. The first continued with sulphasalazine on a daily basis, while the second only took it for a period of fourteen days if they had symptoms of a recurrence. Progress was followed with regular rectal biopsies and these were scored “blind” for evidence of inflammation. At the end of one year there was no statistical difference in the relapse rate between groups. This clearly suggested that “on demand treatment” may be as effective as regular maintenance therapy. This is an important consideration in the management of chronic conditions. Many people find it difficult to take medication daily, especially www.wjgnet.com

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Over 2 yr

Figure 1 Relapse rates amongst patients with ulcerative colitis in remission in a study of delayed release mesalazine compared with placebo[14]

if they feel well. If treatment is only needed during a flareup there is no point pressurising people to take it every day. However, the study was small and may have been of inadequate size to demonstrate any true difference in relapse rates between the groups. Indeed up until this point the whole basis for the long-term management of patients with ulcerative colitis had been based on quantitative research on only 146 patients. Minimal attention was given to any qualitative assessment of the impact of medication on quality of life or to develop a better understanding of patients’ views on taking medication regularly whilst well. In 2002 Sutherland et al reviewed prospective randomised studies of the effect of oral 5-ASA therapy on maintenance of remission in ulcerative colitis between 1981 and 2002[9]. There were 16 studies which looked at the effectiveness of maintenance therapy for 6 mo or longer. Indeed only one study reviewed a period as long as 18 mo[10]. Based on such studies the current Guidelines of the British Society of Gastroenterology state: “Lifelong maintenance therapy is generally recommended for all patients, especially for those with left sided or extensive disease, and those with distal disease who relapse more than once a year”[1]. Interestingly, until recently there were no studies which used the approach adopted in the investigation of the benefits of long term azathioprine therapy in Crohn’s disease[11,12] and ulcerative colitis[13] (Figure 1). In 1999, Ardizzone et al conducted such an investigation amongst 112 patients[14]. Patients were stratified into two groups depending upon whether they had been in remission for under two years or over that period. All patients were then randomised to either active treatment with mesalazine or received a placebo. Mesalazine significantly reduced the relapse rate at 12 mo in the group who had been in remission for less than two years. However, active treatment conveyed no advantage to those who had been in remission for more than two years. How much of this effect can be attributed to the high relapse rate of 49% in the placebo group who had been in remission for less than two years is open to some discussion although the difference was not significant. In this study compliance was monitored through use of tablet counts and noncompliance was defined as failing to take less than 80% of the medication. Long duration of treatment is commonly associated with poor adherence to treatment which can at times reach 40%-50%. However, pill counts may fail to detect the true level of lack of adherence to therapy[15]. Indeed the overall relapse rate at 12 mo for patients who had been in remission for less than two years (38%) was not significantly different to that of those in remission www.wjgnet.com

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for more than two years (22%). Such high rates of relapse certainly raise interesting questions as to patients’ overall adherence to therapy. In view of the fact that maintenance therapy was under investigation the authors used an “intention to treat” approach. This was only the second occasion on which researchers in this field recognised that patient adherence to prolonged therapy could be complicated by their forgetting to take medication or becoming bored or disillusioned with the treatment. Indeed the failure to conduct qualitative research on patients attitude to this therapy meant that the reasons patients might discontinue their treatment were unknown. In this study relapse was defined clinically and based on increased stool frequency and rectal bleeding together with evidence of active disease on sigmoidoscopy. The failure of the investigators to follow the original study plan and recruit eighty six patients compared with the actual sixty one may have led to a falsely significant finding. Indeed, recruitment in the second group was even less near the target size.

SHOULD TREATMENT BE CONTINUOUS OR INTERMITTENT Some support for the role of poor adherence comes from a small study of intermittent therapy from Cambridge[8] and the later study of Bardazzi et al [16]. In this twelve month clinical trial in Florence 25 patients received intermittent treatment with 5-ASA tablets for the first week of each month and 25 received continuous treatment. The relapse free rates at 12 mo were 71% in patients on intermittent therapy and 66% in those on continuous treatment. This difference was not significant and suggested that intermittent therapy might be as effective as continuous therapy. However, a particularly interesting aspect of the study is that the overall relapse rate for the 47 patients who completed it was 32%, and this bears direct comparison with the results from Ardizzone[14] and the 36% relapse rate reported by Dickenson et al[8].

ADHERANCE TO THERAPY AND THE ROLE OF THE PATIENT In addition to raising questions as to how long and how intensive treatment with 5-ASA compounds should be, these two Italian studies again draw attention to the role of patient adherence. The relapse rates of between 20% and 40% suggest that compliance was probably poor. The Cambridge study of on demand therapy with sulphasalazine showed no difference from regular medication. This tends to suggest that this is how patients on regular treatment use their drugs on demand when symptoms remind them of their need to take treatment. Clear confirmation of this view comes from Kane et al’s study in Chicago where the prevalence of medication non-adherence was 60% in patients with quiescent ulcerative colitis[17]. Prescription use by three separate populations from a clinic, pharmacy, and telephone callers was investigated. In total, 94 patients were interviewed by a single investigator. No information is given on the type of interview technique used and the only data collected appeared

Moshkovska T et al . Duration of treatment with 5-ASA compounds

to be demographic. Adherence was defined as “consumption of > 80% of prescribed medication over a six-month period”. This definition was based on the suggestion that loss of more than 20% of patients in a clinical trial makes the results suspect[18]. The choice of this definition for this study was, therefore, arbitrary and not evidence based. Patients who continue to participate in clinical trials may consume less than 80% of prescribed medication. In addition it is “the intention to treat” that is important. Patients may withdraw from a study because an otherwise effective medication has unacceptable side-effects. A better approach would have been to analyse the results at serial levels of consumption e.g. 100%, 90%, 80%, 70% and 60% etc. Univariate analysis was applied to the data and showed non-adherence was significantly associated with male gender, single status, limited disease, and a history of more than four concomitant medications. Of these four factors only the last might be open to clinical alteration. This confirms the limitation of only collecting biodata. No attempt was made to identify those factors which caused men or those with limited disease not to comply with therapy. The simple mantra “Ask the patients” was not utilised by the researchers. In 2003 a study from Canada again failed to follow this simple mantra[19]. The stated purpose of the study was to investigate determinants of non-adherence amongst 153 patients with inflammatory bowel disease. It used a series of questionnaires to examine whether problems with the therapeutic relationship between these patients and their ten doctors was an important factor. The study was based at one centre in Montreal and data on patient-physician discordance, psychological distress, and social support, as well as non-adherence, were collected using validated questionnaires. No preliminary attempt was made to discover from patients why they might not adhere to treatment plans. Although complete data were only available for 77% of patients the demographic, clinical, and psychosocial characteristics of the non-responders were claimed to be similar. A complex statistical analysis was used to show that 51 of 153 (53%) simply forgot to take their medication, 16 (10%) stopped because they felt better and 13 (8%) worse, with 7% overlap between the groups. Non-adherence was again linked to less active disease and recent diagnosis. Interestingly, for patients who forgot to take their medication there was a link with: “less certainty that medication would positively affect health”. This again emphasises the generally held belief that patients with ulcerative colitis should take 5-ASA compounds on a regular long-term basis, despite the fact that evidence for this approach is weak. Again, an opportunity was missed for using qualitative techniques, such as in-depth interviews, to investigate patients’ beliefs about 5-ASA compounds, their actual use of them, and reasons for nonadherence. Such a study would have provided an insight into patient practice and helped clarify whether people in the active wing of long-term maintenance studies really take the medication. Non-adherence has been associated with male gender, being single, and taking multiple concomitant medications. Compliance with an intensive program of medication in ulcerative colitis can prove difficult for a number of pa-

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tients and several other studies have indicated that pills are often forgotten or taken in a disorganised pattern[19-21]. Studies across a wide range of disciplines have shown that patients who are more satisfied and informed about their care are more likely to be adherent to treatment programs[22].

THE CONSEQUENCES OF POOR ADHERENCE The level at which patients adhere to their 5-ASA therapy may have particular importance in the reduction of cancer risk in ulcerative colitis. Early work by Jones et al demonstrated that the development of colorectal cancer as a complication of long-standing ulcerative colitis might be the result of poor compliance with therapy[23]. Patients who developed cancer in their community based surveillance program were the very patients who failed to attend for their regular colonoscopy. In a 10 year study of 175 patients Moody et al found that the crude proportions developing cancer were 5/152 (3%) in the group who took long-term sulphasalazine but 5/16 (31%) in the those who had had their treatment stopped or who did not comply with therapy[24]. Further support for the value of 5-ASA compounds in cancer prevention comes from the work of Eaden et al[25]. In her study of 102 cases of colorectal cancer complicating ulcerative colitis regular 5-ASA therapy reduced cancer risk by 75% (OR 0.25, 95% CI: 0.13-0.48, P < 0.00001). Adjusting for other variables, taking mesalazine regularly was shown to reduce the risk by 81% (OR 0.19, 95% CI: 0.06-0.61, P = 0.006). These studies emphasise the need for adherence to therapy, but how close and how long must that adherence be? In Pinczowski et al’s study from Sweden of 3012 patients with ulcerative colitis, pharmacological therapy, especially sulfasalazine, lasting at least 3 mo was associated with a significant protective effect (RR, 0.38; 95% CI: 0.20-0.69) independent of disease activity[26]. The current evidence is that patients with ulcerative colitis are poorly adherent to 5-ASA therapy and that this situation probably worsens with time. Only limited work has been done on those factors which are associated with lack of adherence but male gender and multiple therapies seem to be important. General work on compliance has suggested that education about the role of drugs may lead to greater adherence. Apart from a reduction in morbidity through reduced frequency of flare-ups greater adherence to 5-ASA therapy is likely to reduce the incidence of colorectal cancer as a complication of long term ulcerative colitis. Greater attention to strategies which are associated with higher adherence would therefore seem worthwhile and it may be that in order to achieve this effect it will not be necessary to make certain that their impact is life-long. Any research on adherence will therefore also need to address the issue of the optimum duration and pattern of treatment.

CONCLUSION The generally held belief that 5-ASA compounds should be taken regularly and life long is based on a small number www.wjgnet.com

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of small quantitative studies. In early trials stratification of the active and control groups for severity, extent, and duration of disease was often omitted from the design. End points have usually been subjective and there was often a failure to adequately blind observers. However, the core issue as to whether these medications are actually taken long term has never been investigated directly with patients. Substitute markers, such as returned pill counts, are inaccurate. There have been no in-depth interviews with patients and we have little knowledge about their attitude towards this form of treatment. This should have been the obvious foundation for research on maintenance therapy in ulcerative colitis and its absence calls into question most of the work done on this topic.

COMMENTS Background

Ulcerative colitis is a life long condition characterised by recurrent attacks of diarrhoea, rectal bleeding and abdominal pain. It predisposes the patient to colorectal cancer. Current therapy suggests that treatment should be continuous and throughout life. This review explores the research background to this clinical approach. It identifies the fact that most clinical trials are for short periods and none have exceeded a period of two years. It investigates the basis for continuous therapy and raises questions as to patients compliance with such a regimen.

Research frontiers

This review draws attention to the need to explore the effectiveness of long term therapy with 5-ASA compounds and to consider alternatives such as intermittent treatment. This work will need to be linked to a better understanding of what motivates patients’ adherence to such a program and the identification of what patients consider important triggers in ensuring good compliance.

Innovations and breakthroughs

In the 21st century it will be increasingly important to build a therapeutic partnership between patients and their clinicians. This requires good communication and means that the information given to patients must be clearly understood. In the field of gastroenterology, especially amongst patients with chronic disease, there has been limited work in this area. It needs a well structured and rigorous research base. This paper has emphasised the fact that the basis for long term use of 5-ASA compounds is weak. Studies have been over relatively short terms and suggest that adherence in the long term is poor. This review has focused on these weaknesses and will hopefully ensure that a broader approach to adherence is now developed. Although once daily preparations may play a part in better compliance with treatment, they are only one element in the approaches we need to consider and evaluate.

Applications

The need to consider the most effective approach to controlling flare-ups and reducing cancer risk in ulcerative colitis is of relevance to all clinicians working in this field and to all patients with this disease. This review draws attention to the need to consider how frequently this medication needs to be taken and to whether adherence can be improved

Peer review

This paper critically investigates the criteria of guidelines for lifelong maintenance therapy with 5-ASA compounds in ulcerative colitis. It is interesting, well written and the criticism raised by the authors is stimulating both for clinical practice and future research.

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REFERENCES 1 2

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Terminology

Ulcerative colitis is an inflammatory condition of the colon. 5-ASA or 5-aminosalicylic acid is a drug which reduces the occurrence of flare-ups and cancer risk. Adherence, congruence, and compliance are terms which describe patients approach to taking medication.

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Carter MJ, Lobo AJ, Travis SP. Guidelines for the management of inflammatory bowel disease in adults. Gut 2004; 53 Suppl 5: V1-V16 Svartz N. Salazopyrin, a new Sulfanilamide preparation. A. Therapeutic results in rheumatic polyarthritis. B. Therapeutic results in ulcerative colitis. C. Toxic manifestations in treatment with sulphanilamide preparations. Acta.Med Scand 1942; 110: 577-598 Cochrane AL. Effectiveness and Efficiency. Random Reflections on Health Services. 1st ed. London Nuffield Provincial Hospitals Trust, 1972: 21-25 BARON JH, Connel AM, Lennard-Jones JE, Jones FA. Sulphasalazine and salicylazosulphadimidine in ulcerative colitis. Lancet 1962; 1: 1094-1096 Dick AP, Grayson MJ, Carpenter RG, Petrie A. Controlled trial of sulphasalazine in the treatment of ulcerative colitis. Gut 1964; 5: 437-442 Riis P, Anthonisen P, Wulff HR, Folkenborg O, Bonnevie O, Binder V. The prophylactic effect of salazosulphapyridine in ulcerative colitis during long-term treatment. A doubleblind trial on patients asymptomatic for one year. Scand J Gastroenterol 1973; 8: 71-74 Dissanayake AS, Truelove SC. A controlled therapeutic trial of long-term maintenance treatment of ulcerative colitis with sulphazalazine (Salazopyrin). Gut 1973; 14: 923-926 Dickinson RJ, King A, Wight DG, Hunter JO, Neale G. Is continuous sulfasalazine necessary in the management of patients with ulcerative colitis? Results of a preliminary study. Dis Colon Rectum 1985; 28: 929-930 Sutherland L, Roth D, Beck P, May G, Makiyama K. Oral 5-aminosalicylic acid for maintenance of remission in ulcerative colitis. Cochrane Database Syst Rev 2002; CD000544 Nilsson A, Danielsson A, Lofberg R, Benno P, Bergman L, Fausa O, Florholmen J, Karvonen AL, Kildebo S, Kollberg B. Olsalazine versus sulphasalazine for relapse prevention in ulcerative colitis: a multicenter study. Am J Gastroenterol 1995; 90: 381-387 O'Donoghue DP, Dawson AM, Powell-Tuck J, Bown RL, Lennard-Jones JE. Double-blind withdrawal trial of azathioprine as maintenance treatment for Crohn's disease. Lancet 1978; 2: 955-957 Vilien M, Dahlerup JF, Munck LK, Norregaard P, Gronbaek K, Fallingborg J. Randomized controlled azathioprine withdrawal after more than two years treatment in Crohn's disease: increased relapse rate the following year. Aliment Pharmacol Ther 2004; 19: 1147-1152 Hawthorne AB, Logan RF, Hawkey CJ, Foster PN, Axon AT, Swarbrick ET, Scott BB, Lennard-Jones JE. Randomised controlled trial of azathioprine withdrawal in ulcerative colitis. BMJ 1992; 305: 20-22 Ardizzone S, Petrillo M, Imbesi V, Cerutti R, Bollani S, Bianchi Porro G. Is maintenance therapy always necessary for patients with ulcerative colitis in remission? Aliment Pharmacol Ther 1999; 13: 373-379 Ley P. Communicating with Patients. Improving Communication Satisfaction and Compliance. 1st ed. London: Routledge, 1988; 59-61 Bardazzi G, d'Albasio G, Bonanomi AG, Trallori G, Messori A, Amorosi A, Bartoletti L, Morettini A, Pacini F. Intermittent versus continuous 5-aminosalicylic acid treatment for maintaining remission in ulcerative colitis. Ital J Gastroenterol 1994; 26: 334-337 Kane SV, Cohen RD, Aikens JE, Hanauer SB. Prevalence of nonadherence with maintenance mesalamine in quiescent ulcerative colitis. Am J Gastroenterol 2001; 96: 2929-2933 Guyatt GH, Sackett DL, Cook DJ. Users' guides to the medical literature. II. How to use an article about therapy or prevention. B. What were the results and will they help me in caring for my patients? Evidence-Based Medicine Working Group. JAMA 1994; 271: 59-63

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Sewitch MJ, Abrahamowicz M, Barkun A, Bitton A, Wild GE, Cohen A, Dobkin PL. Patient nonadherence to medication in inflammatory bowel disease. Am J Gastroenterol 2003; 98: 1535-1544 20 Rubin GP, Hungin AP, Chinn DJ, Dwarakanath D. Quality of life in patients with established inflammatory bowel disease: a UK general practice survey. Aliment Pharmacol Ther 2004; 19: 529-535 21 Stone MA, Mayberry JF, Baker R. Prevalence and management of inflammatory bowel disease: a cross-sectional study from central England. Eur J Gastroenterol Hepatol 2003; 15: 1275-1280 22 Horne R. Adherence to medication: a review of existing research. In: Myers LB. Midence K Adherence to Treatment in Medical Conditions. Amsterdam: Taylor and Francis, 1998:

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285-310 Jones HW, Grogono J, Hoare AM. Acute colitis in a district general hospital. Br Med J (Clin Res Ed) 1987; 294: 683-684 24 Moody GA, Jayanthi V, Probert CS, Mac Kay H, Mayberry JF. Long-term therapy with sulphasalazine protects against colorectal cancer in ulcerative colitis: a retrospective study of colorectal cancer risk and compliance with treatment in Leicestershire. Eur J Gastroenterol Hepatol 1996; 8: 1179-1183 25 Eaden J, Abrams K, Ekbom A, Jackson E, Mayberry J. Colorectal cancer prevention in ulcerative colitis: a casecontrol study. Aliment Pharmacol Ther 2000; 14: 145-153 26 Pinczowski D, Ekbom A, Baron J, Yuen J, Adami HO. Risk factors for colorectal cancer in patients with ulcerative colitis: a case-control study. Gastroenterology 1994; 107: 117-120 23

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World J Gastroenterol 2007 August 28; 13(32): 4316-4320 World Journal of Gastroenterology ISSN 1007-9327 © 2007 WJG. All rights reserved.

GASTRIC CANCER

Results of mass endoscopic examination for gastric cancer in Kamigoto Hospital, Nagasaki Prefecture Satohiro Matsumoto, Kazumi Yamasaki, Kenichiro Tsuji, Satoshi Shirahama Satohiro Matsumoto, Kenichiro Tsuji, Satoshi Shirahama, Department of Internal Medicine, Kamigoto Hospital, 1549-11 Aokata-gou, Shinkamigoto-cho, Minamimatsuura-gun, Nagasaki 857-4404, Japan Kazumi Yamasaki, Department of Internal Medicine, Narao Hospital, 712-3 Narao-gou, Shinkamigoto-cho, Minamimatsuuragun, Nagasaki 853-3101, Japan Correspondence to: Satohiro Matsumoto, MD, Department of Internal Medicine, Kamigoto Hospital, 1549-11 Aokata-gou, Shinkamigoto-cho, Minamimatsuura-gun, Nagasaki 857-4404, Japan. [email protected] Telephone: +81-959-523000 Fax: +81-959-522981 Received: 2007-04-11 Accepted: 2007-04-30

Abstract AIM: To examine how the introduction of endoscopy to gastric cancer screening affected survival prognosis in a regional population. METHODS: The subjects comprised 4261 residents of Kamigoto, Nagasaki Prefecture, who underwent gastric X-ray examination for gastric cancer screening from 1991 to 1995, and all 7178 residents who underwent endoscopic examination for the same purpose from 1996 to 2003. The analysis evaluated trends in age-adjusted gastric cancer mortality rates and standard mortality ratios (SMRs) among the Kamigoto residents. RESULTS: According to demographic statistics, the 1995 and 2000 age-adjusted gastric cancer mortality rates in Nagasaki Prefecture (per 100 000 population) were 42.6 and 37.3 for males and 18.6 and 16.0 for females, while the corresponding rates in Kamigoto before and after the introduction of endoscopic screening were respectively 51.9 and 28.0, and 26.6 and 6.9. The data obtained in this study were divided into those for two periods, 1990-1996 and 1997-2006, and SMRs were calculated separately for males and females. For the first period, the SMR was 1.04 (95% CI 0.50-1.58) for males and 1.54 (95% CI 0.71-2.38) for females, while for the second period the SMR was 0.71 (95% CI 0.33-1.10) for males and 0.62 (95% CI 0.19-1.05) for females. CONCLUSION: Following the introduction of endoscopic examination, gastric cancer death rates decreased in Kamigoto. © 2007 WJG . All rights reserved.

Key words: Endoscopy; Mass screening; Gastric cancer; www.wjgnet.com

Age-adjusted mortality rate; Standard mortality ratio Matsumoto S, Yamasaki K, Tsuji K, Shirahama S. Results of mass endoscopic examination for gastric cancer in Kamigoto Hospital, Nagasaki Prefecture. World J Gastroenterol 2007; 13(32): 4316-4320

http://www.wjgnet.com/1007-9327/13/4316.asp

INTRODUCTION Endoscopic examination is gaining acceptance as a means of gastric cancer screening. The importance of gastric endoscopy is its improvement of post-treatment QOL for the growing number of patients for whom endoscopic mucosal resection (EMR) is indicated, and its most anticipated effect is improvement of survival prognosis. However, evaluation of the effects of endoscopic examination on survival prognosis is incomplete, and a March 2006 report by the Japan Research Group on the Establishment of Appropriate Methodology and Evaluation in Cancer Screening concluded that a level of "I" for endoscopy as part of gastric cancer screening, i.e., implementation in mass screening, could not be recommended. In the Kamigoto treatment area examined in this study, gastric X-ray examination was replaced by endoscopy as a means of gastric cancer screening in 1996. This study examined how the introduction of endoscopy to gastric cancer screening affected survival prognosis of residents of this area.

MATERIALS AND METHODS Regional screening in the Kamigoto treatment area has long been performed by Kamigoto Hospital in partnership with the Kamigoto town office. Of a target screening population of 7400 people, 3200 have been screened, at a rate of approximately 700-1000 screenings per year. Recruiting of applicants for endoscopic screening is performed through public notices and through activities with outpatients, and screening is free of charge to Kamigoto residents age 40 years or older. A significant number of screenings are performed as part of annual or biennial checkups, though the exact number of such screenings is unknown. Kamigoto Hospital is a 186-bed general hospital that performs approximately 3000 upper gastrointestinal

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(Persons) 1200

Table 1 Composition of gastric cancer cases discovered on screening

Female Male

1000 800

Gastric cancer detection Early-stage Invasion depth (m) Indication for EMR1 Gastric cancer recurrence Gastric cancer death

600 400 200 0

'92

'94

'96

'98

'00

X-ray (n = 4261) 2 1/2 1/2 0/2 1 1

Endoscope (n = 7178) 28 25/28 (89.3%) 20/28 (71.4%) 12/28 (42.9%) 0 1

'02 (Yr) 1

Figure 1 Trend in number of gastric cancer screening recipients.

(Persons) 10

(%) 1.5

New gastric cancer Cancer discovery rate

8

1.2

6

0.9

4

0.6

2

0.3

0

'92

'94

'96

'98

'00

'02 (Yr)

0

Figure 2 Trend in gastric cancer cases discovered on screening.

endoscopies annually. Olympus endoscopes (GIF-Q200, GIF-Q240, GIF-XQ240) are used. Although intramuscular injection of anti-cholinergic agent is given immediately before endoscopy, sedation is not generally performed. Sterilization conforms to the Guidelines of the Japan Gastroenterological Endoscopy Society, and peracetic acid preparation is used as a disinfectant. At Kamigoto Hospital, eight physicians perform endoscopic examinations, including five internists and three surgeons. Four of them are physicians who completed residency training less than four years ago and thus have less experience performing endoscopic examinations. Diagnosis is made solely by the performing physician. Gastric cancer is diagnosed on the basis of histological examination of forceps biopsy specimens in Kamigoto Hospital. In 1996, the Kamigoto area replaced its conventional gastric barium X-ray examination for gastric cancer screening by endoscopic examination. The subjects of this study included 4261 patients (1246 males, 3015 females) who underwent gastric X-ray examination for gastric cancer screening from 1991 to 1995, and 7178 patients (2432 males, 4746 females), consisting of all individuals who underwent endoscopic screening from 1996 to 2003. In addition, the regional screening included screening offered by local government only, and did not include screening offered by work site. The mean screening rate for the gastric X-ray examination group was 26.6% and the mean rate for the endoscopy group was 28.2%. To investigate whether reduction of gastric cancer mortality resulted from the introduction of endoscopic examination, gastric cancer cases detected by screening

Indication for EMR (endoscopic mucosal resection) conformed to the March 2001 Gastric Cancer Treatment Guidelines of the Japanese Gastric Cancer Association.

were evaluated in relation to various characteristics of the subjects and by evaluation of outcome (life or death), date of death, and cause of death in patients with gastric cancer for Kamigoto residents, in cooperation with the Kamigoto town office.

RESULTS Following the introduction of endoscopic examination, a decrease was observed in the number of screened individuals (Figure 1). People aged 60 years or older made up 70% of the endoscopic examination group, with this age group exhibiting a slight increase over time in proportion of all individuals screened. Among the 4261 individuals who underwent gastric X-ray examination, 2 (0.05%) cases of gastric cancer were discovered, compared to a Kamigoto population rate of 9.4/year per 100 000 population, and the mean gastric cancer detection rate was 0.05%. Among the 7178 individuals who underwent endoscopic examination, 28 (0.4%) cases of gastric cancer were discovered, compared to 48.8/year per 100 000 population, and the mean gastric cancer detection rate was 0.4%. After the introduction of endoscopic screening, the number of gastric cancer cases detected tended to increase (Figure 2). In the gastric X-ray examination group, the 2 cases noted above were detected during initial screening. In the endoscopic examination group, 13 (46.4%) cases were detected during an annual checkup, 3 (10.7%) cases were detected during a biennial checkup, and 4 (14.3%) cases were detected during initial screening. Table 1 shows the composition of gastric cancer cases detected during screening. Indications for EMR (endoscopic mucosal resection) confor med to the March 2001 Gastric Cancer Treatment Guidelines of the Japanese Gastric Cancer Association. In the gastric X-ray examination group, 1 (0.02%) case of early gastric cancer, 1 (0.02%) case reaching invasive depth M (mucosal layer), and no cases of cancers indicated for EMR were observed. In the endoscopic examination group, the corresponding numbers were 25 (0.35%) cases, 20 (0.28%) cases, and 12 (0.17%) cases. Of the cases detected in the endoscopic examination group, 25 of the 28 (89.3%) were early cancer, suggesting that endoscopy increased the rate of early gastric cancer detection, and 11 of the 28 (39.3%) underwent EMR, 17 of the 28 (60.7%) underwent www.wjgnet.com

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(Persons)

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6 1990-1996 1997-2006

4 2 '94

'96

'98

'00

'02

(Per 100 000 person-year) Male

100

80

80

60

60

40

40

20

20

0

1.041 0.712

Male (0.50-1.58) (0.33-1.10)

1.541 0.622

Female (0.71-2.38) (0.19-1.05)

1

'04

'06 (Yr)

Figure 3 Trend in Town of Kamigoto gastric cancer deaths.

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Table 2 Gastric cancer standard mortality ratios (SMR) Mean 95% Cl

8

0

Volume 13

'94 '96 '98 '00 '02 '04 '06 (Yr)

0

(Per 100 000 person-year) Female Town of Kamigoto Nagasaki Prefecture

'94 '96 '98 '00 '02 '04 '06 (Yr)

Figure 4 Trend in age-adjusted gastric cancer death rates.

definitive surgery and no inoperable case was noted. In one case in the gastric X-ray examination group, the patient subsequently died of gastric cancer, though as of December 2006 gastric cancer death was detected in one case and no recurrences had been noted in the endoscopic examination group (Table 1). The mean number of gastric cancer deaths in Kamigoto fell from a rate of 5.0/year for the 3-year period from 1994 to 1996 preceding the introduction of endoscopic examination to a rate of 2.1/year for the 10-year period from 1997 to 2006 (Figure 3). Thirty-four (94.4%) cases of these gastric cancer deaths involved unscreened individuals. Since all cases of gastric cancer death in 1996 were detected in fiscal year 1995, they were included in the first period. To more accurately characterize findings for Kamigoto, we used data for Nagasaki Prefecture to compare ageadjusted death rates derived from the year-on-year trend in gastric cancer deaths. However, results could not be calculated for males in 1996 or for females in 1994 and 1998 due to lack of confirmation of age at time of death. According to demographic statistics, the age-adjusted gastric cancer death rates (per 100 000 population) for Nagasaki Prefecture were 42.6 for males and 18.6 for females in 1995, and 37.3 for males and 16.0 for females in 2000. In Kamigoto, the corresponding mean values for the 3-year period from 1994 to 1996 preceding the introduction of endoscopic examination were 51.9 for males and 26.6 for females, while mean values for the 10-year period from 1997 to 2006 following the introduction of endoscopic examination were 28.0 for males and 6.9 for females (Figure 4). SMR (standard mortality ratios) were then determined

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1990-1996: Town of Kamigoto population (1995), gastric cancer mortality in Nagasaki Prefecture (1995). 21997-2006: Town of Kamigoto population (2000), gastric cancer mortality in Nagasaki Prefecture (2000). Since all cases of gastric cancer death in 1996 were detected in fiscal year 1995, they were included in the first period.

Table 3 Comparison of discovery costs for 1 gastric cancer case

Total number (a) Cost (b) No. gastric cancers (n) Gross cost (a × b) Cost to detect one case of gastric cancer (a × b/n)

X-ray 4261 ¥3000 2 ¥12 783 000 ¥6 391 500

Endoscope 7178 ¥11 000 28 ¥78 958 000 ¥2 819 928

from the Nagasaki Prefecture data. The data were divided into two groups, representing the 7-year period from 1990 to 1996 and the 10-year period from 1997 to 2006. As noted above, since all cases of gastric cancer death in 1996 were detected in fiscal year 1995, they were included in the first period. For the first period, the 1995 Kamigoto population and the 1995 Nagasaki Prefecture gastric cancer death rates (per 100 000 population) were used; for the second period, the corresponding figures for 2000 were used. The mean SMR values were calculated separately for males and females. For the first period, the results were 1.04 (95% CI: 0.50-1.58) for males and 1.54 (95% CI: 0.71-2.38) for females, while for the second period they were 0.71 (95% CI: 0.33-1.10) for males and 0.62 (95% CI: 0.19-1.05) for females (Table 2). A gastric X-ray examination costs ¥3000 per person, while an endoscopic examination costs ¥11 000 per person. Endoscopic examinations are thus more costly, but the cost incurred per gastric cancer case after discovery was ¥6 391 500 for the gastric X-ray examination group and ¥2 819 928 for the endoscopic examination group, making the cost-benefit ratio superior for the endoscopic examination group (Table 3). In the subject groups considered in this study, no case of incidental disease and endoscopy-related infection discovered by endoscopic examination and requiring medical intervention has yet been discovered.

DISCUSSION The number of gastric cancer cases discovered annually in Japan is 103 685 (estimated, 1999), and the number of deaths from gastric cancer is 49 535 (confirmed, 2003), placing gastric cancer first in Japan among cancers in terms of number of new cases and second only to lung cancer in terms of deaths. In Nagasaki Prefecture in 2000, gastric cancer morbidity was 130.1/100 000 for males and

Matsumoto S et al . Endoscopy in gastric cancer screening

57.5/100 000 for females, compared to mean values of 95.2/100 000 for males and 55.9/100 000 for females in Kamigoto for the 6-year period from 1998 to 2003. Thus, morbidity was significantly lower in males in Kamigoto than in males in Nagasaki Prefecture. Numerous studies have reported that gastric endoscopy is more effective than gastric X-ray examination in discovering small lesions and for qualitative diagnosis, and similarly for assessing gastric cancer detection rates and early gastric cancer ratios. However, according to the fiscal year 2003 Compendium of National Statistics from Gastroenterology Mass Survey, 5 975 956 individuals underwent gastric X-ray examination as part of gastric mass screening, compared to 68 923 who underwent endoscopy, showing that gastric X-ray examination still tends to be used for screening. Gastric cancer detection rate was 0.1% in the gastric X-ray examination group, compared to 0.24% in the endoscopy examination group[1]. In our study, the significant increase in gastric cancer detection after the introduction of endoscopic screening may reflect the aging of our study’s participants. The finding that gastric X-ray screening reduced gastric cancer death rates among both men and women was obtained in case-controlled research by Oshima et al[2], Fukao et al[3], and Abe et al[4], and by a meta-analysis by Tsubono et al (males OR: 0.39, 95% CI: 0.29-0.52; females OR: 0.50, 95% CI: 0.34-0.72)[5]. Mizoue et al[6] also carried out multicenter, cooperative research in a largescale research cohort comprising 30 771 screening group subjects and 56 541 control group subjects who were followed for 8 years. Males demonstrated a 46% reduction in death rate (RR: 0.54, 95% CI: 0.41-0.70), females exhibited a 26% reduction in death rate (RR: 0.74, 95% CI: 0.52-1.07), and among those with a prior history of gastric cancer in either parent, further reduction in death rate was noted (RR: 0.32, 95% CI: 0.12-0.87). A cohort studied by Lee et al[7] comprising 15 189 screening group subjects and 26 961 control group subjects was followed for 13 years and exhibited a 48% reduction in death rate (RR: 0.52, 95% CI: 0.36-0.74). Research by Arisue et al[8] compared a gastric cancer high-screening region to a lowscreening region, and demonstrated a reduction in death rate of 30.2% among males and 36.5% among females in the high-screening region (P < 0.05). Reductions of death rate of 14.1% among males and 22.6% among females were observed in the low-screening region (P < 0.05). With regard to reduction of gastric cancer death rate by endoscopic examination, Riecken et al [9] performed multiple endoscopic screenings from 1989 to 1999 among a Chinese cohort comprising 4394 residents of Linqu County, a gastric cancer cluster area. During a follow-up period extending to 2000, 85 individuals developed gastric cancer, of whom 29 (34%) were found to have early-stage cancer, while 58 (68%) had cancer discovered on screening. Gastric cancer SMRs among these subjects during followup were 1.01 (95% CI: 0.72-1.37), and 1.13 (95% CI: 0.77-1.57) for males and 0.65 (95% CI: 0.26-1.32) for females. The SMRs for two groups representing subjects from 1989-1994 (initial cohort) and those from 1995-2002 (interventional trial cohort) did not differ, at 0.97 (95% CI: 0.50-1.66) for the initial cohort and 1.08 (95% CI: 0.72-1.55)

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for the interventional trial cohort, and the study reported no observation of reduction of death rates. Other than the Riecken study, no studies have evaluated survival prognosis. Rather than compare a screening group and a nonscreening group, our study examined screened individuals and used two methods to analyze outcomes before and after the introduction of endoscopic screening: a direct method, involving age-adjusted death rate, and an indirect method, involving SMR. In each case, Kamigoto subject data were compared to those from Nagasaki Prefecture. Age-adjusted death rates for the Kamigoto females were clearly lower than those for Nagasaki Prefecture. Results for the Kamigoto males were also lower overall, but also reflect poor stability of death rates, a weakness of the direct method attributable to inclusion of a small subject group. We, therefore, also evaluated results by SMR, which is applicable to analysis at the city-, town-, and villageunit levels. Analysis was performed using gastric cancer death rates for Kamigoto and for Nagasaki Prefecture, and a notable reversal was observed at the point at which endoscopic screening was introduced. Although it would be premature to conclude that endoscopic screening decreased gastric cancer deaths, this conclusion has begun to be supported by follow-up data obtained 10 years after this introduction. Issues requiring further attention include detailed identification of high-risk gastric cancer groups, screening intervals, geriatric screening, and screening rates. Detection of H pylori infection, measurement of serum pepsinogen, and other methods have come into use in gastric cancer screening and are expected to provide screening markers for gastric cancer. Increase in screening rate is also a factor determining the effectiveness of gastric cancer screening, and efforts to increase screening rates require the creation of an organized system for screening, as opposed to opportunistic screening. Partnership with local governments is crucial for development of an organized screening system. Since the introduction of gastric endoscopy screening in Kamigoto, the mean screening rate there has been 28.2%, and efforts to communicate these research results, promote screening, and undertake interventions are expected to further increase this rate. In the Kamigoto area, endoscopic screening was implemented in an organized fashion. In the future, we intend to further increase the screening rate in partnership with local governments. The introduction of endoscopic screening increased early gastric cancer detection rates and EMR cases in particular. Since age-adjusted gastric cancer mortality and SMR in Kamigoto also declined after the introduction of endoscopic screening, the results of this study suggest that endoscopic screening probably decreased gastric cancer deaths. We believe our results provide further evidence that use of endoscopic examination for gastric cancer screening should be recommended.

ACKNOWLEDGMENTS An abstract of this paper was presented at the Current Issues in Upper Gastrointestinal Endoscopy Symposium at www.wjgnet.com

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the 70th General Meeting of the Japan Gastroenterological Endoscopy Society (DDW-Japan Kobe).

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REFERENCES

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1

2 3

4

Fiscal year 2003 Compendium of National Statistics from Gastroenterology Mass Survey. Tokyo: Japanese Society of Gastroenterology Mass Survey, National Statistics Committee (in Japanese), 2005: 96-109 Oshima A, Hirata N, Ubukata T, Umeda K, Fujimoto I. Evaluation of a mass screening program for stomach cancer with a case-control study design. Int J Cancer 1986; 38: 829-833 Fukao A, Tsubono Y, Tsuji I, HIsamichi S, Sugahara N, Takano A. The evaluation of screening for gastric cancer in Miyagi Prefecture, Japan: a population-based case-control study. Int J Cancer 1995; 60: 45-48 Abe Y, Mitsushima T, Nagatani K, Ikuma H, Minamihara Y. Epidemiological evaluation of the protective effect for dying of stomach cancer by screening programme for stomach cancer with applying a method of case-control study (in Japanese). Nippon Shokakibyo Gakkai Zasshi (Jpn J Gastroenterol) 1995; 92: 836-845

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8

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Tsubono Y, Hisamichi S. Case-control studies of screening for gastric cancer in Japan (in Japanese). Nippon Shokaki Syudankenshin Gakkai Zasshi (Jpn J Gastroenterol Mass Survey) 1999; 37: 182-185 Mizoue T, Yoshimura T, Tokui N, Hoshiyama Y, Yatsuya H, Sakata K, Kondo T, Kikuchi S, Toyoshima H, Hayakawa N, Tamakoshi A, Ohno Y, Fujino Y, Kaneko S. Prospective study of screening for stomach cancer in Japan. Int J Cancer 2003; 106: 103-107 Lee KJ, Inoue M, Otani T, Iwasaki M, Sasazuki S, Tsugane S. Gastric cancer screening and subsequent risk of gastric cancer: a large-scale population-based cohort study, with a 13-year follow-up in Japan. Int J Cancer 2006; 118: 2315-2321 Arisue T, Tamura K, Yoshida Y, Tebayashi A, Yamaguchi Y, Ikeda S, Ohtuka S. Comparisons of the changes in the mortality from stomach cancer between the model areas of mass screening for stomach cancer and the control areas (in Japanese). Nippon Shokaki Syudankenshin Gakkai Zasshi (Jpn J Gastroenterol Mass Survey) 1986; 73: 26-32 Riecken B, Pfeiffer R, Ma JL, Jin ML, Li JY, Liu WD, Zhang L, Chang YS, Gail MH, You WC. No impact of repeated endoscopic screens on gastric cancer mortality in a prospectively followed Chinese population at high risk. Prev Med 2002; 34: 22-28 S- Editor Zhu LH L- Editor Alpini GD

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E- Editor Liu Y

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World J Gastroenterol 2007 August 28; 13(32): 4321-4327 World Journal of Gastroenterology ISSN 1007-9327 © 2007 WJG. All rights reserved.

GASTRIC CANCER

Syndrome differentiation in traditional Chinese medicine and E-cadherin/ICAM-1 gene protein expression in gastric carcinoma Da-Zhi Sun, Ling Xu, Pin-Kang Wei, Long Liu, Jin He Da-Zhi Sun, Ling Xu, Pin-Kang Wei, Long Liu, Department of Traditional Chinese Medicine, Second Affiliated Hospital to Second Military Medical University, Shanghai 200003, China Jin He, Department of Pathology, Second Affiliated Hospital to Second Military Medical University, Shanghai 200003, China Supported by the National Natural Science Foundation of China, No. 30271626 Correspondence to: Ling Xu, Department of Traditional Chinese Medicine, Second Affiliated Hospital to Second Military Medical University, 415 fengyang Road, Shanghai 200003, China. [email protected] Telephone: +86-21-63610109-73406 Received: 2007-03-03 Accepted: 2007-03-28

Abstract AIM: To explore the syndrome differentiation in traditional Chinese medicine (TCM) and gene protein expression in gastric carcinoma. METHODS: Preoperative data of gastric cancer cases were collected from the General Surgery Department and classified according to the criteria for syndrome differentiation in TCM. E-cadherin (E-cad) and ICAM-1 gene protein expressions were detected in postoperative specimens from these cases by the immunohistochemical EnVision two-step method. RESULTS: The E-cad positive expression rate was 90% in 100 cases of gastric carcinoma. The difference in E-cad expression was significant between the different syndrome differentiation types in TCM (P < 0.01). Further group-group comparison showed that there was a significant difference in E-cad expression between the stagnation of phlegm-damp type and the deficiency in both qi and blood and the deficiency-cold of stomach and spleen types, where E-cad expression was high. There was no significant difference between the internal obstruction of stagnant toxin type and the in-coordination between liver and stomach type, where E-cad expression was relatively low. The ICAM-1 positive expression rate was 58%, and there was no statistically 2 significant difference between the two groups (χ = 8.999, P > 0.05). CONCLUSION: E-cad expression is relatively low in the internal obstruction of stagnant toxin type and the incoordination between liver and stomach type, where

tumor development and metastasis may be associated with low E-cad expression, or with low homogeneous adhesiveness between tumor cells. © 2007 WJG . All rights reserved.

Key words: Gastric carcinoma; Syndrome differentiation in traditional Chinese medicine; Metastasis; E-cadherin; ICAM-1 Sun DZ, Xu L, Wei PK, Liu L, He J. Syndrome differentiation in traditional Chinese medicine and E-cadherin/ICAM-1 gene protein expression in gastric carcinoma. World J Gastroenterol 2007; 13(32): 4321-4327

http://www.wjgnet.com/1007-9327/13/4321.asp

INTRODUCTION Guided by the “yin-yang and five-element theories” and supported by the “viscera-state and meridian doctrines”, traditional Chinese medicine (TCM) elucidates the kinetic laws of human birth, aging, disease and death, and treats illnesses in the light of “syndrome differentiation”. Nevertheless, TCM is unable to explain various human physiopathogical phenomena. Most of its theories and doctrines are abstracted and deduced from direct experience and perception. These theories and doctrines have been playing an undeniable role, but they restrain the advance in TCM science. With the rapid advance in modern science and technology, people have recognized microscopic changes in human organs at cell and molecular levels. However, the potential correlation between TCM theories and microscopic changes has been rarely studied. Syndrome differentiation in TCM, a crucial step in clinical practice of TCM, is a comprehensive analysis of external manifestations of the human body based on TCM theories, and reflects physiopathological changes in the human body (from the TCM viewpoint). Treatment based on syndrome differentiation is the core of TCM therapy for tumors. Traditional Chinese herbal medicines play a unique role in treating tumors, especially in improving the quality of life and prolonging survival of tumor patients. This study was to explore the syndrome differentiation in TCM and E-cadherin (E-cad) and ICAM-1 expression in gastric carcinoma. www.wjgnet.com

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MATERIALS AND METHODS Patients All data were obtained from patients admitted to Changzheng Hospital between November 2002 and Januar y 2005, who were diagnosed with or highly suspected of having gastric carcinoma and scheduled to receive surgical operation on the next day. A total of 500 patients were enrolled in the study, including those who were diagnosed with gastric carcinoma by gastroscopic biopsy and scheduled to receive surgical operation, as well as those who refused to undergo preoperative gastroscopic biopsy, but were highly suspected of having gastric carcinoma by B-ultrasound and CT scan and scheduled to undergo surgical resection. The following patients were excluded from the study, including (1) those who were highly suspected of having gastric carcinoma preoperatively which were excluded from malignant tumors by pathologic study of surgically resected specimens, (2) those who failed to undergo surgical operation on the next day because of various reasons (unable to decide whether there was tumor infiltration or lymph node metastasis), (3) those with non gastric carcinoma, including mesothelioma, gastric lymphoma, gastric metastatic tumor and tumors disseminating from other parts of the body. A total of 81 cases were excluded from the study, including 36 cases of non gastric carcinoma, 21 cases of benign tumor, 6 cases of mesothelioma, 2 cases of tumor spreading from colonal carcinoma and 1 case of pancreatic cancer, 1 case of hamartoma, 32 cases of tumor with unknown pathology and 13 cases who did not receive surgical operation. Finally, a total of 419 cases were included in the present study, including 148 cases of in-coordination between liver and stomach type (IBLS), 84 cases of stagnation of phlegm-damp type (SPD), 74 cases of internal obstruction of stagnant toxin type (IOST), 46 cases of deficiencycold of stomach and spleen type (DCSS), 29 cases of yin impairment due to stomach heat type (YISH), and 38 cases of deficiency in both qi and blood type (DOQB). The details are shown in Table 1. A total of 100 cases were selected randomly for immunohistochemical analysis. Gastric carcinoma was divided into six types according to the six-type differentiation method set by Beijing Coordination Group of the First National Symposium on Gastric Carcinoma (1978). Syndrome differentiation criteria included (1) IBLS with symptoms of stomach fullness, intermittent dull pain which may radiating two flanks, hiccup and vomiting, deep or thin pulse, pink tongue, and thin or light yellow tongue fur; (2) DCSS with symptoms of gastric dull pain which can be relieved by press and warmth, eating in the morning and vomiting in the evening or vice versa, pallor, cold extremities and mental tiredness, loose stool and edema, pale and enlarged tongue, white and moist tongue fur, and deep and slow pulse; (3) IOST with symptoms of gastric stabbing pain, epigastric fullness and rigidity, hemoptysis and melena, dry and scaly skin, dark purplish tongue, deep and thin pulse; (4) YISH with symptoms of intragastric burning heat, oral dryness and thirst, gastric discomfort with acid

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rereflux, postprandial gastric pain, feverish sensation in chest, palms and soles, dry stool, anorexia, thin and wiry pulse, red tongue with little fur or yellow fur with little saliva; (5) SPD with symptoms of chest suffocation and diaphragm fullness, sallow complexion and edema, vomiting and profuse sputum, gastric fullness and loose stool, subcutaneous nodules, pink tongue, and greasy tongue fur; and (6) DOQB with symptoms of generalized hypodynamia, palpitation and shortness of breath, dizziness and halo vision, dim complexion, vexation of deficiency type and insomnia, spontaneous perspiration and night sweating, deficiency in both yin and yang, as well as deep, thin and weak pulse, pale tongue with thin fur. Tumor specimens All tumor specimens were obtained were from the General Surgery Department and paraffin-embedded in the Pathology Department of Changzheng Hospital. Reagents and instruments Mouse anti human E-cad protein primary antibody (a concentrated type: 0 mL, 1:50, serial No. M0536) was purchased for m Fuzhou Maixin Biotechnolog y Development Co., Ltd (Fuzhou, China). Mouse anti human ICAM-1 protein primary antibody (a concentrated type: 0 mL, 1:50, serial No. M0146) was purchased from Shanghai Changdao Biotechnology Co., Ltd (Shanghai, China). PBS buffer solution was prepared for washing before use. EDTA buffer solution was purchased from DAKO. Hematoxylin and DAB color test kit were products of Fuzhou Maixin Biotechnology Development Co., Ltd. Other reagents included 10% neutral formalin, xylene and 3% hydrogen peroxide methanol solution. Main experimental instruments included paraffin section machine (LEICA 2135, Germany), automatic tissue processing machine (SHANDON, UK), electronic balance (MP-110-I, Shanghai No. 2 Balance Instrumentation Factory, Shanghai, China), biological photomicroscope (OLYMPUS, Japan), ZH-3 paraffin section freezing table (Shenyang Zong Heng Hi-technology Co., Ltd, Shenyang, China), water-jacketed electrothermostatic incubator (PYXDHS-40 × 50, Shanghai Yue Jin No. 1 Medical Instruments Factory, Shanghai, China), processing microshaker (75-2A, Shanghai Medical Analytic Instrumentation Factory), and drying box (Shanghai Xin Li Instruments Manufacturer). Immunohistochemistry (using EnVision two-step method) Human gastric tumor specimens fixed in 10% neutral formalin were dehydrated with ethanol, hyalinized with xylene, paraffin-embedded, sliced into 4- μ m sections, deparaffined with xylene, gradient hydrated with alcohol, washed with distilled water, and soaked in 3% hydrogen peroxide methanol solution to block endogenous hydrogen peroxidase. Antigen repair was done with 0.01mol citric acid buffer solution (pH 6.0) in a hyperbaric pot. The specimens were then added into non-immune bovine serum, incubated at 37℃ for 20 min, added into 50 μL concentrated primary antibody E-cad and ICAM-1 (1:50), incubated in a 4℃ refrigerator overnight, added into

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Table 1 Correlation between pathology and syndrome differentiation in 419 cases of gastric carcinoma Items

Syndrome differentiation

All

P

IBLS

SPD

IOST

DCSS

YISH

DOQB

M W

148 99 49

84 64 20

74 57 17

46 34 12

29 11 18

38 21 17

419 286 133

Age (yr)

0.05) (Table 4).

DISCUSSION The significance of syndrome differentiation in TCM was stated as early as in Tang Dynasty as “ The same internal disease may be represented by different external symptoms and signs, and different internal diseases may have the same external manifestations and vice versa. Therefore, sufficiency or deficiency in five-zang organs and six-fu organs, luxuriance or exsiccation and patency or obstruction of blood vessels is not exactly what is observed by the eyes. They should be judged by differential diagnosis”. TCM pays special attention to the integrity and holism of the human body and its interrelationship with nature. The component parts of the human body are

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Table 2 E-cad immunohistochemical expression values in different syndrome differentiation types of gastric carcinoma SD in TCM

n

SPD (1) YISH (2) IOST (3) DOQB (4) DCSS (5) IBLS (6)

17 16 16 17 17 17

E-cad expression value

6 2 0 6 6 0

6 6 0 4 6 12

9 6 6 6 4 2

6 0 12 8 12 0

0 4 6 8 4 2

4 8 0 6 6 3

6 2 3 2 8 2

6 2 0 12 9 1

6 4 3 9 6 6

9 2 3 6 8 4

6 8 2 4 2 1

3 6 0 4 4 6

6 4 8 4 1 2

8 12 1 0 12 6

9 4 4 6 2 2

6 1 4 8 4 4

3

1 0 1 2 0 0

1 1 4 0 0 1

2 1 1 6 0 0

1

9 4 0

SD in TCM: Syndrome differentiation in traditional Chinese medicine.

Table 3 Comparison of E-cad expression values and SD in TCM 2

P

Comparison groups

χ

P

88.434 264.674 0.001 6.021 303.01 46.373 88.107 48.806

< 0.01 < 0.01 > 0.05 > 0.05 < 0.01 < 0.01 < 0.01 < 0.01

2, 6 3, 4 3, 5 3, 6 4, 5 4, 6 5, 6

60.018 265.648 193.162 0.697 5.936 302.395 223.599

< 0.01 < 0.01 < 0.01 > 0.05 > 0.05 < 0.01 < 0.01

χ

Comparison groups 1, 2 1, 3 1, 4 1, 5 1, 6 2, 3 2, 4 2, 5

2

Table 4 ICAM-1 immunhistochemical expression values in different SD types SD type

n

SPD YISH IOST DOQB DCSS IBLS

17 16 16 17 17 17

ICAM-1 0 1 2 0 1 0

0 0 0 0 1 0

0 2 4 1 1 0

1 0 0 0 1 0

1 1 2 1 1 0

1 0 0 0 0 1

6 2 1 1 0 0

0 1 1 1 0 0

0 0 1 0 1 0

1 1 1 0 2 4

1 2 1 1 1 0

2 1 0 0 0 2

1 2 4 0 1 1

1 1 0

2 χ = 8.999, P = 0.109.

inseparable and are functionally coordinative and mutually beneficial while affecting each other pathologically. At the same time, TCM adheres to the basic principle of treatment based on differentiation of symptoms and signs, treats the same disease by different methods and different diseases by the same method, and advocates individualized treatment, which vividly reflects the essence of TCM treatment. Modern biologic research has entered an era of integrating various research technologies and methods to tackle difficult biological problems at biomolecular level as a whole, which is exemplified by studies in the new scientific fields of genomics and proteomics. According to the central dogma of molecular biology and life, the task of molecular biology is to study life phenomena and nature of diseases, governing and controlling the development and progression of phenotype (proteincell-organism) process. Life phenomena, diseases and syndrome differentiation in TCM can obtain the most essential answers from DNA or gene research. Tumor metastasis may occur in all gastric carcinoma

differentiation types, including deficiency syndrome, blood stagnation syndrome and phlegm syndrome, but it is of greater significance to know which differentiation type has a higher metastasis rate. Therefore, to study differences in gene expression between different gastric carcinoma differentiation types and the differentiation nature of gastric carcinoma at gene level and judge the metastatic tendency of different differentiation types are of great significance. This is also the reason why we used E-cad and ICAM-1 expression to study syndrome differentiation of gastric carcinoma in TCM. Cadherins, a group of calcium dependent transmembrane proteins, are members of the cell-cell adhesion molecule family essential for close intercellular connection [2], which mainly mediate adhesion reaction of homologous cells. Cadherins are divided into several subtypes, among which E-cad is a carcinoma inhibitory gene most closely related to tumor invasion and metastasis. Experiments showed that transfection of E-cad to tumor cells could reverse their invasive behavior[3], and use of anti-E-cad antibody could block cell-cell connection, thus

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enabling carcinoma cells to acquire high invasiveness. Introduction of E-cad antisense RNA into tumor cell strain highly expressing calcium adhesion protein may significantly increase its metastatic ability, thus further confirming that E-cad inhibits tumor metastasis [3]. E-cad gene mutation due to loss of cell-cell adhesion during the morula stage leads to fatal impairment to the embryo[4,5], indicating that cadherins play an essential role in the process of histolodifferentiation. Furthermore, E-cad gene has been accepted as a cancer inhibitory gene [6,7]. When E-cad is down-regulated or dysfunctional, adhesion between homogenous cells is lost. For normal tissues, this means that they are unable to develop normally, and for epithelial tumors, this means that they have the tendency to grow invasively and are likely to exfoliate from the primary focus and metastasize to local lymph nodes and distant locations. Down-regulation or dysfunction of E-cad may be an early event in the development and progression of gastric carcinoma[8]. According to Berx et al[9], the type of E-cad mutation in defuse gastric carcinoma is a shearinduced in-frame deletion. Recent studies show that downregulation of E-cad expression in malignant tumor tissue is associated with the degree of differentiation[10-12]. Cai JC et al reported that the E-cad positive expression rate was only 10% in gastric carcinoma group and 100% in control group, indicating that E-cad is lost in gastric carcinoma. It was reported that the E-cad positive rate is 36.96% in gastric carcinoma, and there is a decreasing tendency in malignant tumors [13]. It was also reported that E-cad expression was significantly lower in poorly differentiated[13], especially in metastatic foci, where E-cad expression is reduced or even disappeared [14,15]. In the present study, the E-cad positive expression rate in 100 cases of gastric carcinoma was 90% and E-cad expression was significantly different in different differentiation syndrome types（P < 0.01）and E-cad expression was relatively low in the IOST and IBLS types, suggesting that tumor metastasis in them may be associated with E-cad. ICAM-1 CD54, a single-chain transmemebrane glycoprotein encoded by No. 19 chromosome gene, is a member of the adhesion molecule immunoglobin superfamily (IgSF), which has 5 extracellular immunoglobulin-like sites and a short cytoplasmic trailer structure, with a molecular weight varying with the cell type from 76 000 KD to 114 000 KD. This difference mainly depends on the degree of glycosylation of the molecule. Intercellular adhesion and identification are the necessary processes in immunological surveillance. Antigen dependence and/or non-dependence between target cells and lymphocytes are the initial step of tumor immunological reaction. It was reported that ICAM-1 on the surface of cancer cell or of presenting cell is a kind of co-stimulation molecules[16], enabling T cell receptor mediated cancer cells to combine with T lymphocytes more steadily. ICAM expression on the surface of cancer cells can enhance its susceptibility to lymphocytemediated cytotoxicity. Decreased ICAM expression often leads to lymph node metastasis, suggesting that ICAM-1

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gene transfection can inhibit lymph node metastasis. To investigate whether ICAM-1 gene could treat peritoneal metastasis, Tanaka et al[17] transfected the ICAM-1 cDNA to the gastric carcinoma cell strain OCUM-2MD3 (cancer cells having a high peritoneal metastasis rate), and found that ICAM-1 was significantly expressed on cell surface. The survival of mice peritoneally transplanted with gene transfected 2MD3/ICAM-1 was significantly higher than that of animals transplanted with 2MD3 only, suggesting that ICAM-1 gene transfection therapy can effectively prevent peritoneal metastasis of gastric carcinoma. In the present study, the ICAM-1 positive expression rate was 58%, and there was no significant difference in ICAM-1 expression between the syndrome differentiation types (P > 0.05), demonstrating that the difference in tumor recurrence and metastasis between syndrome differentiation types is not due to ICAM-1 change.

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Rosivatz E, Becker I, Specht K, Fricke E, Luber B, Busch R, Hofler H, Becker KF. Differential expression of the epithelialmesenchymal transition regulators snail, SIP1, and twist in gastric cancer. Am J Pathol 2002; 161: 1881-1891 2 Takeichi M. Cadherin cell adhesion receptors as a morphogenetic regulator. Science 1991; 251: 1451-1455 3 Frixen UH, Behrens J, Sachs M, Eberle G, Voss B, Warda A, Lochner D, Birchmeier W. E-cadherin-mediated cell-cell adhesion prevents invasiveness of human carcinoma cells. J Cell Biol 1991; 113: 173-185 4 Larue L, Ohsugi M, Hirchenhain J, Kemler R. E-cadherin null mutant embryos fail to form a trophectoderm epithelium. Proc Natl Acad Sci USA 1994; 91: 8263-8267 5 Riethmacher D, Brinkmann V, Birchmeier C. A targeted mutation in the mouse E-cadherin gene results in defective preimplantation development. Proc Natl Acad Sci USA 1995; 92: 855-859 Becker KF, Keller G, Hoefler H. The use of molecular biology 6 in diagnosis and prognosis of gastric cancer. Surg Oncol 2000; 9: 5-11 Lee JH, Koh JT, Shin BA, Ahn KY, Roh JH, Kim YJ, Kim KK. 7 Comparative study of angiostatic and anti-invasive gene expressions as prognostic factors in gastric cancer. Int J Oncol 2001; 18: 355-361 Yu J, Ebert MP, Miehlke S, Rost H, Lendeckel U, Leodolter 8 A, Stolte M, Bayerdorffer E, Malfertheiner P. alpha-catenin expression is decreased in human gastric cancers and in the gastric mucosa of first degree relatives. Gut 2000; 46: 639-644 Berx G, Becker KF, Hofler H, van Roy F. Mutations of the 9 human E-cadherin (CDH1) gene. Hum Mutat 1998; 12: 226-237 10 Mayer B, Johnson JP, Leitl F, Jauch KW, Heiss MM, Schildberg FW, Birchmeier W, Funke I. E-cadherin expression in primary and metastatic gastric cancer: down-regulation correlates with cellular dedifferentiation and glandular disintegration. Cancer Res 1993; 53: 1690-1695 11 Karayiannakis AJ, Syrigos KN, Chatzigianni E, Papanikolaou S, Karatzas G. E-cadherin expression as a differentiation marker in gastric cancer. Hepatogastroenterology 1998; 45: 2437-2442 12 Guzman P, Araya J, Villaseca M, Roa I, Melo A, Munoz S, Roa J. Immunohistochemical expression of the E-cadherin-catenin complex in gastric cancer. Rev Med Chil 2006; 134: 1002-1009 13 Cai JC, Jiang SJ, Chen ZP. Relationship between E-cadherin and growth pattern of gastric cancer and degree of cell differentiation. Zhonghua Binglixue Zazhi 1994; 23: 132-134 14 Schuhmacher C, Becker KF, Reich U, Schenk U, Mueller J, Siewert JR, Hofler H. Rapid detection of mutated E-cadherin in peritoneal lavage specimens from patients with diffuse-type

Sun DZ et al. Syndrome differentiation in TCM and gene protein expression in gastric carcinoma gastric carcinoma. Diagn Mol Pathol 1999; 8: 66-70 15 Nabeshima K, Inoue T, Shimao Y, Kataoka H, Koono M. Cohort migration of carcinoma cells: differentiated colorectal carcinoma cells move as coherent cell clusters or sheets. Histol Histopathol 1999; 14: 1183-1197 16 Bertolino P, Schrage A, Bowen DG, Klugewitz K, Ghani S, Eulenburg K, Holz L, Hogg N, McCaughan GW, Hamann A.

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Early intrahepatic antigen-specific retention of naive CD8+ T cells is predominantly ICAM-1/LFA-1 dependent in mice. Hepatology 2005; 42: 1063-1071 17 Tanaka H, Yashiro M, Sunami T, Ohira M, Hirakawa-Y S Chung K. Lipid-mediated gene transfection of intercellular adhesion molecule-1 suppresses the peritoneal metastasis of gastric carcinoma. Int J Mol Med 2002; 10: 613-617 S- Editor Liu Y L- Editor Wang XL

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World J Gastroenterol 2007 August 28; 13(32): 4328-4332 World Journal of Gastroenterology ISSN 1007-9327 © 2007 WJG. All rights reserved.

BASIC RESEARCH

Cystamine ameliorates liver fibrosis induced by carbon tetrachloride via inhibition of tissue transglutaminase Jiang-Feng Qiu, Zhi-Qi Zhang, Wei Chen, Zhi-Yong Wu Jiang-Feng Qiu, Zhi-Qi Zhang, Wei Chen, Zhi-Yong Wu, Department of General Surgery, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200127, China Supported by National Natural Science Foundation of China, No. 30571825 Correspondence to: Zhi-Yong Wu, Professor, Department of General Surgery, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200127, China. [email protected] Telephone: +86-21-50905335 Fax: +86-21-58394262 Received: 2007-03-26 Accepted: 2007-06-18

Abstract AIM: To investigate the anti-fibrosis effect of the tissue transglutaminase (tTG) specific inhibitor cystamine on liver fibrosis. METHODS: Sixty-eight male Sprague Dawley rats were divided into three groups: normal control, liver fibrosis control and cystamine-treated group. Liver fibrosis was induced by intraperitoneal injection of carbon tetrachloride (CCl4), and Cystamine was administrated by intraperitoneal injection starting 2 d before the first administration of CCl 4. Animals in each group were further divided into 2 subgroups according to two time points of 4 wk and 8 wk after treatment. Hepatic function, pathological evaluation (semi-quantitative scoring system, SSS) and liver hydroxyproline (Hyp) content were examined. Real-time PCR was used to detect the expression of tTG, smooth muscle alpha actin (α-SMA), tissue inhibitor of metalloproteinase 1 (TIMP-1) and collagen-1 mRNA. The expressions of tTG and α-SMA protein were detected by Western Blotting. RESULTS: Eight weeks after treatment, the SSS score of liver was significantly less in the cystamine group than that in the fibrosis control group (P < 0.01). The levels of alanine aminotransferase (ALT) and total bile acid (TBA) at the 4 wk and 8 wk time points were decreased in the cystamine group compared with those in fibrosis controls (P < 0.01). Liver hydroxyproline content at the 4 wk and 8 wk time points showed a substantial reduction in the cystamine group compared to fibrosis controls (P < 0.01). The expression of tTG, α-SMA, collagen-1, TIMP-1 mRNA and tTG, as well as α-SMA protein was downregulated in the cystamine group compared to fibrosis controls. CONCLUSION: Cystamine can ameliorate CCl4 induced liver fibrosis and protect hepatic function. The possible

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mechanism is related to the reduced synthesis of the extracellular matrix (ECM) caused by the inhibition of hepatic stellate cell activation and decreased expression of TIMP-1. © 2007 WJG . All rights reserved.

Key words: Tissue transglutaminase; Cystamine; Liver fibrosis; Cross linking; Extracellular matrix Qiu JF, Zhang ZQ, Chen W, Wu ZY. Cystamine ameliorates liver fibrosis induced by carbon tetrachloride via inhibition of tissue transglutaminase. World J Gastroenterol 2007; 13(32): 4328-4332

http://www.wjgnet.com/1007-9327/13/4328.asp

INTRODUCTION Liver fibrosis is characterized by increased synthesis and decreased degradation of the extracellular matrix (ECM) [1] . Decreased ECM degradation results partly from irreversible cross-linking of the ECM and partly from increased expression of tissue inhibitor of metalloproteinase (TIMP) by hepatic stellate cells (HSCs). In the liver, both parenchymal and non-parenchymal cells produce tissue transglutaminase (tTG), and it appears to be released into the extracellular space. tTG is a member of the large transglutaminase (TG) family, which are involved in numerous biochemical processes. tTG not only displays cross-linking activity, but also functions as a GTPase, and it has been shown to be involved in a variety of fibrotic diseases including scleroderma, experimental renal fibrosis and human hepatic fibrosis[2,3]. Indeed, tTG expression is upregulated when there is any injuring process. Studies have demonstrated that liver cell death secondary to ethanol administration occurs by apoptosis, not by liver cell necrosis [4,5], and tTG may be a factor in apoptosis induced by ethanol. Several reports suggest that tTGase activity was increased associated with transforming growth factor beta (TGF-β) and ECM production and α-SMApositive HSC number also increased after CCl4 intoxication in rats[6,7]. These findings have raised the hypothesis that tTG may contribute to the development of liver fibrosis and inhibition of tTG may be effective in treatment of liver fibrosis. In this study, we investigated whether tTG plays a role in the progression of rat hepatic fibrogenesis

Qiu JF et al . Cystamine ameliorates liver fibrosis

induced by carbon tetrachloride (CCl4) via administration of the tTG specific inhibitor cystamine.

MATERIALS AND METHODS Animals and grouping Sixty-eight Sprague Dawley rats weighing 300-350 g (Animal Center, Chinese Academy of Sciences, Shanghai) were studied. The animals were housed in an environmentally controlled vivarium with timed light control (12 h lightdark cycle) and allowed free access to a standard pellet diet and water. The animals were divided into three groups: normal control (N), fibrosis control (F) and cystamine treatment (C). Rats in each group were subdivided into two groups according to two time points of 4 wk and 8 wk as follows: N4 (n = 10), N8 (n = 10); F4 (n = 13), F8 (n = 8), C4 (n = 14), C8 (n = 13). Reagents and instruments Cystamine (tTG specific inhibitor) was purchased from Sigma Co, USA. Mouse monoclonal tTG antibody was purchased from Stratek Scientific, UK (No: CUB 7402), and labelled streptavidin-biotin (LSAB) immunohistochemistry kits and mouse monoclonal α-SMA antibody were from Dako Co, USA. The Beckman Coulter LX 20 automatic biochemical analytic instrument and real time PCR amplifier (ABI Prism 7700 Sequencer Detector, Applied Biosystems, Co) were products from the USA. Model establishment and treatment Normal control rats were injected intraperitoneally with peanut oil (0.2 mL/100 g) twice a week. Liver fibrosis control rats were induced by intraperitoneal injection of 50% CCl 4 solution (CCl 4 :oil = 1:1) at a dose of 0.2 mL/100 g twice a week for 8 wk. One subgroup of rats that had cystamine treatment for the first 4 wk were given cystamine 2 d before the first injection of CCl 4 at a dose of 112 mg/kg per day as in a previous study[8]. The other subgroup of rats that had cystamine treatment for the second 4 wk (8 wk subgroup) were administrated cystamine after injection of CCl4 for 4 wk at the same dose. Rats in each group were sacrificed 3 d after the final administration according to each time point. Blood collected from the aorta was centrifuged and then the serum was stored at -80℃ for hepatic function examination. Liver was removed and fixed in 10% neutral formalin for pathological examination and other assays. Liver function The levels of aspartate aminotransferase (ALT), total bilirubin (TB), and total bile acid (TBA) were tested with Beckman Coulter LX 20 (USA). Liver hydroxyproline content The hydroxyproline kit obtained from Nanjing Jiancheng Biological Engineering Research Institute was used to analyze liver hydroxyproline content (μg/g). Liver histopathological examination Tissues from each liver were fixed in 10% formalin and embedded in paraffin, and then cut into 5 μ m pieces

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and mounted on slides. The samples were stained with hematoxylin and eosin (HE) for histopathological examination and a semi-quantitative scoring system (SSS)[9] was adopted for evaluation of the degree of liver fibrosis. Real-time PCR Taqman real-time polymerase chain reaction (PCR) was used to determine the relative expression of messenger RNAs (mRNAs) for tTG, α-SMA, TIMP-1 and collagen-1 in total RNA extracted from snap-frozen liver by the acidphenol method. All primers were designed by using the Primer Expression 2.0 software (PE Applied Biosystems). Primers synthesized by Shanghai Shenggong Corp. were as follows: (1) tTG, sense: 5'-gtatgatgcgtccttcgtgt-3', antisense: 5'-cagtttgttcaggtggttgg-3'; (2) α-SMA: sense:5'TGGGAAATGCCACAGGTT-3', antisense: 5'-CTGC GGTTCTGGGACTTG-3'; (3) TIMP-1: sense: 5'-atattc tgtctggatcggc-3', antisense: 5'-gcttcgtcatactcctgttt-3'; (4) collagen-1: sense: 5'-tctccaagaggcagggttc-3', antisense: 5'-ggttagcttcggctca tgc-3'; HPRT, sense: 5'-AAAGCCAA GTACAAAGCCTAAA-3', antisense: 5'-CTGTCTGTC TCACAAGGGAAGT-3'. The conditions of the reaction were as follows: initial steps were 95℃ for 10 s, followed by a denaturing step for 5 s at 95℃, and an annealing/ extension step at 60℃ for 30 s performed in 40 cycles. The PCR products were analyzed directly by electrophoresis on 2.0% agarose gels. Relative concentrations were calculated and nor malized according to the expression of the housekeeping gene HPRT. Western blots Total protein was deter mined with a protein assay (BioRad Inc, California, USA) and electrophoresed on a 4%-12% polyacrylamide gradient gel with 10% sodium dodecyl sulfate (SDS), and subsequently transferred to a nitrocellulose membrane. The membrane was blocked for 1 h in 1% to 5% nonfat dry milk in Tween Trisbuffered saline (TTBS). Western blot analysis of liver tissue was performed by using a monoclonal anti-α-SMA and antitTG. Each antibody was used at a concentration of 1:1000. Membranes were incubated overnight with the primary antibody or with nonimmune immunoglobulin G (as a negative control) in TTBS, after which the secondary horseradish-peroxidase-conjugated antibodies were applied in TTBS containing 0.1% nonfat dry milk for 1 h. Reactive bands were identified by using enhanced chemiluminescence (ECL; Amersham, Bucks, UK) and autoradiography according to the manufacturer's instructions. Statistical analysis All data are presented as mean ± SD. Statistical comparison among groups was made by one-way analysis of variance (ANOVA) using SPSS12.0 statistical software (Lead Technologies, Inc, USA). A P value less than 0.05 was considered statistically significant.

RESULTS Histopathological findings Eight weeks after treatment, the liver appeared normal in control rats. The score of SSS evaluation for hepatic fibrosis in cystamine treatment group (4.20 ± 0.81) was www.wjgnet.com

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Hyp content after treatment of cystamine The levels of hydroxyproline (Hyp) in fibrosis controls at 4 wk (77.71 ± 12.47 μg/g) and 8 wk (130.58 ± 25.80 μg/g) were higher than those in normal controls (4 wk, 53.88 ± 10.56 μg/g; 8 wk, 54.67 ± 11.77 μg/g, P < 0.01). The levels of Hyp in cystamine-treated rats at 4 wk (60.64 ± 8.94 μg/g) and 8 wk (69.87 ± 13.48 μg/g) were significantly less than those in fibrosis controls (P < 0.01).

Figure 1 Histologic appearance of representative livers for cystamine treatment and fibrosis control rats. A, B: Cystamine treatment ameliorated liver damage (A × 100, B × 200); C, D: Fibrosis control livers from CCl4-treated rats showed substantial damage including inflammation, fine fibrils and degeneration of hepatocytes (C × 100, D × 200).

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TB in fibrosis control rats were much greater than those in normal control rats (P < 0.01); The levels of ALT and TBA of cystamine-treated rats were significantly less than those in fibrosis controls (P < 0.01); however, there was no significant difference in the levels of TB in the two time point compared with fibrosis controls (Figure 2).

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Effect of cystamine on the expression of tTG, α-SMA, collagen-1 and TIMP-1 The expression of tTG, α-SMA, collagen-1 and TIMP-1 mRNA was upregulated in fibrosis control rats at each time point compared to normal control rats; however, after administration of cystamine, the expression of tTG, α-SMA, collagen-1 and TIMP-1 mRNA was significantly downregulated (P < 0.01, Figure 3). Effect of cystamine on the expression of tTG and α-SMA protein The expression of tTG protein in the liver was decreased after treatment of cystamine. The relative expression difference in each group is shown in Figure 4 after image analysis. The expression of α -SMA protein was also decreased after administration of cystamine (Figure 5).

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significantly less than that in fibrosis control rats (6.46 ± 0.68) (P < 0.01, Figure 1). Effects of cystamine on hepatic function In 4 wk and 8 wk time point, the levels of ALT, TBA and www.wjgnet.com

DISCUSSION Transglutaminases (TGs) are a large family of enzymes that catalyze a calcium-dependent acryl transfer reaction between the γ -carboxamide group of a polypeptide bound lysine residue to form an ε -( γ -glutamyl) lysine isopeptide bond or to form a ( γ -glutamyl) polyamine bond through catalyzing the incorporation of a polyamine into a polypeptide-bound glutamine. Known as “natural biological glues” [10] , TGs can result in covalent and irreversible cross-links between or within proteins and are thus involved in a variety of biochemical processes. Tissue transglutaminase (tTG), one member of the TGs, is widely expressed in endothelial, mesangial, and smooth muscle cells and fibrotic tissue, and localized in the cytosol, plasma membrane, and the nucleus of cells, as well as the extracellular space. tTG is a multifunctional enzyme that has been implicated in several physiological processes and pathological conditions such as maintaining membrane integrity, cell adhesion, cell death or differentiation, signal transduction and tissue remodeling [11]. Recent studies suggest that tTG may contribute to hepatic injury and fibrosis[12,13]. S ch n a b e l a n d c o l l e a g u e s f o u n d a s i g n i f i c a n t upregulation of total tTG at the mRNA and protein level in activated HSCs and ECM during transdifferentiation of quiescent HSCs to collagen producing myofibroblasts

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(MFB) [14] . This finding indicates that tTG plays an important role in the process of HSC activation, and hence, in the mechanism of liver fibrogenesis since the activated HSCs are not only the major source of ECM, but also can continuously upregulate the expression of TIMP-1, which blocks matrix metalloproteinase (MMP) activity and promotes survival of activated HSCs from apoptosis[15,16]. In addition, tTG mediated covalent binding of latent TGF-binding protein 1 (LTBP-1) to the ECM is a prerequisite for the activation of TGF-β, which is stored in its inactive form in the ECM[6,17]. The transdifferentiation of HSCs to ECM-producing MFB is a crucial event in hepatic fibrogenesis, which is stimulated by TGF-β[18]. It is well established that tTG exerts important effects on the progression of liver fibrogenesis through its ECM cross-linking activity. Issa and colleagues [19] confirmed that livers harvested from rats treated for 6 wk with CCl4 (representing reversible fibrosis) showed the histologic appearance of early macronodular cirrhosis uniformly negative for cross-linking. Over a 15 to 28 d recovery

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period, the livers showed a dramatic remodeling of the fibrotic matrix with a return to near-normal histology. However, livers from rats treated with CCl 4 for 12 wk showed a persistent fibrosis with clear evidence of crosslinking even during 366 d of recovery. The data above suggest that tTG mediated matrix cross-linking may be a major factor determining the speed and extent of matrix degradation in hepatic fibrosis. However, there is still controversy about the role of tTG in chronic liver injury and fibrosis. Nardacci and colleagues[20] suggest that tTG is a protective factor against liver fibrosis, they observed that there are greater mortality and more severe hepatic fibrosis after injection of CCl4 in tTG knock-out mice than in wild-type mice. Thus, more studies are needed to elucidate the importance of tTG during hepatic fibrosis. In the present study, we have established a liver injury and fibrosis rat model induced by intraperitoneal injection www.wjgnet.com

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of CCl4. Two cohorts of rats according to two different stages were administrated the tTG specific inhibitor cystamine for the purpose of observing the role of tTG in the development of liver fibrogenesis. The first stage was when rats were injected with CCl4 for a total of 4 wk, and cystamine treatment was started one day before the first injection of CCl4. This was carried out in order to investigate the effects of cystamine on the activation of HSCs and hepatic function during the early period of liver injury. The second stage was when rats were given cystamine intraperitoneously after 4 wk of CCl4 treatment for 4 wk to observe the effects of cystamine on activated HSCs and the progression of hepatic fibrosis. Our results showed that cystamine significantly downregulated the expression of tTG mRNA and protein, inhibited the activation of HSCs associated with a diminution of α -SMA, collagen-1 and TIMP-1 mRNA expression, as well as α-SMA protein, and there was a decrease in hydroxyproline content and dramatic improvement of liver pathologic evaluation. Furthermore, after administration of cystamine, the hepatic function of cystamine-treated rats was robustly improved. The results of this study strongly suggest that cystamine was beneficial in treatment of liver injury and fibrosis. The possible mechanism might be related to the inhibition of HSC activation, decreased expression of TIMP-1, reduced synthesis of ECM, and protection of hepatic cells against injury. In this study, we analyzed the changes in histology, the expression of tTG mRNA and protein, the activation of HSCs and the hydroxyproline content, as well as hepatic function after administration of cystamine in CCl4 induced fibrosis. In the current study, we did not undertake experiments for detection of in situ HSC apoptosis and ECM cross-linking, and therefore, to some extent this affects the interpretation of our observations. However, based on our results we confirmed that cystamine might ameliorate CCl4 induced liver fibrosis via inhibition of tTG. We are currently carrying out experiments to investigate the molecular mechanism of HSC apoptosis and ECM cross-linking in advanced fibrosis (12 wk of CCl 4 treatment) through tTG RNA interfere to further study the role of tTG in the progression of hepatic fibrosis. In summary, tTG plays an important role in the development of hepatic fibrosis. Cystamine, a specific inhibitor of tTG, can attenuate CCl4-induced liver fibrosis and protect hepatic function via inhibiting activation of HSCs and downregulating expression of TIMP-1 which result in the reduction of the synthesis of ECM. Better understanding of the role of tTG in pathogenesis of liver fibrosis could facilitate the development of new therapeutic strategies.

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Sarem M, Znaidak R, Macias M, Rey R. Hepatic stellate cells: it's role in normal and pathological conditions. Gastroenterol Hepatol 2006; 29: 93-101 Johnson TS, Griffin M, Thomas GL, Skill J, Cox A, Yang B, Nicholas B, Birckbichler PJ, Muchaneta-Kubara C, Meguid El Nahas A. The role of transglutaminase in the rat subtotal nephrectomy model of renal fibrosis. J Clin Invest 1997; 99:

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2950-2960 Grenard P, Bresson-Hadni S, El Alaoui S, Chevallier M, Vuitton DA, Ricard-Blum S. Transglutaminase-mediated cross-linking is involved in the stabilization of extracellular matrix in human liver fibrosis. J Hepatol 2001; 35: 367-375 Lamb RG, Koch JC, Snyder JW, Huband SM, Bush SR. An in vitro model of ethanol-dependent liver cell injury. Hepatology 1994; 19: 174-182 Kurose I, Higuchi H, Miura S, Saito H, Watanabe N, Hokari R, Hirokawa M, Takaishi M, Zeki S, Nakamura T, Ebinuma H, Kato S, Ishii H. Oxidative stress-mediated apoptosis of hepatocytes exposed to acute ethanol intoxication. Hepatology 1997; 25: 368-378 Nunes I, Gleizes PE, Metz CN, Rifkin DB. Latent transforming growth factor-beta binding protein domains involved in activation and transglutaminase-dependent cross-linking of latent transforming growth factor-beta. J Cell Biol 1997; 136: 1151-1163 Gressner AM, Weiskirchen R, Breitkopf K, Dooley S. Roles of TGF-beta in hepatic fibrosis. Front Biosci 2002; 7: d793-d807 Dedeoglu A, Kubilus JK, Jeitner TM, Matson SA, Bogdanov M, Kowall NW, Matson WR, Cooper AJ, Ratan RR, Beal MF, Hersch SM, Ferrante RJ. Therapeutic effects of cystamine in a murine model of Huntington's disease. J Neurosci 2002; 22: 8942-8950 Zeng MD, Wang TL, Wang BE.. Consensus on evaluation of the diagnosis and efficacy of hepatic fibrosis. Zhonghua Ganzangbing Zazhi 2002; 10: 327-328 Griffin M, Casadio R, Bergamini CM. Transglutaminases: nature's biological glues. Biochem J 2002; 368: 377-396 Lorand L, Graham RM. Transglutaminases: crosslinking enzymes with pleiotropic functions. Nat Rev Mol Cell Biol 2003; 4: 140-156 Wu J, Zern MA. Tissue transglutaminase, a key enzyme involved in liver diseases. Hepatol Res 2004; 29: 1-8 Mirza A, Liu SL, Frizell E, Zhu J, Maddukuri S, Martinez J, Davies P, Schwarting R, Norton P, Zern MA. A role for tissue transglutaminase in hepatic injury and fibrogenesis, and its regulation by NF-kappaB. Am J Physiol 1997; 272: G281-G288 Schnabel C, Sawitza I, Tag CG, Lahme B, Gressner AM, Breitkopf K. Expression of cytosolic and membrane associated tissue transglutaminase in rat hepatic stellate cells and its upregulation during transdifferentiation to myofibroblasts in culture. Hepatol Res 2004; 28: 140-145 Yoshiji H, Kuriyama S, Yoshii J, Ikenaka Y, Noguchi R, Nakatani T, Tsujinoue H, Yanase K, Namisaki T, Imazu H, Fukui H. Tissue inhibitor of metalloproteinases-1 attenuates spontaneous liver fibrosis resolution in the transgenic mouse. Hepatology 2002; 36: 850-860 Parsons CJ, Bradford BU, Pan CQ, Cheung E, Schauer M, Knorr A, Krebs B, Kraft S, Zahn S, Brocks B, Feirt N, Mei B, Cho MS, Ramamoorthi R, Roldan G, Ng P, Lum P, HirthDietrich C, Tomkinson A, Brenner DA. Antifibrotic effects of a tissue inhibitor of metalloproteinase-1 antibody on established liver fibrosis in rats. Hepatology 2004; 40: 1106-1115 Nunes I, Shapiro RL, Rifkin DB. Characterization of latent TGF-beta activation by murine peritoneal macrophages. J Immunol 1995; 155: 1450-1459 Gressner AM, Weiskirchen R, Breitkopf K, Dooley S. Roles of TGF-beta in hepatic fibrosis. Front Biosci 2002; 7: d793-d807 Issa R, Zhou X, Constandinou CM, Fallowfield J, Millward-Sadler H, Gaca MD, Sands E, Suliman I, Trim N, Knorr A, Arthur MJ, Benyon RC, Iredale JP. Spontaneous recovery from micronodular cirrhosis: evidence for incomplete resolution associated with matrix cross-linking. Gastroenterology 2004; 126: 1795-1808 Nardacci R, Lo Iacono O, Ciccosanti F, Falasca L, Addesso M, Amendola A, Antonucci G, Craxi A, Fimia GM, Iadevaia V, Melino G, Ruco L, Tocci G, Ippolito G, Piacentini M. Transglutaminase type II plays a protective role in hepatic injury. Am J Pathol 2003; 162: 1293-1303 S- Editor Liu Y L- Editor McGowan D E- Editor Yin DH

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Clinical presentation and endoscopic management of Dieulafoy’s lesions in an urban community hospital Srikrishna Nagri, Suryanarayan Anand, Yashpal Arya Srikrishna Nagri, Suryanarayan Anand, Department of Gastroenterology, Brooklyn Hospital Center, Brooklyn, NY 11201, United States Yashpal Arya, Wyckoff Heights Medical Center, Department of Gastroenterology, Wyckoff Heights Medical Center, 374 Stockholm St, Brooklyn, NY 11237, United States Correspondence to: Srikrishna Nagri, Department of Gastroenterology, Brooklyn Hospital Center, Brooklyn, NY 11201, United States. [email protected] Telephone: +1-347-4264451 Received: 2007-05-06 Accepted: 2007-05-31

Abstract AIM: To identify rates of occurrence, common clinical and endoscopic features, and to review the outcome of endoscopic management of Dieulafoy’s lesions in the upper gastrointestinal (GI) tract in an urban community hospital setting. METHODS: Endoscopic data from esophagogastroduo denoscopies (EGDs), done at Wyckoff Heights Medical Center, Brooklyn, NY between 2000 and 2006 were reviewed to identify patients with Dieulafoy’s lesions. Demographic data, medical history, examination findings, lab data, endoscopic findings and details of therapy for patients treated for Dieulafoy’s lesions were reviewed retrospectively. RESULTS: Dieulafoy’s lesions were documented to be the cause of bleeding in approximately 1% of patients presenting with upper gastrointestinal bleeding, while they were detected in only 2 patients when the indications for EGDs were different from active GI bleeding. When we analyzed EGDs performed in patients above age 65 years presenting with gastrointestinal bleeding, prevalence of Dieulafoy’s lesions approached 10 percent. The most common location of the lesion was the body of stomach (7), followed by the cardia (4) and the esophagus (2). One patient had this lesion in the fundus and one patient in the duodenal apex. All patients were initially treated endoscopically with epinephrine injection, in eight cases heater probe was applied following epinephrine and endoscopic clips were applied in two cases. All but one of the patients did well in near and intermediate term follow-up (average followup period of 18 mo). One patient died of multi-organ failure during the same hospital stay. Average length hospital stay was 7 d.

CONCLUSION: Community hospital gastroenterologists and endoscopists should be aware that Dieulafoy's lesions are an uncommon cause of upper GI bleeding among elderly patients. Early accurate diagnosis through emergent endoscopy and endoscopic therapy, especially in patients with multiple co-morbid conditions, can be very effective and life saving. © 2007 WJG . All rights reserved. Key words: Dieulafoy’s lesion; Gastrointestinal bleeding; Community Hospital; Endoscopic treatment; Obscure GI bleeding Nagri S, Anand S, Arya Y. Clinical presentation and endoscopic management of Dieulafoy’s lesions in an urban community hospital World J Gastroenterol 2007; 13(32): 4333-4335

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INTRODUCTION Dieulafoy’s lesion consists of a small submucosal artery that protrudes through a tiny mucosal defect anywhere in gastrointestinal tract and is reported to be an uncommon, but important cause of major gastrointestinal bleeding especially in the elderly. Dieulafoy’s lesion was first described by Gallard in 1884 and later named for the French surgeon Dieulafoy. The majority of Dieulafoy’s lesions occur in the proximal stomach, but they have also been reported in the esophagus, small and large bowel[1-3]. Typically, diagnosis is made by endoscopy where therapeutic endoscopy is the treatment of choice[4-7]. This study describes the clinical presentation and endoscopic management of 15 consecutive cases of Dieulafoy’s lesion in Wyckoff Heights Medical Center in Brooklyn NY, an urban community hospital, between years 2000 and 2006. This study shows that Dieulafoy’s lesion is a frequent finding among elderly patients presenting with massive upper gastrointestinal bleeding in a community hospital setting and outcomes of management are similar to published results from tertiary care centers.

MATERIALS AND METHODS Endoscopic data from emergent esophagogastroduod enoscopies (EGDs) done at Wyckoff Heights Medical www.wjgnet.com

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Table 1 Analysis of EGDs findings performed at Wyckoff Heights Medical Center between June 2000 and September 2006 Findings AVM Esophagitis Gastritis MW tear Tumor Ulcer Varices Dieulafoy’s Lesion

Hematochezia All Indications Hematemesis Melena (14 000) and or Melena (Total 460) (Total 330) (Total 834) 3 10 2 N/A 206 122 63 N/A 589 360 281 N/A 18 22 2 N/A 9 4 0 N/A 245 198 71 N/A 128 45 15 N/A 11

1

1

RESULTS Results of analysis of EGDs perfor med for acute gastrointestinal bleeding in Wyckoff Heights Medical Center between June 2000 and September 2006 are shown in Table 1. Dieulafoy’s lesion was documented to be the cause of bleeding in approximately 1% of patients presenting with upper gastrointestinal bleeding, while it was detected in only 2 patients when the indication was other than active GI bleeding. When we analyzed EGDs performed in patients above age 65 years presenting with gastrointestinal bleeding, prevalence of Dieulafoy’s lesions approached 10 percent (12/123). Mean age of the patients was 79 (SD 8.4), the youngest patient was 61 years old, while the oldest was 92. Six of the 15 patients were female. Thirteen of the 15 patients presented with overt acute gastrointestinal bleeding with hypotension and tachycardia requiring blood transfusion. Mean transfusion requirement was three units of packed red cells. Nine patients presented with hematemesis, one had melena, two had melena and hematemesis, and one had hematochezia alone. Two had anemia with occult gastrointestinal bleeding with no history of overt bleeding. Average hemoglobin at presentation was 9.5 gm (SD 1.5). Summary of patient characteristics and endoscopic findings are shown in

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Table 2 Summary of 15 cases with Dieulafoy’s Lesion treated at Wyckoff Heights Medical Center between June 2000 and September 2006 Patient Age (yr)

Hgb at Presentation (gm/dL) 7.6

Location of Dieulafoy’s Lesion Esophagus

1

76

2

81

7.9

3

87

11.2

4

90

5

92

10

6

78

11.2

7

68

7

Body of Stomach

8

73

10.3

Body of Stomach

9

79

11.3

Body of Stomach

10

71

9.9

Body of Stomach

11

61

11.2

Body of Stomach

12

84

8.8

Body of Stomach

13 14 15

80 82 86

7.8 8.7 9

Duodenum Fundus Fundus

9.8

15

Center, Brooklyn, NY between 2000 and 2006 for GI bleeding were reviewed. All the cases were performed by 3 board certified gastroenterologists with more than 10 years of experience. EGD report database was searched for endoscopic findings of Dieulafoy’s lesion during this period. The study was reviewed and approved by our Institutional review board. Endoscopic diagnosis of Dieulafoy’s lesion is based on following published criteria[8], (a) active arterial spurting, oozing of blood from minute mucosal defect (less than 3 mm), (b) visualization of protruding vessel with or without active bleeding within a minute mucosal defect or normal appearing mucosa or, (c) densely adherent clot with a narrow point of attachment to a minute mucosal defect or normal appearing mucosa. Demographic data, medical history, examination findings, lab data, endoscopic findings and details of therapy of patients with Dieulafoy’s lesions obtained from their medical charts were reviewed retrospectively.

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Esophagus Cardia of Stomach Body of Stomach Cardia of Stomach Fundus of Stomach

Endoscopic Treatment Epinephrine Epi + Heater Probe Epinephrine Epinephrine Epinephrine Epi + Heater Probe Epi + Heater Probe Epi + Heater Probe Epi + Heater Probe Heater Probe Epi + Heater Probe Epi + Heater Probe Epinephrine Epi + Endoclip Epi + Endoclip

Table 2. The most common location of the lesion was the body of stomach (7), followed by cardia (4) and esophagus (2). One patient had this lesion in the fundus and one patient in the duodenal apex. All the cases were identified on index endoscopy. All of the patients were initially treated endoscopically with epinephrine injection (1:10000 dilution) and in 8 cases heater probe was applied following epinephrine. In two patients endoclips were used. Five patients underwent repeat procedures during the same hospital admission, two of them for rebleeding and the others to confirm the initial findings. Both of the rebleeding patients had heater probe applied during the first endoscopy and probe application was repeated during the subsequent endoscopy to successfully control bleeding. The most often reported additional findings were hiatal hernia (4) and antral erythema (8). All the patients had comorbidities, most common being hypertentsion followed by coronary artery disease and type 2 diabetes. All but one of the patients did well in near and intermediate term follow-up (average follow-up period of 18 mo). Follow up data was also obtained from chart-review. One patient died of multiorgan failure during the same hospital stay. Average length hospital stay was 7 d.

DISCUSSION The pathogenesis of Dieulafoy’s lesion is not clearly understood, but elongation and tortuosity of a submucosal artery associated with aging may be a factor. Histological description of a Dieulafoy’s lesion shows an artery of 1-3 mm in diameter which is ten times the diameter of a normal submucosal capillary[9].

Nagri S et al . Dieulafoy’s lesions in a community hospital setting

Dieulafoy’s lesion is an important, but uncommon cause of massive GI bleeding especially from the upper GI tract. These lesions can easily be missed on endoscopy unless the lesion is actively bleeding because these vessels are normally retracted. Studies have described Dieulafoy’s lesion as the cause of upper GI bleeding in 1%-5.8% of cases. In our series, 0.1% of EGDs showed this lesion when all the indications for EGD were included (15 of 14 000) as opposed to about 1% in patients presenting with upper GI bleeding (13 of approximately 1200). Patients affected are typically elderly with significant comorbid conditions. Gender variation was noted (slight male to female predominance). There have been several endoscopic modalities used successfully to treat Dieulafoy’s lesions. Thermal and/or mechanical methods, hemoclipping and heater probe had better results in retrospective as well as prospective trials[10-12]. Endoscopic hemoclip application, endoscopic band ligation, heater probe application, Nd:YAG, with or without injection therapy have all been shown to be effective in various studies. Initial hemoclip therapy was mostly successful (94.1%-95.2%) with a low recurrence rate[13-15]. Endoscopic heater probe and endoscopic band ligation had similar results[16]. Endoscopic sclerotherapy also had good results in one study[17]. If endoscopic therapy fails, management with other options like angiography with embolization or surgery is indicated. Wedge resection is the surgical procedure of choice. Recurrences can occur, though very rare[13-16]. Long-term results were excellent for endoscopic methods of therapy[16-19]. Mortality was generally low with prompt diagnosis and treatment but one study reported higher mortality due to comorbidities and higher age[20]. Clinical features and management outcome of our series of patients with Dieulafoy’s lesion from an urban community hospital were similar to results reported in the literature. One percent of patients presenting with upper GI bleeding were diagnosed with this condition. The patients were elderly with multiple comorbid conditions. The diagnosis of this lesion was increased sharply if the indication was active upper GI bleeding in elderly patients. Standard therapy with epinephrine and heater probe coagulation of the lesion was very effective and resulted in a good patient outcome. Although injection therapy alone with epinephrine was used in five patients with successful outcome, it is considered suboptimal treatment based on current literature and is not recommended. Based upon our experience endoclip application is the optimal treatment modality for these lesions. Initial injection with epinephrine did not interfere with the application of endoclips. Based upon these results we conclude that community hospital gastroenterologists and endoscopists should be aware that Dieulafoy's lesions are an uncommon, but important finding among elderly patients with upper GI bleeding. Early accurate diagnosis through emergent endoscopy and therapy, especially in patients with multiple comorbid conditions, can be very effective and life saving.

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REFERENCES 1

Romaozinho JM, Pontes JM, Lerias C, Ferreira M, Freitas D. Dieulafoy's lesion: management and long-term outcome. Endoscopy 2004; 36: 416-420 2 Enns R. Dieulafoy's lesions of the rectum: a rare cause of lower gastrointestinal bleeding. Can J Gastroenterol 2001; 15: 541-545 3 Blecker D, Bansal M, Zimmerman RL, Fogt F, Lewis J, Stein R, Kochman ML. Dieulafoy's lesion of the small bowel causing massive gastrointestinal bleeding: two case reports and literature review. Am J Gastroenterol 2001; 96: 902-905 Stark ME, Gostout CJ, Balm RK. Clinical features and 4 endoscopic management of Dieulafoy's disease. Gastrointest Endosc 1992; 38: 545-550 5 Parra-Blanco A, Takahashi H, Mendez Jerez PV, Kojima T, Aksoz K, Kirihara K, Palmerin J, Takekuma Y, Fuijta R. Endoscopic management of Dieulafoy lesions of the stomach: a case study of 26 patients. Endoscopy 1997; 29: 834-839 Reilly HF 3rd, al-Kawas FH. Dieulafoy's lesion. Diagnosis and 6 management. Dig Dis Sci 1991; 36: 1702-1707 Lin HJ, Lee FY, Tsai YT, Lee SD, Lee CH, Kang WM. Thera7 peutic endoscopy for Dieulafoy's disease. J Clin Gastroenterol 1989; 11: 507-510 Norton ID, Petersen BT, Sorbi D, Balm RK, Alexander 8 GL, Gostout CJ. Management and long-term prognosis of Dieulafoy lesion. Gastrointest Endosc 1999; 50: 762-767 Lee YT, Walmsley RS, Leong RW, Sung JJ. Dieulafoy's lesion. 9 Gastrointest Endosc 2003; 58: 236-243 10 Chung IK, Kim EJ, Lee MS, Kim HS, Park SH, Lee MH, Kim SJ, Cho MS. Bleeding Dieulafoy's lesions and the choice of endoscopic method: comparing the hemostatic efficacy of mechanical and injection methods. Gastrointest Endosc 2000; 52: 721-724 11 Kasapidis P, Georgopoulos P, Delis V, Balatsos V, Konstantinidis A, Skandalis N. Endoscopic management and longterm follow-up of Dieulafoy's lesions in the upper GI tract. Gastrointest Endosc 2002; 55: 527-531 12 Cheng CL, Liu NJ, Lee CS, Chen PC, Ho YP, Tang JH, Yang C, Sung KF, Lin CH, Chiu CT. Endoscopic management of Dieulafoy lesions in acute nonvariceal upper gastrointestinal bleeding. Dig Dis Sci 2004; 49: 1139-1144 13 Ljubicic N. Efficacy of endoscopic clipping and long-term follow-up of bleeding Dieulafoy's lesions in the upper gastrointestinal tract. Hepatogastroenterology 2006; 53: 224-227 14 Katsinelos P, Paroutoglou G, Mimidis K, Beltsis A, Papaziogas B, Gelas G, Kountouras Y. Endoscopic treatment and followup of gastrointestinal Dieulafoy's lesions. World J Gastroenterol 2005; 11: 6022-6026 15 Yamaguchi Y, Yamato T, Katsumi N, Imao Y, Aoki K, Morita Y, Miura M, Morozumi K, Ishida H, Takahashi S. Short-term and long-term benefits of endoscopic hemoclip application for Dieulafoy's lesion in the upper GI tract. Gastrointest Endosc 2003; 57: 653-656 16 Park CH, Joo YE, Kim HS, Choi SK, Rew JS, Kim SJ. A prospective, randomized trial of endoscopic band ligation versus endoscopic hemoclip placement for bleeding gastric Dieulafoy's lesions. Endoscopy 2004; 36: 677-681 17 Yilmaz M, Ozutemiz O, Karasu Z, Ersoz G, Gunsar F, Batur Y, Aydin A, Tekesin O, Yonetci N, Ilter T. Endoscopic injection therapy of bleeding Dieulafoy lesion of the stomach. Hepatogastroenterology 2005; 52: 1622-1625 18 Sone Y, Kumada T, Toyoda H, Hisanaga Y, Kiriyama S, Tanikawa M. Endoscopic management and follow up of Dieulafoy lesion in the upper gastrointestinal tract. Endoscopy 2005; 37: 449-453 19 Skok P. Endoscopic hemostasis in exulceratio simplexDieulafoy's disease hemorrhage: a review of 25 cases. Endoscopy 1998; 30: 590-594 20 Pointner R, Schwab G, Konigsrainer A, Dietze O. Endoscopic treatment of Dieulafoy's disease. Gastroenterology 1988; 94: 563-566 S- Editor Liu Y L- Editor Rippe RA E- Editor Yin DH

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World J Gastroenterol 2007 August 28; 13(32): 4336-4339 World Journal of Gastroenterology ISSN 1007-9327 © 2007 WJG. All rights reserved.

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Effects of long term hydrophilic bile acid therapy on in vitro contraction of gallbladder muscle strips in patients with cholesterol gallstones Mehmet Refik Mas, Bilgin Comert, Nuket Mas, Levent Yamanel, Haluk Ozotuk, Ilker Tasci, Riadh P Jazrawi Mehmet Refik Mas, Bilgin Comert, Levent Yamanel, Haluk Ozotuk, Ilker Tasci, Department of Internal Medicine, Gülhane School of Medicine, Ankara 06018, Turkey Nuket Mas, Department of Anatomy, Baskent University, Ankara, Turkey Riadh P Jazrawi, Charterhouse Clinical Research Unit, Ravenscourt Park Hospital, Ravenscourt Park, London W6 0TN, United Kingdom Correspondence to: Dr. Mehmet Refik Mas, Department of Internal Medicine, Gülhane School of Medicine, GATA Ic Hastaliklari B.D., Etlik 06018 Ankara, Turkey. [email protected] Telephone: +90-312-3044018 Fax: +90-312-3044000 Received: 2006-12-22 Accepted: 2007-03-01

Key words: Ursodeoxycholic acid; Hydrophilic bile acid; Gallbladder; Cholesterol gallstone Mas MR, Comert B, Mas N, Yamanel L, Ozotuk H, Tasci I, Jazrawi RP. Effects of long term hydrophilic bile acid therapy on in vitro contraction of gallbladder muscle strips in patients with cholesterol gallstones. World J Gastroenterol 2007; 13(32): 4336-4339

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INTRODUCTION Abstract AIM: To evaluate ursodeoxycholic acid (UDCA) therapy on the in vitro contraction of gallbladder smooth muscle strips from cholesterol gallstone patients. METHODS: The contraction forces of gallbladder smooth muscle strips from 28 patients with cholesterol gallstones treated with UDCA were compared with contraction forces from 14 untreated patients. The strips were stimulated with increasing concentrations of cholecystokinin-8 (CCK-8). RESULTS: Al though the contraction forces that developed in response to CCK-8 were higher in strips from specimens of UDCA treated patients compared to untreated patients, longer treatment periods (6-wk) caused more contraction responses than the short treatment period of 3-wk (F = 19.297, 1.85 ± 0.22 g vs 1.70 ± 0.10 g, P < 0.01). Contraction forces developed with maximal stimulation with KCl in the 6-wk treatment group were also higher than contraction forces in the untreated group (F = 4.274, 3.77 ± 0.45 g vs 3.30 ± 0.30 g, P < 0.05). CONCLUSION: Six-week UDCA treatment caused an increase in contractions of muscle strips from patients with cholesterol gallstones when compared to shorter treatment administration or controls. We suggest that extending UDCA treatment periods may cause more effective contractions in the gallbladder, and thereby increase the rate of response to treatment. © 2007 WJG . All rights reserved.

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Surgery is the standard treatment for symptomatic patients with gallstone disease. However, since surgical procedures have the risk of morbidity and mortality, noninvasive treatments are still under investigation, especially for use in high-risk patients. Ursodeoxycholic acid (3 alpha, 7 beta-dihydroxy-5 beta-cholanoic acid-UDCA) is a safe and efficacious drug that has been widely used for oral gallstone dissolution therapy[1]. At least three pathologies must occur together for the formation of cholesterol gallstones; (1) high cholesterol saturation in the gallbladder, (2) decreased motility of the gallbladder, and (3) acceleration of nidus formation. There are controversial results concerning the effects of UDCA on gallbladder contraction. While some authors have reported impaired gallbladder emptying in patients treated with UDCA[2,3] this was not confirmed by others[4]. However, these studies have used indirect methods, such as ultrasonography and Tc-HIDA scans for measuring gallbladder emptying. We, therefore, searched for a clinical in vitro model to determine whether UDCA treatment increases gallbladder contractions in muscle strips from patients with cholesterol gallstones. Although a similar effect was previously reported[5], our main purpose was to establish a time-response rate relationship.

MATERIALS AND METHODS Forty-two patients with cholesterol gallstones who were selected for elective cholecystectomy were enrolled in the study. The inclusion criteria were: (1) presence of gallstones and/or sludge by gallbladder ultrasonography, patent cystic bile duct, absence of choledocholithiasis,

Mas MR et al . Long term hydrophilic bile acid therapy and in vitro contraction of gallbladder muscle

and < 4 mm gallbladder wall thickness, (2) absence of calcification on the plain film of the abdomen, and (3) visualization of the gallbladder by oral cholecystography. The patients were randomized into three groups at the time of enrollment. All three groups had similar age (66.4 ± 4.3, 67.4 ± 3.9, and 67.5 ± 4.1) and gender (male/female ratio: 6/8, 5/9, and 5/9, respectively) distributions. All patients and controls gave informed consent to participate in the study and the Ethics Committee of Gulhane School of Medicine approved the protocol. GroupⅠ(n = 14) was treated with UDCA (10 mg/ kg/day) with a single dose at bedtime starting three weeks before surgery. Group Ⅱ (n = 14) received the same treatment but for six weeks prior to surgery. The patients in Group Ⅲ (n = 14) who served as controls received no treatment and underwent surgery after enrollment (Group Ⅲ). In the treatment groups, the last dose of UDCA was given on the day before surgery. No patient had clinical or laboratory signs of acute colecystitis when they underwent surgery. Ultrasonographic examination just before the surgery indicated no difference from the initial ultrasonography in terms of size and number of gallstones, sludge, gallbladder wall thickness, and absence of choledocholithiasis. After cholecystectomy, for in vitro analysis, all gallbladder tissues were subjected to a series of manipulations as described by Behar et al with modifications [6] . A 10 mm × 20 mm specimen parallel to the long axis of the gallbladder was put into cold Krebs solution (NaCl: 118.4 mmol/L, KCl: 4.7 mmol/L, CaCl2: 1.9 mmol/L, NaHCO 3: 25 mmol/L, MgSO 4: 1.2 mmol/L, Glucose: 11.7 mmol/L). The specimens were washed with Krebs solution again and 3 mm × 10 mm strips were prepared. The serous membrane on the outer surfaces of the strips was cleaned and the gallbladder strips were hung with a 6.0 silk suture into a 10 mm tissue bath (pH: 7.4 and 37℃). The other ends of the strips were tied to an isometric force transducer (Force Transducer, Type 45196 A Nec San-ei Instruments Ltd, Japan). The force transducer was connected to a polygraph writer (360 Polygraph Model 2 G. 66 Nec San-ei Instruments Ltd, Japan). The tissue bath was perfused with 95% oxygen and 5% carbon dioxide throughout the study. An equilibrium period of two hours was used prior to the start of the experiment. During this period the Krebs solution in the tissue bath was changed regularly every 15 min. After a 2 h equilibrium period, tension in the strip was changed into a basal situation. At this time periodic contractions of 1-2 min occurred. Later, by studying one agonist in each of the gallbladder strips, contraction responses to cholecystokinin octapeptide (CCK-8; 10-10, 10-9, 10-8, 10-7, 10-6 concentrations) and KCl (0.25 mol/L) were recorded. The differences between basal and CCK-8 induced tensions of the strips were calculated ( ∆ tension) and the mean ± SD were obtained. Later, the ∆ tensions as a percentage of the maximum tension created by KCl were calculated. Results were expressed as mean ± SD. Oneway ANOVA and Tukey HSD tests were used for statistical analysis. A P value of less than 5% was accepted as

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Table 1 Response of gallbladder strips against KCl and increasing CCK-8 concentrations (mean ± SD) Agent CCK-8 10-10 M 10-9 M 10-8 M 10-7 M 10-6 M KCl (0.25 mol/L) Basal

GroupⅠ (n = 14)

Group Ⅱ (n = 14)

Group Ⅲ (n = 14)

0.21 ± 0.02 g 0.32 ± 0.02 g 0.42 ± 0.04 g 1.20 ± 0.04 g 1.70 ± 0.010 g 3.50 ± 0.50 g 0.13 ± 0.03 g

0.25 ± 0.02 g 0.37 ± 0.04 g 0.48 ± 0.05 g 1.35 ± 0.10 g 1.85 ± 0.22 g 3.77 ± 0.45 g 0.14 ± 0.02 g

0.20 ± 0.01 g 0.26 ± 0.02 g 0.36 ± 0.04 g 1.04 ± 0.06 g 1.50 ± 0.10 g 3.30 ± 0.30 g 0.12 ± 0.05 g

P < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.03 > 0.05

G: gram; KCl: potassium chloride; CCK: cholecystokinine.

significant. All statistical measurements were made by using SPSS PC ver. 11.05 (SPSS Inc. USA).

RESULTS Strips prepared from gallbladder tissue showed two contraction patterns in the equilibrium period: (1) contractions as fibrillation, which caused no significant increase in tension, and (2) rhythmic contractions, which had a frequency of 1/min and caused 0.10-0.17 g ∆ tension. Basal active tensions were 0.13 ± 0.03 g in GroupⅠ, 0.14 ± 0.02 g in Group Ⅱ, and 0.12 ± 0.05 g in Group Ⅲ (P > 0.05); no difference was observed between treatment and control groups regarding basal active tensions. Responses of the treatment and the control groups as ∆ tension to increasing concentration of CCK-8 and KCl are shown in Table 1. Contraction responses of gallbladder strips were higher with increasing concentrations of CCK-8. UDCA treatment before the colecystectomy improved in vitro contraction responses of gallbladder strips to CCK-8. Contraction responses to different CCK-8 concentrations were significantly higher in the 6-wk treatment group when compared to the 3-wk treatment or the control group. Moreover, except for CCK-8-10, contraction responses to different CCK-8 concentrations in the 3-wk treatment group were significantly higher than the controls. Potassium provokes extracellular calcium entrance through the voltage dependent calcium channels and causes smooth muscle contraction[5]. In this study, KCl was used to induce maximum strip contraction. Contraction responses to maximal stimulation with KCl in the 6-wk treatment group were also higher than the 3-wk group, but the difference was not statistically significant. In the 3-wk treatment group, contraction responses were also higher, but not statistically significant than the controls. The only significant difference in maximal stimulation with KCl was obtained for the 6-wk treatment group when compared with the untreated controls (3.77 ± 0.45 g vs 3.30 ± 0.30 g, P < 0.05).

DISCUSSION The results of the present study indicate that UDCA treatment increases gallbladder contractions in response to www.wjgnet.com

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CCK-8, and this effect is more pronounced as the duration of treatment increases. Moreover, UDCA results in a net increase in the maximal contraction capacity of the gallbladder strips. The results confirm previous findings on gallbladder muscle strips from patients with cholesterol gallstone disease, which is a 3-wk UDCA treatment prior to the operation increases contractility [5] . Moreover, in the present investigation, we used KCl for maximal stress, and maximal contraction response in muscle strips from the 6-wk treatment group was significantly higher than the control group. On the other hand, in a previous report, this effect was not observed in the 3-wk treatment group[5]. These results suggest that long term treatment with UDCA may be more beneficial in terms of improving gallbladder contractile functions. Though some clinical studies investigating the effect of UDCA on gallbladder contraction failed to show a significant improvement in gallbladder contractions, this may be associated with the methods used to measure contraction responses of the g allbladder, such as scintigraphy or ultrasonography[2,3,7]. For instance, in one of these investigations, gallbladder emptying was found to be improved in patients with successful litholysis, whereas no improvement was observed in patients with unsuccessful litholysis, which might be explained by gallbladder dysfunction at baseline[2]. Accordingly, some authors reported significant improvements in gallbladder emptying function after UDCA treatment with similar methods [4,8], which is supported by the in vitro data of the present work. In addition, van Der Werf et al found a negative correlation between the cholesterol saturation index (CSI) and gallbladder emptying function[9] suggesting that a decrease in gallbladder contractility may not be possible after UDCA treatment, which decreases CSI. It was previously reported that CCK-8 induced muscle strip contractions in patients with cholesterol gallstones is less than that in patients with pigment gallstones[6]. It was also observed that spontaneous cyclic contractions were less than in muscle strips of patients with pigment gallstones, which may be caused by the myotoxic effect of cholesterol in the gallbladder. This may be explained, at least in part, by the mechanism of action of UDCA on muscle strips in our study in that UDCA alters the composition of the bile, which results in improvement of the contraction response. Also, excess cholesterol has been suggested to reduce membrane fluidity and deteriorate subsequent cellular functions in smooth muscle cells in the gallbladder wall[10]. In a recent study by Xiao et al, UDCA treatment in guinea pigs with acute cholecystitis prevented gallbladder muscle dysfunction and improved oxidative stress markers when compared with chenodeoxycholic acid treated guinea pigs[11]. These results indicate that UDCA treatment may improve contraction even in a gallbladder with inflammation but without stone. In conclusion, in the present investigation, UDCA caused an increase in contraction in gallbladder smooth muscle strips from patients with cholesterol gallstones when compared to untreated controls, not only with a 3-wk but also with a 6-wk treatment period prior to www.wjgnet.com

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colecystectomy. Extension of the UDCA treatment period appears to cause more effective contractions in the gallbladder, and thereby may increase the rate of response to treatment.

COMMENTS Background

There are discrepancies between the results of the studies searching the effects of ursodeoxycholic acid on gallbladder contractions. Frequently, indirect methods have been used to measure contractile responses to date. Pretreatment with ursodeoxycholic acid for 3 wk was shown to improve contractility of the muscle strips prepared after cholecystectomy in humans. The present study address the effect of ursodeoxycholic acid in a more extended period as well as the maximal contractility capacity in response to a different inducer.

Research frontiers

Ursodeoxycholic acid is a naturally occurring hydrophilic bile acid. It has been used for gallstone dissolution clinically with favorable or unfavorable effects. Though it is known to change the composition of the bile, gallbladder emptying has been reported to be impaired in patients treated with ursodeoxycholic acid.

Innovations and breakthroughs

Strips from gallbladders extracted from the patients treated with ursodeoxycholic acid for 3- or 6-wk prior to cholecystectomy displayed marked improvements in contractile responses. More beneficial results as well as increased maximal contractility capacity were obtained in the 6-wk treatment groups. Ursodeoxycholic acid may be an agent having no adverse action on gallbladder emptying.

Applications

Response to ursodeoxycholic acid treatment may be delayed in the clinical setting, and treatment should be kept on as long as possible especially for the patients who are not eligible for surgery.

Terminology

Ursodeoxycholic acid: a hydrophilic bile acid which is the drug of choice for the treatment of primary biliary cirrhosis. It has also been used for other cholestatic disorders, hepatostetosis as well as gallstone dissolution in selected cases.Muscle strip: gallbladder tissue sample prepared for in vitro test.Cholecystochinine-8: an enzyme causing contraction of the smooth muscle cells in the gallbladder wall. Potassium chloride (KCl): used to measure maximal contractility capacity in the muscle strips.

Peer review

The study is interesting and aims to answer a relevant issue in the pathogenesis and treatment of impaired gall bladder contractions in gallstone disease.

REFERENCES 1 2 3

4

5

Kowdley KV. Ursodeoxycholic acid therapy in hepatobiliary disease. Am J Med 2000; 108: 481-486 Sylwestrowicz TA, Shaffer EA. Gallbladder function during gallstone dissolution. Effect of bile acid therapy in patients with gallstones. Gastroenterology 1988; 95: 740-748 Festi D, Frabboni R, Bazzoli F, Sangermano A, Ronchi M, Rossi L, Parini P, Orsini M, Primerano AM, Mazzella G. Gallbladder motility in cholesterol gallstone disease. Effect of ursodeoxycholic acid administration and gallstone dissolution. Gastroenterology 1990; 99: 1779-1785 van Erpecum KJ, van Berge Henegouwen GP, Stolk MF, Hopman WP, Jansen JB, Lamers CB. Effects of ursodeoxycholic acid on gallbladder contraction and cholecystokinin release in gallstone patients and normal subjects. Gastroenterology 1990; 99: 836-842 van de Heijning BJ, van de Meeberg PC, Portincasa P, Doornewaard H, Hoebers FJ, van Erpecum KJ, VanbergeHenegouwen GP. Effects of ursodeoxycholic acid therapy on in vitro gallbladder contractility in patients with cholesterol

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gallstones. Dig Dis Sci 1999; 44: 190-196 Behar J, Lee KY, Thompson WR, Biancani P. Gallbladder contraction in patients with pigment and cholesterol stones. Gastroenterology 1989; 97: 1479-1484 Forgacs IC, Maisey MN, Murphy GM, Dowling RH. Influence of gallstones and ursodeoxycholic acid therapy on gallbladder emptying. Gastroenterology 1984; 87: 299-307 Realini S, Reiner M, Pescia R. Study of gallbladder emptying using 99m Tc-HIDA in acalculous cholecystopathy. Schweiz Med Wochenschr 1987; 117: 1217-1220

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van der Werf SD, van Berge Henegouwen GP, Palsma DM, Ruben AT. Motor function of the gallbladder and cholesterol saturation of duodenal bile. Neth J Med 1987; 30: 160-171 10 Chen Q, Amaral J, Oh S, Biancani P, Behar J. Gallbladder relaxation in patients with pigment and cholesterol stones. Gastroenterology 1997; 113: 930-937 11 Xiao ZL, Biancani P, Carey MC, Behar J. Hydrophilic but not hydrophobic bile acids prevent gallbladder muscle dysfunction in acute cholecystitis. Hepatology 2003; 37: 1442-1450 S- Editor Wang J

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Effect of vitamin E on oxidative stress status in small intestine of diabetic rat A Shirpoor, MH Khadem Ansari, S Salami, F Ghaderi Pakdel, Y Rasmi A Shirpoor, F Ghaderi Pakdel, Department of Physiology, Faculty of Medicine, Jaddeh Nazloo, Urmia, Iran MH Khadem Ansari, S Salami, Y Rasmi, Department of Biochemistry, Faculty of Medicine, Jaddeh Nazloo, Urmia, Iran Correspondence to: A Shirpoor, Department of Physiology, Faculty of Medicine, Jaddeh Nazloo, Urmia, Iran. [email protected] Telephone: +98-441-2770698 Fax: +98-441-2780801 Received: 2007-05-09 Accepted: 2007-05-28

Abstract AIM: To investigate the effect of vitamin E on oxidative stress status in the small intestine of diabetic rats. METHODS: Twenty-four male Wistar rats were randomly divided into three groups: Control (C), non-treated diabetic (NTD) and vitamin E-treated diabetic (V ETD) groups. The increases in lipid peroxidation, protein oxidation and superoxide dismutase (SOD) in these three groups was compared after 6 wk. RESULTS: There was no significant difference in catalase activity between NTD and control rats. Compared to NTD rats, the treatment with vitamin E significantly decreased lipid peroxidation and protein oxidation, and also increased catalase activity and SOD. CONCLUSION: The results revealed the occurrence of oxidative stress in the small intestine of diabetic rats. Vitamin E, as an antioxidant, attenuates lipid peroxidation and protein oxidation, and increases antioxidant defense mechanism. © 2007 WJG . All rights reserved.

Key words: Diabetes mellitus; Small intestine; Rat; Vitamin E; Oxidative stress Shirpoor A, Ansari MHK, Salami S, Pakdel FG, Rasmi Y. Effect of vitamin E on oxidative stress status in small intestine of diabetic rat. World J Gastroenterol 2007; 13(32): 4340-4344 http://www.wjgnet.com/1007-9327/13/4340.asp

INTRODUCTION Gastrointestinal (GI) disorders are common among human beings[1,2]. The entire GI tract from the esophagus to www.wjgnet.com

the anorectal region can be affected by diabetes mellitus[3]. Common complaints include hyperplasia and hypertrophy of the epithelial cells[4], elevated levels of digestive enzymes [5], increased absorption of sugars and amino acids[6], enhanced endogenous synthesis of cholesterol and triglycerides[6,7] and decreased fluidity of the brush border membrane[8]. The intestinal mucosa is also vulnerable to oxidative stress and reactive oxygen species (ROS) generated by several conditions, such as ischemia/reperfusion, inflammatory bowel disease[9], surgical stress[10] and diabetes[11]. Recently, several studies have examined the role of oxidative stress on developmental diabetic-mediated disorders, possibly via the formation of free radicals[11-13]. Free radicals or ROS generated during oxidative metabolism can inflict damage on all classes of cellular macromolecular components (e.g., mitochondria, endoplasmic reticulum, protein, etc.), eventually leading to cell death[14]. The driving force behind the destructive nature of ROS is the unpaired electron residing within their structures, making them unstable and highly reactive. It is well known that metabolic changes brought about by diabetes increase production of ROS (e.g., nitrosonium cation [OH˚], lipid peroxides [ROO˚] and hydrogen peroxides [H2O2]) as well as reactive nitrogen species (e.g., nitrosonium cation [NO+], nitroxyl anion [NO-] and peroxinitrite [ONOO-])[15,16]. These free radicals and non-radical species react with several amino acid residues altering their structures and, by extension, the tertiary structures of the parent protein. These free radicals also degrade the phospholipids of cellular membranes through the process of lipid peroxidation. One promising aspect of understanding the role of oxidative stress in diabetes-mediated disorders is the ability of antioxidant supplementation to attenuate diabetes’s adverse effects. Antioxidants, such as ascorbic acid, α-tocopherol (vitamin E), endogenous glutathione peroxidase and the pineal hormone melatonin, have all been tested for efficacy in defending against free-radical-mediated tissue injuries. Melatonin, for example, has been shown to be an effective scavenger of the hydroxyl radical, as well as other radicals such as superoxide, nitric oxide and peroxynitrite, that protects against lipid peroxidation in the brain[17-19]. Vitamin E comprises eight naturally occurring fat-soluble vitamins of which the most predominant, essential and with the highest biological activity is α-tocopherol[20]. Vitamin E is a major antioxidant in biological systems acting as a powerful chain-breaking agent through the scavenging of peroxyl radicals[21]. Vitamin E terminates the chain reaction of lipid peroxidation in membranes and lipoproteins. Thus, a number of studies have been carried out to determine the

Shirpoor A et al . Preventive effects of vit E on oxidative stress

protective effects of vitamin E in different biological models of injury[22]. Currently, there is considerable interest in the roles of vitamin E in the protection of membranes lipids against oxidative stress[23]. The present study was, therefore, undertaken to determine whether the small intestine is subjected to oxidative damage during diabetes as well as to examine the accompanying changes in antioxidant status, lipid peroxidation and protein oxidation in order to understand its role in the pathogenesis of the disease. Also, in this study, a possible protective effect of vitamin E against diabetes-induced alterations of enzymatic and oxidative components of antioxidant defense systems in mucosal layer of rat small intestine was investigated.

MATERIALS AND METHODS All procedures on rats were followed according to “Principles of Laboratory Animal Care” (NIH publication no. 85-23, revised 1985), as well as specific rules of “Animal Care and Use Committee”, National Medical and Health Service. Sixteen male Wistar rats, weighing 220-240 g, were made diabetic by intraperitoneal injection of streptozotocin (STZ, 60 mg/kg body weight in 0.05 mol/L citrate buffer, pH 4.5). Age- and weight-matched normal control group was injected intraperitoneally with an equivalent amount of buffer. Glucose was determined by glucose oxidase using Biosystem kit (Barcelon, Spain) on blood samples obtained from tail veins 48 h after injection of STZ. Rats with blood glucose higher than 3 g/L were included into the study as diabetic. Three groups (n = 8) of rats were studied for 6 wk after the entry: (1) Control (non-diabetic); (2) untreated diabetic; and (3) vitamin E-treated diabetic (VETD) groups. Rats in VETD group received 300 mg vitamin E (Merck-Germany) in tap water beside regular diet daily. Food was supplied ad libitum in all groups throughout the experiment. After 6 wk, all rats were anesthetized by 10% chloral hydrate (5 mL/kg body weight). The body weight was measured at the end of the experiment. Blood samples were directly obtained from the heart of the rats by syringe. Then the abdominal cavity was opened and the whole small intestine was harvested. The small intestine was segmented and each segment was flushed with chilled 115 g/L KCL solution and the mucosa was scraped. A 100 g/L homogenate was prepared in 50 nmol/L phosphate buffer (pH 7.4) and centrifuged at 10 000 g for 10 min at 4℃ in a refrigerated centrifuge (Hermel Germany). The obtained supernatant was used for all the assays[11]. The protein content was determined by the method of Bradford[24] using bovine serum albumin as the standard. HbA1C or glycosylated hemoglobin was analyzed by HPLC using automated D-10 BioRad hemoglobin analyzers. Protein carbonyl contents were measured in the supernatant using Cayman (Cayman Co. USA) kit. Briefly, 2, 4-dinitropheylhyrayine (DNPH) reacts with protein carbonyls forming a Schiff base to produce the corresponding hydrazone, which can be analyzed spectrophotometrically. Catalase activity was determined in the supernatant using Cayman (Cayman, Co, USA) kit. The method was based on the reaction enzyme with methanol in the presence of optimal concentration of H2O2. The formaldehyde produced was

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measured at 540 nm by spectrophotometry with 4-amino3-hydrazino-5-mercapto-1, 2, 4-triazole as the chromogen. Tissue SOD activity was determined using a Ransod kit (Randox Laboratories, Crumlin, UK). Briefly, the method uses Xanthine and Xanthine oxidase to generate superoxide radicals, which react with 2-(4-iodophenyl)-3-(4nitrophenonal)-5-phenyltetrazolium chloride to form a formazan dye. Using spectrophotometer (Perkin-Elmer, Germany), the SOD activity was measured by the degree of inhibition of the reaction. The results were expressed in units per miligram of protein. The 8-isoprotane assay was based on the competition between 8-isoprostane and 8-isoprostane-acetylcholinesterase (AchE) conjugate with specific rabbit antiserum, and then Ellman’s reagent was added. The final product gave distinct yellow color which was measured at 412 nm. One way analysis of variance (ANOVA) was used to compare the values among groups. In each test, the data were expressed as mean ± SE and P < 0.05 was considered statistically significant.

RESULTS Table 1 shows body mass gain, blood glucose levels in the normal, 48 h after STZ injection and at the end of study, HbA1C and total protein of rats in control, non-treated diabetic and VETD groups. Body gain was significantly decreased in the non-treated diabetic rats compared to the control (P = 0.002); however, there is no significant difference between VETD and control (P = 0.09) groups. As shown in Table 1, blood glucose level was significantly increased after 48 h of STZ injection compared to control rats (P = 0.005). In the end of study, blood glucose in the VETD rats significantly decreased compared to the nontreated diabetic rats and also after 48 h of STZ injection (P < 0.05). HbA1c was significantly elevated in the non-treated diabetic rats compared to the control rats (P = 0.005), but it was normalized in the VETD group (Table 1). The intestinal levels of total protein found in the control and non-treated diabetic rats did not differ significantly (Table 1). Total protein level in the VETD rats elevated significantly compared to the control rats (P < 0.05). As shown in Figure 1, 8-isoprostanoide level was significant increased in the non-treated diabetic rats compared to the control rats (P = 0.005), while it was significantly decreased in VETD rats compared to the control and non-treated diabetic rats (P < 0.05). VETD group showed a significant increase in the small intestine SOD activities as compared to the control (P = 0.005) and non-treated diabetic rats (P < 0.01) (Figure 1B). Catalase activity in the non-treated diabetic rats was significantly decreased compared to the control rats (P < 0.05) (Figure 1C), but it was normalized in the VETD rats compared with the control mice (P < 0.5). In addition, there was a significant increase in protein carbonyl contents in the non-treated diabetic rats compared to the control rats (P = 0.0005). In the VETD rats, protein carbonyl was significantly reduced below the level of the control rats (P = 0.004).

DISCUSSION Our study demonstrated that vitamin E treatment can improve oxidative stress status via normalization of lipid perwww.wjgnet.com

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Table 1 Body gain, blood glucose, HbA1C and protein content in the three groups (mean ± SEM) Body gain (g)

Blood glucose (mg/L) Initial

Control Non-treated diabetic VETD

48 h after STZ

HbA1C%

Total protein

End of study

6.2 ± 3  -70.2 ± 7a

1450 ± 150 1480 ± 170

1560 ± 130 4925 ± 210a

1510 ± 120 6044.6 ± 480a

5.7 ± 0.3 28.8 ± 1a

 -17 ± 12

1440 ± 200

4580 ± 280

3803 ± 650c

4 ± 0.3c

7.2 ± 0.22 7.5 ± 0.18 8.02 ± 1a

a

P < 0.05 vs control; cP < 0.05 vs non-treated diabetic.

A

B

SOD (U/mg protein)

1.2 1 0.8 0.6 0.4 0.2 0

C

Control

NTD

VETD

50 40 30 20 10

Control

NTD

VETD

8 7 6 5 4 3 2 1 0

D Protein carbonyl (nmol/mg protein)

8-isoprotane (pg/mg protein)

60

0

9 Catalase (nm/min per mg protein)

1.4

Control

NTD

VETD

0.25

0.2

0.15

0.10

0.05

0.00 Control

NTD

VETD

Figure 1 Biochemical markers which indicate status of oxidative stress. Significant high levels of superoxide dismutase activity (A) were observed in the non-treated diabetic rats instead of low level of catalase activity (B); 8-isoprostan (C) and protein carbonyl (D) contents of the small intestine of the non-treated diabetic rats were also significantly high. Vitamin E-treated diabetic rats showed elevated levels of superoxide dismutase (A) and catalase activities (B) accompanying with significant decrease in 8-isoprostan (C) and protein carbonyl (D) contents.

oxidation, protein oxidation and partially glycemic control in diabetic Wistar rats. Since the oxidative stress status did not alter in non-treated diabetic rats, the vitamin E treatment probably exerts its effects by protecting the small intestine from the toxic effects of ROS produced under hyperglycemic condition. Our previous study and others showed that the diabetes-induced small intestine morphologic changes include increasing of weight, length, crypt depth and villus height in diabetic rats[11,25,26]. Our previous[26] study also showed that vitamin E treatment restored all morphologic changes induced by diabetes in all parts of the small intestine. Vitamin E or α-tocopherol is highly soluble in lipids, so that it is the main antioxidant of lipoproteins and cell membrane. Some studies[27,28] have shown that the control of diabetes improves with the administrawww.wjgnet.com

tion of vitamin E to patients, since it protects the fatty acids of cell membrane and thereby preserves their reaction with respect to insulin. Our results showed a significant increment of lipid peroxidation in the small intestine in the non-treated diabetic rats. The observed increases in lipid peroxidation levels in the small intestine are in agreement with similar finding in other tissues[29]. Lipid peroxidation may bring about protein damage and inactivation of membrane-bound enzymes either through direct attachment by free radicals or through chemical modification by its end products, malondialdehyde and 4-hydroxynonenal[9]. It is also known to decrease the fluidity of the intestinal brush border membrane[30]. Therefore, the observed increase in lipid peroxidation could provide an additional explanation for the previously reported decrease in fluidity

Shirpoor A et al . Preventive effects of vit E on oxidative stress

of the intestinal brush border membrane during diabetes attributed to changes in lipid composition alone[31]. In this study, significantly higher levels of protein-bound carbonyls were found in the small intestine of the non-treated diabetic rats. Recently, it has been proposed that carbonyl stress, i.e. the increase in reactive carbonyl compounds derived from oxidative and non-oxidative reactions, leads to increased chemical modification of proteins and, at a later stage, to oxidative stress and tissue damage. A deficit in the detoxification of carbonyl compounds by the enzymes of glyoxalase pathway and aldose reductase is believed to be partly responsible for carbonyl stress and consequent oxidative stress[32]. In the present study, VETD rats showed significant decrement in lipid peroxidation and protein oxidation compared to the non-treated diabetic and control rats. Elimination of lipid and protein oxidation as two important free radical generation sources by vitamin E may be resulted in recovery of cell membrane to its normal physiologic state, and thus the insulin binds the cell readily. Our result also showed significant decrement in blood glucose and HbA1c levels in the VETD rats as compared with the non-treated diabetic rats. It is well known that hyperglycemia leads to autoxidation of glucose, lipid peroxidation and protein oxidation, that are three major ROS generation sources and consequently oxidative stress in diabetic subjects[33-35]. HbA1c was found to increase in patients with diabetes mellitus and the amount of increase was directly proportional to the fasting glucose level[36]. During diabetes mellitus, the excess glucose present in the blood reacts with hemoglobin to form HbA1c[37]. HbA1c is used as a marker for estimating the degree of protein glycation in diabetes mellitus[38]. Administration of vitamin E to diabetic rats reduced the glycation of hemoglobin, and thus decreased the levels of glycosylated hemoglobin in diabetic rats. This normalization of glycosylated hemoglobin indicates decreased glycation of protein. The activity of SOD was increased in the small intestine of non-treated diabetic and VETD rats. It is known that diabetes induces oxidative stress by production of superoxide anion radicals[35] and it is reasonable to expect an increased activity of SOD[39]. In physiological conditions, SOD is an important intracellular antioxidant which catalyses the conversion of the superoxide anion radical to molecular oxygen and hydrogen peroxide (H2O2) and thus protects against superoxide-induced damage[40]. Compared with the control rats, catalase activity significantly decreased in the non-treated diabetic rats. Vitamin E treatment normalized the catalase activity in the control group. In contrast to our results, increases in catalase activity in the small intestine of diabetic rats had been reported[11]. Giron et al[41] reported a lack of changes in the activity of intestinal catalase of diabetic rats fed with diets containing different fat supplements. This inconsistency in the reported literature might be due to the difference in the strain of the animals used, the duration of the experiment and/or severity of diabetes. Moreover, vitamin E has a number of effects at the cellular level that are not dependent on its antioxidant activity and may potentially contribute to improved insulin action. For example, vitamin E inhibits protein kinase C by a non-antioxidant mechanism[42]. Vitamin E also accelerates diacylglycerol kinase activity, thereby decreasing levels of diacylglycerol, which is an allosteric activator or protein

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kinase C[43]. Increased protein kinase C activity apparently impairs insulin action by phosphorylating serine or threonine residues on insulin receptor and insulin receptor-1 proteins[44]. This decreases insulin-stimulated, phosphatidylinositol 3-kinase-catalyzed phosphorylation of tyrosine residues in these proteins, which is required for effective insulin action. Recent evidence suggests that vitamin E may influence the activity of these enzymes by decreasing the curvature of plasma membranes[24]. In conclusion, our results clearly demonstrate that vitamin E administration improves type 1 diabetes-induced oxidative stress via decreasing lipid peroxidation and protein oxidation as a free radical generation sources and elevating antioxidant defense system enzymes like SOD and catalase activities.

COMMENTS Background

The entire GI tract from the esophagus to the anorectal region can be affected by diabetes mellitus. Recently, several studies have examined the role of oxidative stress on developmental diabetic-mediated disorders, possibly via the formation of free radicals. Free radicals or reactive oxygen species are generated during oxidative metabolism and can inflict damage on all classes of cellular macromolecular components (e.g. mitochondria, endoplasmic reticulum, protein, etc.), eventually leading to cell death. One promising aspect of understanding the role of oxidative stress in diabetes-mediated disorders is the ability of antioxidant supplementation to attenuate adverse effect of diabetes. Antioxidants, such as ascorbic acid, α-tocopherol (vitamin E), endogenous glutathione peroxidase and the pineal hormone melatonin, have all been tested for efficacy in defending against free-radical-mediated tissue injuries.

Research frontiers

Currently, there is considerable interest in the roles of vitamin E in the protection of membranes lipids, proteins or other biomolecules against oxidative stress as well as precise molecular mechanism by which they may occur.

Related publications

For more information about each section, please refer to articles which included in reference list.

Innovations and breakthrough

This study tried to open new horizons on molecular base of protective effects of vitamin E on diabetes-induced injuries in the small intestine.

Applications

The results of current study launch a new view on pathogenesis of diabetes mellitus; hence, further studies may be designed to elucidate more informative details.

Terminology

Free radicals or reactive oxygen species are the molecules which generated during oxidative metabolism and can inflict damage on all classes of cellular macromolecular components called oxidative damage.

Peer review

The authors in this manuscript use a streptozotocin model of type I diabetes to examine the effects of vitamin E on the gut. Improved glucose control, less weight loss, and reduced oxidative injury in the gut were observed. The authors conclude that vitamin E may play a beneficial role in preventing oxidative injury associated with diabetes.

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Role of serum interleukin-18 as a prognostic factor in patients with hepatocellular carcinoma Pisit Tangkijvanich, Duangporn Thong-ngam, Varocha Mahachai, Apiradee Theamboonlers, Yong Poovorawan Pisit Tangkijvanich, Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand Duangporn Thong-ngam, Department of Physiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand Varocha Mahachai, Department of Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand Apiradee Theamboonlers, Yong Poovorawan, Center of Excellence in Viral Hepatitis Research, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand Supported by the Rajadapiseksompoj research grant, Faculty of Medicine, Chulalongkorn University Correspondence to: Yong Poovorawan, Professor, MD, Viral Hepatitis Research Unit, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand. [email protected] Telephone: +662-256-4909 Fax: +662-256-4929 Received: 2007-02-12 Accepted: 2007-03-15

Abstract AIM: To determine whether serum interleukin-18 (IL-18) levels correlated with clinicopathologic features and prognosis in patients with hepatocellular carcinoma (HCC). METHODS: Serum IL-18, IL-6 and IL-12 levels were measured by enzyme-linked immunosorbent assay (ELISA) from 70 patients with HCC and 10 healthy controls. RESULTS: Serum IL-18, IL-6 and IL-12 levels of patients with HCC were significantly higher that those of the controls. The levels of IL-18 correlated significantly with the presence of venous invasion and advanced tumor stages classified by Okuda’s criteria. Patients with high 5 serum IL-18 levels (≥ 10 pg/mL) had a poorer survival 5 than those with low serum IL-18 levels (< 10 pg/mL) (4 and 11 mo, respectively, P = 0.015). Multivariate analyses showed that serum IL-18 level, but not IL-6 and IL-12 levels, was a significant and independent prognostic factor of survival. CONCLUSION: These findings demonstrate that serum IL-8 may a useful biological marker of tumor invasiveness and an independent prognostic factor of survival for patients with HCC. Thus, the detailed mechanisms of IL-18 involving in tumor progression should be further investigated.

© 2007 WJG . All rights reserved.

Key words: Hepatocellular carcinoma; Interleukin-18; Serum marker; Prognosis

Tangkijvanich P, Thong-ngam D, Mahachai V, Theamboonlers A, Poovorawan Y. Role of serum interleukin-18 as a prognostic factor in patients with hepatocellular carcinoma. World J Gastroenterol 2007; 13(32): 4345-4349

http://www.wjgnet.com/1007-9327/13/4345.asp

INTRODUCTION Hepatocellular carcinoma (HCC) represents one of the most common cancers worldwide with a particularly high prevalence in sub-Saharan Africa and Southeast Asia where hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are common[1]. Although recent advances in the detection and treatment of HCC have improved the survival, the prognosis of most patients is somewhat unsatisfactory due to rapid clinical deterioration after the initial diagnosis and high incidence of recurrence after surgical resection[2]. In general, the natural history of HCC depends on the severity of the underlying liver disease, tumor characteristics and the efficacy of treatment interventions[3]. Besides these features, a number of biological markers including cytokines and growth factors have been demonstrated to be increased in the sera of patients with HCC and may be associated with a poor prognosis. Interleukin-18 (IL-18), originally known as interferon-γ (IFN-γ)-inducing factor (IGIF), is a cytokine that shares structural and functional properties with interleukin-1 (IL-1)[4,5]. This cytokine is mainly produced by activated macrophages, but may also be expressed by Kupffer cells, T cells, B cells, keratinocytes, astrocytes, and osteoblasts[6]. Like IL-1, IL-18 is synthesized as an inactive precursor (pro-IL-18, 24 kDa), which is cleaved by interleukin-1 β-converting enzyme (ICE or caspase-1) into an active 18 kDa mature form [6-8]. IL-18 has multiple biological activities via its capacity to stimulate innate immunity and both Th1 and Th2 mediated responses[6,8]. It also exerts anti-tumor effects that are mediated by enhancement of NK cell activity, reduction of tumorigenesis, induction of apoptosis and inhibition of angiogenesis in tumor cells[9,10]. In addition, recent data have been suggested that inappropriate production of

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IL-18 contributes to the pathogenesis of cancers and may influence the clinical outcome of patients[11]. Specifically, it has been demonstrated that serum IL-18 level may have prognostic significance in some types of cancer including colonic carcinoma, gastric carcinoma, esophageal carcinoma, breast cancer, and hematologic malignancies[12-16]. However, the prognostic role of serum IL-18 level in patients with HCC has never been investigated. Therefore, in this study, we determined whether serum IL-18 level correlated with clinicopathologic features and prognosis of patients with HCC.

MATERIALS AND METHODS Patients and blood samples For the purpose of this study, 70 patients with HCC were randomly selected from a pool of patients with chronic liver disease who were seen and followed at King Chulalongkorn Memorial Hospital (Bangkok, Thailand) between August 1997 and September 2003. The control group comprised 10 healthy adults from the blood bank. Serum samples were collected from each subject at the time of their clinical evaluation and stored at -70℃ until further tested. The study was approved by the Ethical Committee of the Faculty of Medicine, Chulalongkorn University. Informed consent was obtained according to the regulations of the committee. The diagnosis of HCC was based on histopathology and/or a combination of mass lesions in the liver on hepatic imaging and serum alpha-fetoprotein (AFP) levels above 400 ng/mL. All demographic and clinical data were extracted from patients’ files. The authors collected the data including sex and age, as well as clinical data such as liver function tests, severity of liver disease graded as the Child-Pugh status, Okuda staging, etiologic factors (HBsAg, Anti-HCV or alcohol abuse), serum AFP levels at the time of diagnosis, and presence of venous invasion diagnosed by CT scan. Hepatitis B surface antigen (HBsAg), hepatitis C virus antibody (anti-HCV) and AFP level were determined by enzyme-linked immunosorbent assays (ELISA) using commercially available kits (Auszyme Ⅱ, Abbott Laboratories, IL for HBsAg; ELISA Ⅱ, Ortho Diagnostic Systems, Chiron Corp., CA for anti-HCV; and Cobus  Core, Roche Diagnostics, Basel, Switzerland for AFP). Biochemical liver function tests were determined by automated chemical analyzer (Hitachi 911) at the central laboratory of the hospital. Measurement of serum IL-6, IL-12 and IL-18 levels Serum IL-6, IL-12 and IL-18 were determined by using ELISA kits (R&D systems, Inc., Minneapolis, MN). ELISA was performed according to the manufacturer’ instructions. Statistical analysis Data are expressed as percentage, mean and standard deviation. Comparisons between groups were analyzed by the χ2 or Fisher’s exact test for categorical variables and by the Mann-Whitney test or Student’s t test when appropriate for quantitative variables. Survival curves were constructed

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Table 1 Clinical and demographic data of patients with HCC at the time of the diagnosis Clinical features Age (yr) Sex (male:female) Etiology Alcohol dependence HBsAg-positive Anti-HCV-positive HBsAg- and anti-HCV-positive Unknown Liver function test Total bilirubin (mg%) Albumin (g/dL) AST (IU/L) ALT (IU/L) Alkaline phosphatase (IU/L) Prothrombin time (s) Child-Pugh classification A B C Okuda staging system 1 2 3 Venous invasion Extrahepatic metastasis AFP (≥ 400 ng/mL)

mean ± SD or percentage (%) 55.0 ± 13.6 (range, 26-89) 60:10 9/70 (12.8) 39/70 (55.7) 7/70 (10) 2/70 (2.9) 13/70 (18.6) 2.3 ± 3.5 3.5 ± 0.7 150.9 ± 130.4 82.9 ± 84.1 512.0 ± 317.9 14.4 ± 4.2 43/70 (61.4) 23/70 (32.9) 4/70 (5.7) 19/70 (27.2) 46/70 (65.7) 5/70 (7.1) 17/70 (24.3) 12/70 (17.1) 27/70 (38.6)

using the Kaplan-Meier method and difference between curves was testing by the log-rank test. The Cox regression analysis was performed to identify which independent factors have a significant influence on the overall survival. P values below 0.05 for a two-tailed test were considered statistically significant. All statistical analyses were performed using the SPSS software for windows 10.0 (SPSS Inc., Chicago, IL).

RESULTS The clinical data of patients with HCC The clinical and demographic data of patients with HCC in this study are shown in Table 1. Among the 70 recruited patients, 60 were men and 10 were women. The average age of the patients was 55.0 ± 13.6 years (ranged 26-89 years). All patients had underlying cirrhosis. Seventeen patients (24.3%) had venous invasion. Extrahepatic metastasis was found in 12 patients (17.1%). The mean total bilirubin (TB), serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), albumin, alkaline phosphatase (AP), and prothrombin time (PT) were 2.3 ± 3.4 mg/dL, 150.9 ± 130.4 IU/L, 82.9 ± 84.1 IU/L, 3.5 ± 0.7 g/dL, 512.6 ± 317.9 IU/L, and 14.4 ± 4.2 sec, respectively. Twenty-seven patients (38.6%) had serum AFP higher than 400 ng/mL. According to Okuda staging system, there were 19 patients (27.2%) in stage 1, 46 patients (65.7%) in stage 2, and 5 patients (7.1%) in stage 3. For predisposing etiologic factors, 9 patients (12.8%) were associated with alcohol-dependent. Thirty-nine patients (55.7%) were associated with HBsAg-positive, and 7 patients (10%) were associated with anti-HCV-positive.

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Table 2 Serum levels of interleukins in patients with HCC and in healthy controls

IL-6 (pg/mL) IL-12 (pg/mL) IL-18 (pg/mL)

Healthy controls (n = 10) 2.9 ± 13.4 1.5 ± 0.9 38.5 ± 22.4

HCC patients (n = 70) 31.2 ± 52.2 6.2 ± 9.6 104.6 ± 65.8

P 0.01 0.03 0.002

Two patients (2.9%) had both HBsAg-positive and antiHCV-positive. The predisposing factors could not be determined in 13 patients (18.6%). Seven patients (10%) had undergone surgical resection, 19 patients (27.1%) had been treated with transarterial chemoembolization (TACE), and the remaining 44 patients (62.9%) had received no specific treatment because of their advanced tumor stage or refusal to therapy. Serum IL-18 levels of patients with HCC and the survival As shown in Table 2, serum IL-18 levels in patients with HCC were significantly elevated compared with those of the controls (104.6 ± 65.8 vs 38.5 ± 22.4 pg/mL, P = 0.002). Similarly, the levels of serum IL-6 and IL-12 in patients with HCC were significantly increased compared with healthy subjects (31.2 ± 52.2 vs 2.9 ± 13.4 pg/mL, P = 0.01, and 6.2 ± 9.6 vs 1.5±0.9 pg/mL, P = 0.02, respectively). Serum IL-18 levels also exhibited a positive correlation with serum IL-6 and IL-12 levels (P = 0.021; Pearson r = 0.276 and P = 0.002; Pearson r = 0.369, respectively). In order to evaluate the association between serum IL-18 and the survival, the patients with HCC were further categorized into two groups according to their serum IL-18 levels. In this respect, the cut point of 105 pg/mL, which represented the mean serum IL-18 level in the whole group, was used. There were 41 patients with serum IL-18 < 105 pg/mL and 29 patients with serum IL-18 ≥ 105 pg/mL. There was no statistically significant difference in age, gender, serum ALT, AST, AFP, PT and extrahepatic metastasis between these two groups (Table 3). However, patients with high serum IL-18 levels had significantly lower mean serum albumin level (P = 0.01), but had significantly higher mean total bilirubin (P = 0.03), serum AP levels (P = 0.04), exhibited more advanced tumor stages classified by Okuda’s criteria (P = 0.03), and had higher percentage of venous invasion (P = 0.02) than patients with low serum IL-18 levels. Kaplan-Meier survival curves revealed that the median survival of patients with low serum IL-18 and the other were 10.5 and 5.0 mo, respectively (Figure 1A). By using log-rank test, there was a statistically significant difference in the median survival between these two groups (P = 0.007). Among patients who were treated with surgery or TACE, the medial overall survival for the low and high serum IL-18 groups were 18.5 and 10.0 mo, respectively (P = 0.021) (Figure 1B). In untreated cases, the medial overall survival for the low and high serum IL-18 groups were 4.5 and 2.7 mo, respectively (P = 0.043) (Figure 1C). Serum IL-18, IL-6 and IL-12 levels were entered into a Cox regression analysis together with other variables that may influence prognosis. These included age, gender, serum

Table 3 Comparison of clinical data of patients with HCC according to serum IL-18 levels Clinical features

5 5 IL-18 < 10 IL-18 ≥ 10 pg/mL pg/mL (n = 41) (n = 29) 54.9 ± 15.3 55.2 ± 11.1 34:7 26:3

Age (yr ) Sex (male:female) Liver function test Total bilirubin (mg%) 1.4 ± 0.7 Albumin (g/dL) 3.8 ± 0.7 AST (IU/L) 136.8 ± 116.4 ALT (IU/L) 66.5 ± 39.2 Alkaline phosphatase (IU/L) 436.9 ± 275.2 Prothrombin time (s) 13.9 ± 2.4 Okuda staging (1:2:3) 15:25:1 Venous invasion (+:-) 6:35 Extrahepatic metastasis (+:-) 6:35 AFP (< 400 ng/mL: ≥ 400 ng/mL) 25:16

3.6 ± 4.9 3.3 ± 0.7 170.8 ± 148.3 106.2 ± 119.7 612.1 ± 348.2 14.9 ± 5.8 4:21:4 11:18 6:29 18:11

P

NS NS 0.03 0.01 NS NS 0.04 NS 0.03 0.02 NS NS

AFP level, HBsAg status, tumor size, tumor number, venous invasion, extrahepatic metastasis, Child-Pugh classification, Okuda staging, and therapy of HCC. Multivariate analyses revealed that independent prognostic factors of overall survival included high serum IL-18 level, venous invasion and no receiving therapy for HCC (Table 4).

DISCUSSION Enhanced expression of proinflammatory, hematopoietic and angiogenic cytokines has been demonstrated in several human tumors[17]. Some of these cytokines may act as autocrine or paracrine tumor cell growth factors, inhibitors of apoptosis, attractors of immune cells, and promoters of angiogenesis[18,19]. Accordingly, it is likely that the deregulation of these cytokines may contribute to the development or progression of the malignant process. Currently, serum levels of several cytokines have been found to be increased in patients with HCC and may be correlated with clinical outcomes. For instance, higher level IL-10 was observed in patients with HCC[20], and increased IL-10 values were associated with a poor prognosis in patients undergoing surgical resection[20], as well as in patients with unresectable tumor[21] Similarly, serum IL-8 was shown to be a useful biological marker of tumor invasiveness and an independent prognostic factor of survival for patients with HCC[22]. To the best of our knowledge, this is the first study demonstrating that the levels of serum IL-18 were markedly elevated in patients with HCC compared with healthy controls. In addition, our data showed that a high-serum IL-18 level was significantly correlated with advanced tumor stage classified by Okuda’s criteria. Furthermore, serum IL-18 levels were significantly correlated with venous invasion, a pathobiological feature indicative of tumor aggressiveness. These data suggest that serum IL-18 might be useful in the clinical setting to predict tumor invasiveness and stage. A high-serum IL-18 level was also a significant prognostic factor in terms of overall survival, as demonstrated by multivariate analysis. These results were in agreement with previous data suggesting that serum IL-18 levels are related to the prognosis of patients with various www.wjgnet.com

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Figure 1 Overall survival of HCC patients with low serum IL-18 level (< 105 pg/mL) or high IL-18 level (≥ 105 pg/mL). A: All patients; B: Patients who were treated with surgery or TOCE; C: Untreated patients.

Table 4 Multivariate analysis of prognostic factors of survival with Cox’s proportional hazards model Factors High serum IL-18 level Presence of venous invasion No receiving therapy

Risk ratio (95% CI) 1.86 (1.11-3.11) 2.09 (1.19-3.67) 5.01 (2.49-10.06)

P 0.019 0.010 < 0.001

malignant diseases in the gastrointestinal tract, including colonic, gastric and esophageal carcinoma [12,13,16] . In agreement with our results, it has been shown recently that the expression of IL-18 receptor in tumor tissues was found to be a significant predictor of a poor outcome in HCVassociated HCC patients[23]. The precise mechanisms underlying the positive correlation between serum IL-18 levels and advanced tumor stages are unclear. As previously mentioned, IL-18 exerts anti-tumor activity via several mechanisms including enhancement of NK cell function, induction of apoptosis via Fas/Fas ligand interaction and inhibition of angiogenesis[9,10]. Indeed, recent data have demonstrated that positive IL-18 immunoreactivity is significantly higher in the surrounding hepatocytes compared with the tumor portion from the same individual[24]. It has been shown that decreasing IL-18 production in tumor cells may be related to the down regulation of ICE gene expression, as demonstrated in colon and ovarian carcinoma[13,25]. In contrast, IL-18 and ICE transcripts have been detected in the corresponding normal colon and ovarian epithelium suggesting the bioactive IL-18 is most likely produced by the adjacent normal cells [13,25]. Taken together, it is speculated that IL-18 production by the normal adjacent hepatocytes may reflect the degree of defense mechanisms against tumor growth and dissemination of HCC. IL-12, also known as NK cell stimulatory factor or cytotoxic lymphocyte maturation factor, is a multifunctional cytokine produced primarily by antigen-presenting cells (APC), such as monocytes and NK cells[26]. This cytokine augments proliferation, cytokine production and the development of Th1. Furthermore, IL-12, in combination with IL-18, induces anti-tumor effects against a variety of tumor cells via the activity of IFN-γ[27,28]. It has been shown that serum IL-12 levels are significantly higher in www.wjgnet.com

patients with gastric and esophageal cancers compared with healthy controls [12,29]. In patients with esophageal carcinoma, increasing serum IL-12 and IL-18 levels correlate with tumor growth and progression[12]. In contrast, serum IL-12 levels in patients with far-advanced gastric cancer are significantly lower that those with less-advanced stages[29]. In this study, we demonstrated that serum IL-12 levels were significantly higher in patients with HCC than in healthy controls. Furthermore, we found that its levels were correlation with IL-18 levels, suggesting that these cytokines may act synergistically in the anti-tumor activity. However, unlike IL-18, IL-12 levels were not confirmed as a prognostic factor in multivariate analysis. IL-6 is a pleiotropic cytokine that was originally identified as a T cell-derived lymphokine inducing final maturation of B cells into antibody-producing cells[30]. This cytokine plays an important role in hematopoiesis, acute-phase responses and host defense mechanisms[31]. In addition, IL-6 has also shown to act as an autocrine growth factor in malignancy[30]. Increased serum levels of IL-6 have been demonstrated in patients with a variety of cancers and may be associated with a poor outcome[32-34]. However, the clinical significance of serum IL-6 levels in patients with HCC remains to be established[20,35,36]. In this study, we found that though levels of IL-6 were significantly higher in patients with HCC than in healthy subjects, its levels were not an independent prognostic factor in multivariate analysis. Thus, our results were in agreement with the reports conducted by Chau et al and Parasole et al[20,36]. In summary, our data demonstrated that serum IL-18 levels in patients with HCC correlated with advanced tumor stage classified by Okuda’s criteria and the presence of venous invasion. Serum IL-18 level also exhibited an independent predictor of prognosis in patients with HCC. These data suggest that IL-18 contributes an important role in the pathogenesis and disease progression of HCC. If confirmed in additional longitudinal studies, the immuno-modulation of this cytokine may have therapeutic potential in the future.

ACKNOWLEDGMENTS This study was supported by the Rajadapiseksompoj research grant. We also thank Thailand Research Fund

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and Center of Excellence, Viral Hepatitis Research Unit, Chulalongkorn University.

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E- Editor Liu Y

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World J Gastroenterol 2007 August 28; 13(32): 4350-4354 World Journal of Gastroenterology ISSN 1007-9327 © 2007 WJG. All rights reserved.

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Genetic polymorphism of UL144 open reading frame of human cytomegalovirus DNA detected in colon samples from infants with Hirschsprung’s disease Zhi-Qin Mao, Ying Huang, Mei Sun, Qiang Ruan, Ying Qi, Rong He, Yu-Jing Huang, Yan-Ping Ma, Yao-Hua Ji, Zheng-Rong Sun, Hong Gao Zhi-Qin Mao, Mei Sun, Department of Pediatrics, Second Affiliated Hospital, China Medical University, Shenyang 110004, Liaoning Province, China Ying Huang, Hong Gao, Department of Pediatric Surgery, Second Affiliated Hospital, China Medical University, Shenyang 110004, Liaoning Province, China Qiang Ruan, Rong He, Ying Qi, Yu-Jing Huang, Yan-Ping Ma, Yao-Hua Ji, Zheng-Rong Sun, Virus Laboratory, Second Affiliated Hospital, China Medical University, Shenyang 110004, Liaoning Province, China Supported by the National Natural Science Foundation of China, No.30170986 Correspondence to: Dr. Zhi-Qin Mao, Department of Pediatrics, Second Affiliated Hospital, China Medical University, Shenyang 110004, Liaoning Province, China. [email protected] Telephone: +86-24-83955570 Received: 2007-04-03 Accepted: 2007-04-26

Abstract AIM: To explore the genetic diversities of UL144 open reading frame (ORF) of cytomegalovirus DNA detected in colon tissue from infants with Hirschsprung’s disease (HD) by sequencing UL144 DNA in 23 aganglionic colon tissue and 4 urine samples from 25 HD infants. METHODS: Nest PCR was performed for amplification of the UL144 gene. The UL144 gene was analyzed with softwares, such as DNAclub, BioEdit, PROSITE database, and DNAstar. RESULTS: The strains from HD patients were distributed among three genotypes of UL144: group 1A (64%), group 2 (24%), and group 3 (12%). The UL144 genotypes between strains from HD and control group 2 were compared by chi square test (χ = 1.870, P = 0.393). Strains from the colon were sporadically distributed in UL144 genotypes. CONCLUSION: There are genetic diversities of UL144 ORF in colon tissue of infants with HD. However, cytomegalovirus UL144 genotypes are not associated with clinical manifestations of HD. © 2007 WJG. All rights reserved.

Key words: Hirschsprung’s disease; Cytomegalovirus; UL144 gene; Polymorphism www.wjgnet.com

Mao ZQ, Huang Y, Sun M, Ruan Q, Qi Y, He R, Huang YJ, Ma YP, Ji YH, Sun ZR, Gao H. Genetic polymorphism of UL144 open reading frame of human cytomegalovirus DNA detected in colon samples from infants with Hirschsprung’s disease. World J Gastroenterol 2007; 13(32): 4350-4354

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INTRODUCTION Human cytomegalovirus (HCMV) is composed of a large and complicated genome. HCMV infection can cause various clinical manifestations, such as jaundice, hepatomegaly, splenomegaly, purpura, pneumonia, central nervous system impairment. HCMV infection is mild or asymptomatic in immunocompetent hosts. However, serious outcomes may occur in immunocompromised hosts and congenitally-infected infants. It has been shown that glycoprotein B (gB) is associated with the severity and/or tissue-tropism of human cytomegalovir usinfected hosts[1-6], other studies have shown contradictory results[7-9]. HCMV UL144 gene encodes a transmembrane glycoprotein and is a homologue of the herpes simplex virus entry mediator (HveA or HveM), a member of tumor necrosis factor receptor (TNFR) superfamily[10]. Therefore, the locus may play an important role in determining the biological behavior of wild-type HCMV strains. Lurain et al [11] have shown significant strain-specific sequence variability of UL144 by sequencing 45 low passaged clinical isolates from solid organism graft and AIDS adults. The nucleotide sequences of cytomegalovirus UL144 gene can be divided into group 1, group 2 and group 3. Group 1 can be divided into three distinct subgroups (A, B and C). Bale et al[12] have confirmed these results by using isolates from 28 healthy children and 16 congenitally-infected infants subsequently, suggesting that the UL144 genotype is associated with neither the outcomes of intrauterine HCMV infection nor the development and severity of HCMV disease at birth. However, Arav-Bogger et al[13] reported different results with those of Bale and Lurain. Hirschsprung’s disease (HD) is one of the common malformations in children. Its etiology is obscured. It was reported that congenital HCMV infection is correlated with development of HD[14-17]. To further understand the relationship between genetic diversities of HCMV UL144

Mao ZQ et al . Polymorphism of CMV UL144 gene in infants with HD

ORF in colon samples and clinical outcomes of HD, we sequenced UL144 DNA in 23 aganglionic colon tissue and 4 urine samples from 25 HD infants.

MATERIALS AND METHODS Patients and samples Fifty-five infants with Hirschsprung’s disease were selected from July 2001 to May 2004. The youngest infant was 19- day old and the eldest one was 1.5- year old. Their average age was 8.71 ± 6.8 mo on operation. All patients were diagnosed as HD based on clinical analysis, barium enema examination, and biopsy specimens. HE staining showed that nerve plexus proliferated and no ganglion cells were found on colon mucous membrane under light microscope. Veins became large and congested, and leukomonocyte infiltration was observed on colon mucous membrane of the narrowed segment in some HD infants. A total of 53 aganglionic colon tissue and 4 urine samples were obtained from 55 infants with HD. Of which, 3 were total colonic type, and 4 long segment HD type, 46 sigmoid rectal type. The specimens were stored at -70℃ until use. Sixteen urine samples from 11 asymptomatic patients, 3 from thrombocytopenia patients, 1 from congenital heart disease patient, and 1 from renal disease infant, were investigated after cytomegalovirus DNA was detected by quantitative PCR[18]. All infants were admitted to Second Affiliated Hospital of China Medical University from March 2001 to September 2004. Cytomegalovirus was detected in all urine samples and 16 of 53 narrow segment colon specimens from HD patients by quantitative PCR. The study was approved by the local ethical committee. PCR amplification Virus DNA in urine was isolated from the precipitated cells by boiling them in lysis buffer for 15 min. Tissue DNA was extracted with xylene, proteinase K, and phenol chloroform protocol, followed by ethanol precipitation. All extracted DNA preparations were diluted in water as templates used in PCR amplification. The PCR reaction mixture containing 1 × buffer, 1.5 mmol/L MgCl2, 0.2 mmol/L dNTPs, 150 ng up and down primer respectively, 0.5 U of Taq polymerase (Promega, Madison city, USA), 3.5 µL sample, and ddH2O was added to a final volume of 50 µL. Nest PCR was performed to amplify UL144 when the outside primers yielded either negative or weak results. The sequences of outside primer set designated by Lurain et al[11] as UL144B are as follows: forward (UL144Ca) 5’-CGTATTACAAACCGCGGA GAGGAT-3’, reverse (UL144Cb) 5’-ACTCAGACAC GGTTCCGTAA-3’. The inner primers were designed based on the Toledo sequence (GenBank accession No. AY446871) using Primer premier 5.0: forward (UL144Ca2) 5’-AGACACCGTTCGGCCCTAT-3’, reverse (UL144Cb2) 5’-TTTAGTGCAGGAATTGGAA-3’. A 681 bp fragment containing UL144 coding sequence was amplified. The conditions for amplification with all primer sets were at 95℃ for 5 min, followed by 30 cycles at 95℃ for 45 s, at 54℃ for 1 min, and at 72℃ for 1 min and 30 s, and a

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single extension cycle at 72℃ for 10 min. PCR products were detected on a 1.5% agarose gel stained with ethidium bromide under UV illumination. DNA sequencing PCR products including the whole length of UL144 open reading frame (ORF) were gel-purified using PCR fragment purification kit (TaKaRa, Dalian city, China) according to the manufacturer’s instructions, and then sequenced directly with the BigDye terminator cycle sequencing kit (Perkin Elmer, Foster city, USA). Sequencing was usually performed on both DNA strands, using the UL144Ca2 and UL144Cb2 primers. Sequencing reactions were performed with a Perkin-Elmer Gene Amp PCR system 2400 (Perkin Elmer, Foster city, USA) at 96℃ for 10 s, at 50℃ for 5 s, and at 60 ℃ for 4 min for a total of 30 cycles. The sequencing products were analyzed on an ABI 3700 automated sequencer. Cloning To get accurate sequence data for clinical strain M20, in which the sequence represents lapped spike, UL144 PCR products of clinical strain M20 were cloned into PGEM-T vector (Promega, Madison city, USA) and the UL144 was sequenced using standard M13 + primer. Sequence analysis Nucleotide and amino acid sequences were compared using Program of BioEdit 5.0. Multiple-alignment algorithm in the Megalign program package was used in phylogenetic analysis (Lasergene; DNAstar). Functional motifs were identified from the PROSITE database. Nucleotide sequence accession numbers Twenty-seven strains from HD infants and 16 strains from urine sample were sequenced. UL144 ORF DNA sequences from these strains were submitted to GenBank by using program Sequin. The accession number of strains from HD patients is AY999272-AY999296, AY818285, AY818293, respectively. The accession number of strains from control group is AY818269-270, AY818272, AY818276, AY818280, AY818283, AY818286, AY818292, AY818295, AY818302-303, AY818305-306, AF447377, AF447388-89, respectively. Statistical analysis Descriptive statistics were carried out by chi square test or Fisher's exact test with SPSS 10.0 software package. P ≤ 0.05 was considered statistically significant.

RESULTS UL144 variability Twenty-three operational and 4 urine samples from HD infants and 16 urine samples from control group yielded positive results of the predicted size when amplified with the UL144Ca/Cb and UL144Ca2/Cb2 primer sets. All the amplified products were sequenced. UL144 ORF was detected using DNAclub. Then, UL144 ORFs of all strains were compared using Clustal W. Alignment comparison www.wjgnet.com

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U253 M8 M24 M12 M15 U167 pt-20G1a M16 G1A 64% M1 M4 M48 M17 M11 M20 M42 M22 M5 -To-UL144 0.1

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Table 1 Distribution of UL144 genes and source of the HCMV strains n 25 16

1A (%) 1B (%) 2 (%) 16 (64.0) 0 (-) 6 (24.0) 6 (37.5) 1 (6.3) 5 (31.2)

3 (%) 3 (12.0) 4 (25.0)

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Relationship between UL144 clades and clinical phenotypes of HD Three UL144 genotypes (group 1A, group 2, and group 3) were found among strains in colon samples from infants with Hirschsprung’s disease. With respect to clinical features, strains from Hirschsprung’s disease patients were scattered in the UL144 genotypes. Because the number of samples of the long segment and total colon type was limited, no relationship was found between their clinical phenotypes and UL144 genotypes.

0.393

C-group: Control group.

revealed that the variations dispersed over the whole ORF but concentrated in the 5’ half. UL144 sequence of various clinical strains was found in 80.4%-99.4% of nucleotides and 79%-100% of amino acids sequence identities compared with that of Toledo. Strains from HD patients presented cytomegalovirus UL144 hypervariability. The cytomegalovirus UL144 sequences from the same patients, but different samples (M25 and U296, M27 and U298) were completely identical. Based on the phylogenetic analysis, the UL144 sequences of strains from HD patients were categorized into three major groups according to the schema classified by Lurain et al [11], referred to as group 1A, group 2, and group 3 (Figure 1). Our results showed that most of the strains belonged to group 1A, amounting to 64% (16 of 25). The minority of infants (3 of 25, 12%) showed strains that conformed to group 3. Twenty-four percent (6 of 25) of the strains were distributed in group 2. No strains were found in group 1B and group 1C. The variability within each genotype ranged 0%-0.6%. Compared with the corresponding groups (groups 1A, 2, and 3) remarked by Lurain[11], the homology was 97.5%-98.9%, 97.9%-98.1%, and 99.1%-99.2%, respectively. The distribution of UL144 genotypes in urine samples from infants with other diseases except for Hirschsprung’s www.wjgnet.com

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disease (control group) was found in 6 (37.5%), 1 (6.3%), 5 (31.2%), 4 (25.0%) of genotype group 1A, group 1B, group 2, and group 3, respectively. The UL144 genotypes between strains from infants with Hirschsprung’s disease and those from control group were compared by chi square test (χ2 = 1.870 and P = 0.393, Table 1). Analysis of posttranslational modification sites of predicted UL144 protein showed that most of the important motifs, such as tumor necrosis factor-like receptor (TNFR), N-myristoylation (MYR), protein kinase C phosphorylaction site (PKC), and N-glycosylation (ASN) were shared by strains from all groups. However, because valine (V) and serine (S) substituted by glycine (G) at 19 and 53 amino acid residues in strains belong to group 2, another two MYR functional sites were added, respectively, in 19-24 and 53-59 amino acid residues. Compared with those in the other two groups, only strains in group 3 had a specific acyl carrier protein site (ACP) but MYR was absent motif in their UL144 putative protein (Figure 2).

M27 M39 M10 G2 24% M41 M13 M26 pt 16-G2 M37 M45 G3 12% M25 pt 10-G3

Figure 1 Phylogenetic analysis based on UL144 nucleotide sequence of clinical strains from 25 HD infants. Pt 10, pt 16, and pt 20 represented respectively different UL144 genotypes of Lurain data; TO: Toledo strain; M: clinical strains from narrow bowel tissue of HD; U: clinical strains from urine samples of HD.

Source HD C-group

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DISCUSSION Hirschsprung’s disease has been proved to be a disease of neural crest. It is a developmental disorder of the enteric nervous system (ENS) characterized by the absence of ganglion cells in the myenteric and submucosal plexuses along a variable portion of the distal intestine. It was reported that different genes such as RET, GDNT, EDNRB and EDN3 are involved in the pathogenesis of Hirschsprung’s disease[19]. However, microenvironment factors can also play a role in the pathogenesis of aganglionosis. It was reported that one of the etiologies of Hirschsprung’s disease is cytomegalovirus infection deter mined by immuno-histochemical and in situ hybridization test, and cytomegalovirus DNA has been detected either in ganglion cells or within myenteric and submucosal plexus along small and large intestine[17,20,21]. It has been widely accepted that Hirschsprung’s Disease is associated with a narrowed bowel segment. In this study, we explored the genetic diversities of cytomegalovirus UL144 ORF in narrowed colon tissue samples from infants with Hirschsprung’s disease. Based on the nucleotide sequences of UL144 gene, cytomegalovirus strains have been divided into five genotypes by Lurain et al[11]. In their study, the predominant genotype of UL144 was group 3. In the current study, samples were obtained directly from narrow segment colon tissue and urine in patients with Hirschsprung’s disease, without any passage in cell culture. Hence, the strains in bowel tissue specimens from patients could authentically

Mao ZQ et al . Polymorphism of CMV UL144 gene in infants with HD

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Figure 2 Alignment of amino acid sequences of clinical strains from 25 HD infants with the Toledo as a reference. The posttranslational modification motifs were marked in abbreviation. TNFR: tumor necrosis factor-like receptor; MYR: N-myristoylation; PKC: protein kinase C phosphorylaction site; ASN: N-glycosylation; ACP: Acyl carrier protein site.

reflect the natural genetic composition of cytomegalovirus infection in end organ. The UL144 gene sequences of cytomegalovirus strains from Hirschsprung’s disease patients could be categorized into three major groups according to the schema of Lurain et al[11], referred to as group 1A in 16 of 25(64%), group 2 in 6 of 25 (24%), and group 3 in 3 of 25 (12%), no strains were found in group 1B and group 1C in patients with Hirschsprung’s disease. Compared with the results of Lurain [11] , Bale [12] , and Murayama et al[22], the predominant genotype of UL144 in our studied strains was group 1A (64%). The discrepancy may be due to the difference in the studied population. A number of studies have attempted to correlate c y t o m e g a l o v i r u s g e n e t i c va r i a n t s w i t h s p e c i f i c manifestations of cytomegalovirus disease or sites of infection [7,9,23]. However, no definitive association has been established yet. Whether the outcomes of congenital cytomegalovirus infection and tissue tropism are related to UL144 genotype is still controversial. No association has been found between the gene variation and particular

diseases[11,12,24]. However, Ara-Boger et al[13] showed that the relatively rare UL144 genotypes A and C, but not the most common genotype B, may be associated with the most serious outcome of cytomegalovirus congenital infection. Genotypes A-C correspond to group 1A, group 3 and group 2 in Lurains’ study, respectively. It was reported that polymorphism of gB genotypes is related to geographic and demographic composition[25]. However, the results of our previous study[26] are consistent with the reported data. Nonetheless, the proportion of UL144 group 1A is higher in strains from infants with congenital cytomegalovirus infection[13,27], suggesting that cytomegalovirus strains in group 1A have a much stronger pathogenicity in congenital cytomegalovirus infection. In the current study, the predominant UL144 genotype in intestinal tissue samples from infants with Hirschsprung’s disease was group 1A (64%), but the comparison of distribution of gene composition of strains between infants with Hirschsprung’s disease and control group did not reach statistical significance (P = 0.393), suggesting www.wjgnet.com

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that UL144 gene diversities are not related to the particular disease. With respect to clinical features, strains from 18 infants with Hirschsprung’s disease of sigmoid rectal type were scattered in all UL144 genotypes. Since the number of samples was small, the association has not been ascertained between UL144 genotype and clinical phenotype of HD. Moreover, the identical sequences of cytomegalovirus UL144 gene from the same patients (M25 and U296, M27 and U298) suggested that DNA sequences in urine samples can reflect cytomegalovirus UL144 genotype of infected organs. In the current study, analysis of posttranslational modification sites of predicted UL144 protein showed that most of the important motifs, including TNFR, MYR, PKC, and ASN were conserved in all strains, suggesting that it is necessary for these motifs to maintain the HCMV biological functions. However, only the strains in group 2 had two MYR motifs in 19-24 and 53-59 amino acid residues, meanwhile, only the strains in group 3 had a specific ACP but MYR was absent motif in their UL144 putative protein in comparison with those in the other two groups. Further study is required on the relationship between these changes and cytomegalovirus infection, replication and clinical sequelae.

ACKNOWLEDGMENTS The authors thank Lian-Ying Wang and Ya-Luo Dong for their help in obtaining clinical samples and statistic analysis.

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REFERENCES

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Torok-Storb B, Boeckh M, Hoy C, Leisenring W, Myerson D, Gooley T. Association of specific cytomegalovirus genotypes with death from myelosuppression after marrow transplantation. Blood 1997; 90: 2097-2102 Rosen HR, Corless CL, Rabkin J, Chou S. Association of cytomegalovirus genotype with graft rejection after liver transplantation. Transplantation 1998; 66: 1627-1631 Shepp DH, Match ME, Ashraf AB, Lipson SM, Millan C, Pergolizzi R. Cytomegalovirus glycoprotein B groups associated with retinitis in AIDS. J Infect Dis 1996; 174: 184-187 Klein M, Schoppel K, Amvrossiadis N, Mach M. Strainspecific neutralization of human cytomegalovirus isolates by human sera. J Virol 1999; 73: 878-886 Rasmussen L, Hong C, Zipeto D, Morris S, Sherman D, Chou S, Miner R, Drew WL, Wolitz R, Dowling A, Warford A, Merigan TC. Cytomegalovirus gB genotype distribution differs in human immunodeficiency virus-infected patients and immunocompromised allograft recipients. J Infect Dis 1997; 175: 179-184 Bongarts A, Von Laer D, Vogelberg C, Ebert K, Van Lunzen J, Garweg J, Vaith P, Hufert FT, Haller O, Meyer-Konig U. Glycoprotein B genotype of human cytomegalovirus: distribution in HIV-infected patients. Scand J Infect Dis 1996; 28: 447-449 Fries BC, Chou S, Boeckh M, Torok-Storb B. Frequency distribution of cytomegalovirus envelope glycoprotein genotypes in bone marrow transplant recipients. J Infect Dis 1994; 169: 769-774

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Peek R, Verbraak F, Bruinenberg M, Van der Lelij A, Van den Horn G, Kijlstra A. Cytomegalovirus glycoprotein B genotyping in ocular fluids and blood of AIDS patients with cytomegalovirus retinitis. Invest Ophthalmol Vis Sci 1998; 39: 1183-1187 Chern KC, Chandler DB, Martin DF, Kuppermann BD, Wolitz RA, Margolis TP. Glycoprotein B subtyping of cytomegalovirus (CMV) in the vitreous of patients with AIDS and CMV retinitis. J Infect Dis 1998; 178: 1149-1153 Benedict CA, Butrovich KD, Lurain NS, Corbeil J, Rooney I, Schneider P, Tschopp J, Ware CF. Cutting edge: a novel viral TNF receptor superfamily member in virulent strains of human cytomegalovirus. J Immunol 1999; 162: 6967-6970 Lurain NS, Kapell KS, Huang DD, Short JA, Paintsil J, Winkfield E, Benedict CA, Ware CF, Bremer JW. Human cytomegalovirus UL144 open reading frame: sequence hypervariability in low-passage clinical isolates. J Virol 1999; 73: 10040-10050 Bale JF Jr, Petheram SJ, Robertson M, Murph JR, Demmler G. Human cytomegalovirus a sequence and UL144 variability in strains from infected children. J Med Virol 2001; 65: 90-96 Arav-Boger R, Willoughby RE, Pass RF, Zong JC, Jang WJ, Alcendor D, Hayward GS. Polymorphisms of the cytomegalovirus (CMV)-encoded tumor necrosis factor-alpha and beta-chemokine receptors in congenital CMV disease. J Infect Dis 2002; 186: 1057-1064 Tam PK, Quint WG, van Velzen D. Hirschsprung's disease: a viral etiology? Pediatr Pathol 1992; 12: 807-810 Dechelotte PJ, Mulliez NM, Bouvier RJ, Vanlieferinghen PC, Lemery DJ. Pseudo-meconium ileus due to cytomegalovirus infection: a report of three cases. Pediatr Pathol 1992; 12: 73-82 Dimmick JE, Bove KE. Cytomegalovirus infection of the bowel in infancy: pathogenetic and diagnostic significance. Pediatr Pathol 1984; 2: 95-102 Asabe K, Nagasaki A, Sato K, Nakayama M. Intestinal obstruction caused by congenital cytomegalovirus infection: report of a case. Surg Today 2003; 33: 764-767 He R, Liu LQ, Lu SM, Ruan Q. Quantitative detection of HUMAN CYTOMEGALOVIRUS-DNA from urine in infants by FQ-PCR. Zhonghua Xiaoer neike Zazhi 2001; 12: 739-742. In China. Martucciello G, Ceccherini I, Lerone M, Jasonni V. Pathogenesis of Hirschsprung's disease. J Pediatr Surg 2000; 35: 1017-1025 Wang Lianying, Li Zheng, Ruan Qiang. Congenital megacolon and cytomegalovirus infection in infants. Zhonghua Xiaoer Waike Zazhi 1996; 17: 3-5 Chen Leiling, Hu Tingzhe, Liu Jihong. Human cytomegalovirus infection and Hirschsprung’s disease. Zhonghua Xiaoer Waike Zazhi 2002; 23: 225-227 Murayama T, Takegoshi M, Tanuma J, Eizuru Y. Analysis of human cytomegalovirus UL144 variability in low-passage clinical isolates in Japan. Intervirology 2005; 48: 201-206 Pignatelli S, Dal Monte P, Rossini G, Landini MP. Genetic polymorphisms among human cytomegalovirus (HCMV) wild-type strains. Rev Med Virol 2004; 14: 383-410 Picone O, Costa JM, Chaix ML, Ville Y, Rouzioux C, LeruezVille M. Human cytomegalovirus UL144 gene polymorphisms in congenital infections. J Clin Microbiol 2005; 43: 25-29 Zipeto D, Hong C, Gerna G, Zavattoni M, Katzenstein D, Merigan TC, Rasmussen L. Geographic and demographic differences in the frequency of human cytomegalovirus gB genotypes 1-4 in immunocompromised patients. AIDS Res Hum Retroviruses 1998; 14: 533-536 He R, Ruan Q, Xia C, Liu LQ, Lu SM, Lu Y, Qi Y, Ma YP, Liu Q, Ji YH. Sequence variability of human cytomegalovirus UL144 open reading frame in low-passage clinical isolates. Chin Med Sci J 2004; 19: 293-297 Tanaka K, Numazaki K, Tsutsumi H. Human cytomegalovirus genetic variability in strains isolated from Japanese children during 1983-2003. J Med Virol 2005; 76: 356-360 S- Editor Liu Y L- Editor Wang XL

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Association of high expression in rat gastric mucosal heat shock protein 70 induced by moxibustion pretreatment with protection against stress injury Xiao-Rong Chang, La Peng, Shou-Xiang Yi, Yan Peng, Jie Yan Xiao-Rong Chang, Shou-Xiang Yi, Yan Peng, Jie Yan, College of Acupuncture and Massage, Hunan University of Traditional Chinese Medicine, Changsha 410007, Hunan Province, China La Peng, Hunan Yi-Er-Kang Healthcare Limited Company, Changsha 41001, Hunan Province, China Supported by National Natural Science Foundation, No. 30572310, and Hunan Natural Science Foundation, No. 05JJ 4008 Correspondence to: Xiao-Rong Chang, Professor, Hunan University of Traditional Chinese Medicine, College of Acupuncture and Massage, 113 Shaoshan Mid-Road, Changsha 410007, Hunan Province, China. [email protected] Telephone: +86-735-5381163 Fax: +86-735-5557891 Received: 2007-03-07 Accepted: 2007-03-31

Abstract AIM: To study the effect of moxibustion on Zusanli or Liangmeng point on gastric mucosa injury in stressinduced ulcer rats and its correlation with the expression of heat shock protein 70 (HSP70). METHODS: Sixty healthy SD rats (30 males, 30 females) were divided into control group, injury model group, Zushanli point group, Liangmeng point group. Stress gastric ulcer model was induced by binding cold stress method. Gastric mucosa ulcer injury (UI) index was calculated by Guth method. Gastric mucosa blood flow (GMBF) was recorded with a biological signal analyzer. Protein content and gene expression in gastric mucosal HSP70 were detected by immunohistochemistry (IHC) and reverse transcription polymerase chain reaction (RT-PCR). Thiobarbital method was used to determine malondialdehyde (MDA) content. Gastric mucosal endothelin (ET) and prostaglandin E 2 (PGE 2 ) were analyzed by radioimmunoassay. RESULTS: High gastric mucosal UI index, high HSP70 expression, low GMBF and PGF2, elevated MDA and ET were observed in gastric mucosa of rats subjected to cold stress. Moxibustion on Zusanli or Liangmeng point decreased rat gastric mucosal UI index, MDA and ET. Conversely, the expression of HSP70, GMBF, and PGE2 was elevated in gastric mucosa after pretreatment with moxibustion on Zusanli or Liangmeng point. The observed parameters were significantly different between Zusanli and Liangmeng points. CONCLUSION: Pretreatment with moxibustion on

Zusanli or Liangmeng point protects gastric mucosa against stress injury. This protection is associated with the higher expression of HSP70 mRNA and protein, leading to release of PGE2 and inhibition of MDA and ET, impairment of gastric mucosal index. © 2007 WJG . All rights reserved.

Key words: Moxibustion; Zusanli; Liangmeng; Stress ulcer; Gastric mucosa protection; Heat shock protein 70; Gastric mucosal blood flow; Prostaglandin E 2 ; Malondialdehyde; Endothelin Chang XR, Peng L, Yi SX, Peng Y, Yan J. Association of high expression in rat gastric mucosal heat shock protein 70 induced by moxibustion pretreatment with protection against stress injury. World J Gastroenterol 2007; 13(32): 4355-4359 http://www.wjgnet.com/1007-9327/13/4355.asp

INTRODUCTION Recent studies indicate that moxibustion can protect gastric mucosa against injury [1-3]. However, its exact mechanism remains unclear. Several strategies have been proposed recently. Heat shock protein (HSP), the cellular selfprotective defense factor, is one of the hot spots. HSP70 is one of the important HSPs in gastric mucosal protection[4]. To explore the involvement of HSP70 in gastric mucosal protection, we studied the effects of moxibustion pretreatment on the expression of HSP70 mRNA expression and protein with the purpose of clarifying its endogenous protective mechanism. We believe that the experiment results provide a useful tool to induce HSPs via traditional Chinese medicine.

MATERIALS AND METHODS Materials Sixty healthy SD rats (30 males, 30 females), weighing 200-250 g, were provided by College of Animal Sciences & Technology, Hunan Agriculture University. Endothelin (ET) and malondialdehyde (MDA) detection kits were provided by Radioimmunoassay Institute of Scientific and Technological Development Center of PLA General www.wjgnet.com

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Hospital. Other regents were of analytical purity. Rabbit anti-rat HSP70 affinity-purified antibody and DEPC were purchased from Wuhan Boshide Company. SP detection kit and DAB yellowbrown color detection kit were purchased from Beijing Zhongshan Golden Bridge Biotech Limited Company. Trizol reagent kit was from Invitrogen (USA). AMV reverse transcription enzyme, RNasin, dNTPs, Taq DNA polymerase, 100 bp DNA ladder were from Promega (USA). PGE2 detection kit was from Institute of Blood, Suzhou University. Aizhu was purchased from Suzhu Oriental Aiyong Institute (“Shenjiu 300 Jiu”, Oriental type 1). Experiment design The patients were divided into control group (Ⅰ), injury model group (Ⅱ), Zushanli point group (Ⅲ), Liangmeng point group (Ⅳ). The control point[5] was located 1 cm from Liangmeng point and no specific point was located between knee joint and Zusanli. Rats in groups Ⅰ, Ⅱ were not treated with moxibustion, whereas rats in groups Ⅲ , Ⅳ were treated with moxibustion (4 times a day, 30 min each time, for 8 d) on the points located at one side. After the hair was removed, AiZhu was pasted on the points located on both sides (4 times a day, 30 min each time, for 8 d). Seven days after moxibustion pretreatment, acute stress gastric ulcer model was established as previously described[6]. Rats in groups Ⅱ, Ⅲ, Ⅳ were treated for six days with no access to food and water for 24 h on the last day. These rats were submerged into 20℃ water at the level of bottom of breastbone and released 10 h after submersion. All animals were anesthetized with 10% urethane intraperitoneal injection 24 h after establishment of stress model. Five rats in each group were used for immunohistochemical analysis. The pylorus portion of stomach was ligated and 3 mL of 4% formaldehyde was infused into the stomach from esophagus. The cardiac portion of stomach was ligated after the needle was pulled out. The esophagus and duodenum were cut off from the ligated sites. The stomach was extracted and dissected along the gastric greater curvature after 10 min. The stomach content was washed out with normal saline and fixed with 4% formaldehyde for 24 h. Paraffin sections were prepared. The other rats were anesthetized and dissected as described above. A small piece of mucosa on the gastric sinas portion was prepared (about 50 mg), washed three times with 0.1% DEPC solution, and stored in liquid nitrogen. The other portions of gastric mucosa were washed with ice cold normal saline. Gastric mucosa ulcer injury (UI) index was calculated and the gastric mucosa was weighed with an analytical balance. A certain amount of normal saline was added to the 1.5 mL/400 mg concentration. Gastric mucosal tissue homogenate was prepared with a glassglass homogenizer and centrifuged at 35 000 r/min for 15 min at 4℃. Supernatant was extracted and stored at -20℃ for assay. According to GUTH method, the summary of gastric mucosal impairment site length was calculated as UI index and expressed as millimeter. The standard score was: www.wjgnet.com

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Figure 1 RNA quality analysis.

1 point: < or = 1 mm; 2 points: 1 mm < damage < or = 2 mm; 3 points: 2 mm < damage < or = 3 mm; 4 points: 3 mm < damage < or = 4 mm; 5 points: > 4 mm. The UI index should be doubled if the width of injury was more than 2 mm. HSP immunohistochemical assay Paraffin sections (4 μm thick) were prepared. Tissue sections were deparaffinized and hydrated in xylenes and graded alcohol. The sections were incubated with primary anti-HSP70 diluted in buffer. PBS was used as negative control. The positive results were analyzed with the MIAS medical imaging analytical system. Five scopes on each section were quantified under microscope. The average density of each section was calculated. HSP70 mRNA expression RT-PCT technique was used to analyze the expression of HSP70 mRNA in rat gastric mucosa. Total RNA was extracted from rat gastric mucosa with TRIzol method. Ten μL of RNA sample was taken to analyze the purity and completeness of RNA (Figure 1). Three mol/L and 1/10 volume of NaAc (pH 5.2) and ethanol were dehydrated 3 times and stored at -20℃ or -70℃. Three μg of total RNA was used for RT-PCR (20 μL total volume). Three μg RNA, 0.5 μg oligo (dT), 18 primers, 20 U RNasin, 10 mmol/L dNTPs, 5 × RT buffer, and 10 U AMV reverse transcriptase were mixed and heated at 42℃ for 1 h. The cDNA product was used as PCR reaction source of gene for analysis. The primeres of rat HSP70 were synthesized by Invitrogen Biotech Company. The routine PCR reaction (25 μL total volume) included: 2.5 μL 10 × PCR buffer, 1.5 mmol/L MgCl2, 200 μmol/L dNTPs, 2 μL cDNA, 0.1 μmol/L specific primers, 2 U Taq DNA polymer, and paraffin oil for cover. The HSP70 cDNA in PCR products was 268 bp. The reaction conditions were set at 94℃ for 2 min, at 94℃ for 30 s, at 56℃ for 30 s, at 72℃ for 30 s, for 35 cycles. The cDNA in PCR products was 426 bp. The reaction conditions were set at 4℃ for 2 min, at 94℃ for 30 s, at 56℃ for 30 s, at 72℃ 30 s, for 25 cycles. Ten μL of HSP70 cDNA and 10 μL of GAPDH were loaded onto 1.5 % agarose gel for electrophoresis. The absorbance (A) of gel electrophoresis products was scanned and read. The ratio of HSP70 cDNA/ GAPDH cDNA was used for observed parameters. GMBF Rats were anesthetized and operated along the mid-line of abdomen to expose the stomach. Laser Doppler blood flow meter and miniature surface probes (Biopac Inc. USA) were used to record the blood flow. The acquired signal was converted to blood perfusion unit (BPU) and input into a computer. The curve was recorded with

Chang XR et al . Moxibustion-induced HSP70 expression

UI 12.0 ± 5.9d 26.8 ± 9.8b 14.1 ± 5.4 26.2 ± 7.7b

Ⅰ Ⅱ Ⅲ Ⅳ

ET (ng/L)

PGE2 (ng/L)

173 ± 70c 345 ± 285a 1485 ± 69 196 ± 124

1108 ± 486c 6227 ± 169a 1064 ± 437 693 ± 426a

a

P < 0.05, bP < 0.01 vs group Ⅲ; cP < 0.05, dP < 0.01 vs group Ⅱ.

bp 500 400

←400 bp Ⅰ

Ⅲ

Ⅳ

B bp 500 400

← 426 bp Ⅰ

Table 2 Efects of moxibustion on gastric mucosal GMBF, MDA and HSP70 (mean ± SD)

Ⅱ

← ←

Groups

A

← ←

Table 1 Effects of moxibustion on UI,ET and PGE2 of gastric mucosa (mean ± SD)

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Ⅱ

Ⅲ

Ⅳ

Figure 2 HSP70 (A) and GAPDH cDNA (B) in gastric mucosa.

Groups GMBF (mL/min) MDA (mol/L) HSP70 (A) HSP70mRNA (A) Ⅰ Ⅱ Ⅲ Ⅳ

364 ± 169d 139 ± 33a 280 ± 173 142 ± 58a

2925 ± 625c 3906 ± 768a 2586 ± 252 3464 ± 1502a

0.021 ± 0.010c 0.077 ± 0.057b 0.133 ± 0.035 0.059 ± 0.038a

0.616 ± 0.023b,d 0.669 ± 0.007b 0.799 ± 0.042 0.733 ± 0.025b,d

a

P < 0.05, bP < 0.01 vs group Ⅲ; cP < 0.05, dP < 0.01 vs group Ⅱ.

Acqknowledge v3.5 software. A 2 cm opening cut was made at the greater curvature of stomach. Laser probes were inserted into the sinus, bottom, greater and lesser curvature of stomach. The data were obtained only after the measurement curve on the display became stable. Measurement was performed three times (15 s for each time) on each point for every rat. The average value was designated as the 10 s stabling curve. The final observed parameters were obtained from the average value of four different points. Measurement of ET, PGE2, and MDAin gastric mucosa Radioimmunoassay was performed to detect ET and PGE2 in gastric mucosa according to the instructions of ready-use reagent from the Radioimmunoassay Institute of Scientific and Technological Development Center of PLA General Hospital, and Institute of Blood, Suzhou University, China. Thiobarbital was used to determine malondialdehyde (MDA) following the instructions from Radioimmunoassay Institute of Scientific and Technological Development Center of PLA General Hospital. Statistical analysis The data were analyzed with SPSS 11.5 software and one way analysis of variance (ANOVA), and expressed as mean ± SD. P < 0.05 was considered statistically significant.

RESULTS UI index, ET and PGE2 in gastric mucosa As shown in Table 1, UI index was significantly lower in group Ⅲ than in groups Ⅱ, Ⅳ (P < 0.01) and no difference was observed between groups Ⅲ andⅠ (P > 0.05). UI Index was higher in group Ⅱ than in groupⅠ(P < 0.01). ET was markedly lower in group Ⅲ than in group Ⅱ (P < 0.05) and there was no difference between groups Ⅲ andⅠ (P > 0.05). ET was elevated in group Ⅱ after stress stimulation (P < 0.05 vs groupⅠ). PGE2 was significantly

increased in group Ⅲ after moxibustion (P < 0.05 vs group Ⅱ). No difference was observed between groups Ⅲ andⅠ(P > 0.05). PGE2 was lower in group Ⅱ than in groupⅠ(P < 0.05). GMBF and MDA in gastric mucosa As shown in Table 2, MDA was significantly lower in group Ⅲ than in group Ⅱ (P < 0.05) and no difference was observed between groups Ⅲ andⅠ(P > 0.05). MDA was higher in group Ⅱ than in groupⅠ(P < 0.05). Moxibustion also improved GMBF (P < 0.05 vs group Ⅱ) and no difference was observed between groups Ⅲ andⅠ. Cold stress stimulation alone attenuated GMBF (P < 0.01 vs groupⅠ). HSP70 and HSPmRNA expression in gastric mucosa The immunohistochemical results indicated that HSP70 positive portion (yellow-brown color) was detected in cytoplasm, mostly in sinus of stomach. Moxibustion on points significantly enhanced the expression of HSP70 (P < 0.01 vs group Ⅱ; P < 0.05 vs group Ⅳ). No difference was observed between groups Ⅲ andⅠ) (P > 0.05). Interestingly, HSP70 expression was markedly higher in group Ⅱ than in groupⅠ(P < 0.05) (Table 2). As shown in Figure 2, HSP70 gene expression was significantly elevated in group Ⅲ (P < 0.01 vs groupsⅠ, Ⅱ, and Ⅳ). Correspondingly, HSP70 gene expression also enhanced in group Ⅱ (P < 0.01 vs groupⅠ). A panel of representative photomicrographs showed HSP70 staining in gastric mucosa from rats in different groups (Figure 3 A-D).

DISCUSSION HSPs are a group of highly conserved stress proteins and play an important role in maintaining body self-stability. The main functions of HSPs are to promote cellular tolerance against stress factors, to maintain cellular normal physiological function, and to increase defense against and adaptation of cells to deadly stimulation [7]. Overexpression of HSP70 has been observed in rats with acute and chronic gastric ulcer, chronic atrophic gastritis, and in patients with gastric cancer. The most leading over-expression of HSP70 has been found in apparent pathological portion[8-10]. Over-expression of HSP70 promotes ulcer healing by increasing gastric mucosal blood flow, and cell multiplication[11-13]. Expression of HSP70 can also be inwww.wjgnet.com

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A

DAB × 400

B

DAB × 400

C

DAB × 400

D

DAB × 400

duced with heat pretreatment, long term stimulation with low ethanol, and long term administration of aspirin. The expression of HSP70 is associated with adaptive cellular protection-tolerances to stimulation of hyperthermia, high concentration of alcohol, and high doses of aspirin[14-16]. Moxibustion acts as a kind of physiological mild stimulator to induce HSP70 production. This effect connects its therapeutic application to immunogenic property for activation of immunological system[17,18]. Our study indicates that cold stress stimulation could increase HSP70 protein and gene expressions in rat gastric mucosa (P < 0.05 vs control group), suggesting that pretreatment with moxibustion can significantly enhance the expressions of HSP70 protein (P < 0.01 vs stress model group). Stress gastric injury is attributed to some factors that decrease the function of gastric mucosa defense system and relatively increase the function of impairment system. Endogenous prostaglandins are a kind of protective substances in gastric mucosa and can increase secretion of gastric media and biocarbonate, promoting surface hydrophobity and gastric mucosal blood flow. Conversely, endothelin, a potent vasoconstrictor, constricts gastric mucosal blood flow leading to dysfunction of gastric circulation and damage to gastric mucosa[19]. Our results showed higher PGE2 and lower ET in group Ⅲ (moxibustion, P < 0.05), which were related to the improvement of gastric mucosal damage (P < 0.01). Oxidative stress plays an important role in stress-induced gastric mucosal injury and involves cellular apoptosis. It was reported that MDA increases 3 h after stress and reaches its peak within 6-12 h[20]. Free radical generation increases after long time exposure to stress. The free radical scavenger system is insufficient to remove the newly generated free radicals and leads to damage to gastric mucosa[21]. Electro-acupunture on Zusanli point decreases plasma MDA and improves oxygen free radical www.wjgnet.com

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Figure 3 Positive expression of HSP70 in blank rat gastric parietal cell cytoplasm (A), HSP70 mRNA expression in cytoplasm of model rat stomach and gastric mucosa (B), positive expression of HSP70 and nuclear expression in cytoplasm (C), positive expression of HSP70 in a small amount of cytoplasm (D).

metabolism. These effects are related to its protective effects against stress[22]. In our study, gastric mucosal MDA was higher in group Ⅱ than in groupsⅠand Ⅲ (P < 0.05), indicating that moxibustion decreases gastric mucosal MDA and protects gastric mucosa against oxidative injury. Channel connection with Zang and Fu organs (viscera) is one of the important theories in traditional Chinese medicine. “Twelve channels connect with arms and body surface outwardly and associate with Zang and Fu organs inwardly”. This theory has been described in detail in . Foot Yangming Wei Channel is a channel full of Qi and blood, and participates in the circulation of 14 channels and provides the sources for Qi and blood. It is one of the essential elements of Channel theory. Stomach (Wei) is the “field of water and food” and converts ingestant into nutrients and blood. Spleen and stomach (PiWei) are the essential parts for growth and development. Zusanli point, a co-point in bottom of stomach (Wei) and Wei Channel, is a first selection point for stomach disease. Liangmeng point is another point in foot Zusanli stomach channel. The present study indicated that moxibustion pretreatment on Zusanli and Liangmeng points increased HSP70 protein and gene expressions and PGE2 in gastric mucosa (P < 0.05 or 0.01 vs moxibustion pretreatment on no specific point). Moxibustion reduced damage to gastric mucosa (P < 0.01) and the enhanced MDA induced by gastric mucosal stress and exerted antioxidation, suggesting that Zusanli and Liangmeng points protect gastric mucosa against stress injury by a point-specific mechanism. These results provide strong evidence for the treatment of digestive illness with acupuncture method. Moxibustion can prevent and cure diseases through mild warm stimulation and point-channel action. Modern clinical and experimental studies have confirmed that

Chang XR et al . Moxibustion-induced HSP70 expression

moxibustion has analgesic effects and improves blood circulation. Metabolism dysfunction, activity of Zang and Fu organs, and immunological function can also be improved with moxibustion[23]. In summary, moxibustion protects gastric mucosa against stress injury, which is related to the induction of HSP70 protein and mRNA expression. These results provide an alternative pathway to trigger endogenous protective substances with acupuncture. Effective induction of HSPs with means of traditional Chinese medicine is a useful tool to provoke endogenous defense system for prevention and treatment of diseases. However, whether moxibustion-induced HSP70 expression can protect other tissues or organs against stress injury remains to be further studied.

REFERENCES 1

2

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4 5 6 7 8 9 10

Hu G, Chen H, Hou Y, He J, Cheng Z, Wang R. A study on the clinical effect and immunological mechanism in the treatment of Hashimoto's thyroiditis by moxibustion. J Tradit Chin Med 1993; 13: 14-18 Waldum HL, Marvik R, Gronbech JE, Sandvik AK, Aase S. Oxyntic lesions may be provoked in the rat both by the process of acid secretion and also by gastric acidity. Aliment Pharmacol Ther 2000; 14: 135-141 Liu J, Yu RC, Tang WJ. Influence of combined therapy of guben yiliu III, moxibustion and chemotherapy on immune function and blood coagulation mechanism in patients with mid-late stage mailgnant tumor. Zhongguo Zhongxiyi Jiehe Zazhi 2002; 22: 104-106 Welch WJ. Mammalian stress response: cell physiology, structure/function of stress proteins, and implications for medicine and disease. Physiol Rev 1992; 72: 1063-1081 Li ZR. Experimental Acupuncture. Beijing: Chinese Medicine Press, 2003; 327-329 Kwiecien S, Brzozowski T, Konturek SJ. Effects of reactive oxygen species action on gastric mucosa in various models of mucosal injury. J Physiol Pharmacol 2002; 53: 39-50 Takumida M, Anniko M. Heat shock protein 70 delays gentamicin-induced vestibular hair cell death. Acta Otolaryngol 2005; 125: 23-28 Shichijo K, Ihara M, Matsuu M, Ito M, Okumura Y, Sekine I. Overexpression of heat shock protein 70 in stomach of stressinduced gastric ulcer-resistant rats. Dig Dis Sci 2003; 48: 340-348 Shen X. Effects of cyclooxygenase-2 on formation and healing of acetic acid-induced gastric ulcer in rats. Zhonghua Yixue Zazhi 2001; 81: 1380-1383 Svestka T, Krechler T, Bruha R, Jablonska M. [Protective mechanism of the gastric mucosa. Cas Lek Cesk 2005; 144 Suppl

4359 1: 40-43 Shichijo K, Ihara M, Matsuu M, Ito M, Okumura Y, Sekine I. Overexpression of heat shock protein 70 in stomach of stress-induced gastric ulcer-resistant rats. Dig Dis Sci 2003; 48: 340-348 12 T s u k i m i Y , N a k a i H , I t o h S , A m a g a s e K , O k a b e S . Involvement of heat shock proteins in the healing of acetic acid-induced gastric ulcers in rats. J Physiol Pharmacol 2001; 52: 391-406 13 Isomoto H, Oka M, Yano Y, Kanazawa Y, Soda H, Terada R, Yasutake T, Nakayama T, Shikuwa S, Takeshima F, Udono H, Murata I, Ohtsuka K, Kohno S. Expression of heat shock protein (Hsp) 70 and Hsp 40 in gastric cancer. Cancer Lett 2003; 198: 219-228 14 Al Moutaery AR. Protective effect of ketoconazole against experimentally induced gastric ulcers in rats. Res Commun Mol Pathol Pharmacol 2003; 113-114: 5-23 15 Tsukimi Y, Okabe S. Recent advances in gastrointestinal pathophysiology: role of heat shock proteins in mucosal defense and ulcer healing. Biol Pharm Bull 2001; 24: 1-9 16 Jin M, Otaka M, Okuyama A, Itoh S, Otani S, Odashima M, Iwabuchi A, Konishi N, Wada I, Pacheco I, Itoh H, Tashima Y, Masamune O, Watanabe S. Association of 72-kDa heat shock protein expression with adaptation to aspirin in rat gastric mucosa. Dig Dis Sci 1999; 44: 1401-1407 17 Nikaido H, Tsunoda H, Nishimura Y, Kirino T, Tanaka T. Potential role for heat shock protein 72 in antagonizing cerebral vasospasm after rat subarachnoid hemorrhage. Circulation 2004; 110: 1839-1846 18 Kobayashi K. Induction of heat-shock protein (hsp) by moxibustion. Am J Chin Med 1995; 23: 327-330 19 Li ZK. Gastric mucosa injury and protection- basic and clinical study. Shanghai: Scientific and Technological Publishing House, 2004: 255-269 20 Chung H, Kim E, Lee DH, Seo S, Ju S, Lee D, Kim H, Park S. Ghrelin inhibits apoptosis in hypothalamic neuronal cells during oxygen-glucose deprivation. Endocrinology 2007; 148: 148-159 21 Kwiecien S, Brzozowski T, Konturek PC, Pawlik MW, Pawlik WW, Kwiecien N, Konturek SJ. Gastroprotection by pentoxyfilline against stress-induced gastric damage. Role of lipid peroxidation, antioxidizing enzymes and proinflammatory cytokines. J Physiol Pharmacol 2004; 55: 337-355 22 Katsuyama M, Fan C, Arakawa N, Nishinaka T, Miyagishi M, Taira K, Yabe-Nishimura C. Essential role of ATF-1 in induction of NOX1, a catalytic subunit of NADPH oxidase: involvement of mitochondrial respiratory chain. Biochem J 2005; 386: 255-261 23 Meng Z, Zou Y, Luo E, Zou L. The development of clinical application of the rejuvenator and a study of its mechanism for the treatment of functional erectile dysfunction. Shengwu Yixue Gongchengxue Zazhi 2001; 18: 658-660 11

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World J Gastroenterol 2007 August 28; 13(32): 4360-4364 World Journal of Gastroenterology ISSN 1007-9327 © 2007 WJG. All rights reserved.

RAPID COMMUNICATION

Effects of body-resistance strengthening and tumorsuppressing granules on immune adhesion function of red blood cells and expression of metastasis protein CD44 in tumor cells of patients with esophageal carcinoma Jian-Xiong Zhao, Xue-Feng Li, Xue-Xi Wang Jian-Xiong Zhao, Xue-Feng Li, Xue-Xi Wang, Institute of Combined Western and Chinese Traditional Medicine, Lanzhou University Medical School, Lanzhou 730000, Gansu Province, China Supported by the Major Science and Technology Program of Ministry of Education, China, No. 01128 Correspondence to: Jian-Xiong Zhao, MD, Institute of Combined Western and Chinese Traditional Medicine, Lanzhou University Medical School, 99 Donggangxi Road, Lanzhou 730000, Gansu Province, China. [email protected] Telephone: +86-931-8915184 Received: 2007-01-23 Accepted: 2007-03-21

Abstract AIM: To investigate the effects of Fuzheng Yiliu granules (body-resistance strengthening and tumor-suppressing granules) in patients with esophageal carcinoma. METHODS: We compared the immune adherent properties of red blood cells (RBCs), the expression of metastasis protein CD44, and the metastasis inhibition factor nm23, in esophageal carcinoma tumor cells of patients before and after radiotherapy in the presence and absence of orally administered Fuzheng Yiliu granules. Sixty-three hospitalized patients with esophageal carcinoma were treated with standard radiotherapy and randomly divided into treatment group (n = 30) treated with both radiotherapy and Fuzheng Yiliu granules and control group (n = 33) given radiotherapy only. Blood samples and tumor tissue were obtained before and after 21 d of treatment. The rosette rates for complement receptor type 3b (C3bRR) and immune complex receptor (ICRR) on RBCs were measured by erythrocyte immunological methods. Expression of CD44 and nm23 in tumor tissue sections was determined by immunohistochemical staining with monoclonal antibodies CD44v6 ad nm23H-1, respectively. RESULTS: The positivity of RBC-C3bRR before and after 21 d of treatment increased from 7.78% ± 1.59% to 10.03% ± 2.01% in the double treatment group, while it changed only slightly from 7.18% ± 1.29% to 7.46% ± 1.12% in the radiotherapy group. The positive rate for RBC-ICRR decreased from 37.68% ± 2.51% to 22.55% ± 1.65% after the double treatment, and from www.wjgnet.com

37.28% ± 2.41% to 24.69% ± 1.91% in radiotherapy group at the same time points. The difference in erythrocyte immune adherent function between the two + groups was significant (P < 0.01, t -test). The CD44 cases were reduced from 21 (70.00%) to 12 (40.00%) after treatment with Fuzheng Yiliu granules, whereas + the CD44 -cases (69.70%) in the radiotherapy group remained unchanged. The difference between the treatment (40.00%) and control (69.70%) groups was + significant (P < 0.05). Although the nm23 -cases were increased from 4 (13.33%) to 6 (20.00%) in the double treatment group and from 6 (18.18%) to 7 (21.21%) in the radiotherapy group, the difference was not significant (P > 0.05). CONCLUSION: Fuzheng Yiliu granules enhance the immune adhesion function of RBCs and reduce the + number of CD44 -cells in esophageal carcinoma patients, suggesting a potential role of these Chinese herbals in suppression of invasion and metastasis of malignant cells. However, this anti-metastatic effect has yet to be validated in vivo . © 2007 WJG . All rights reserved.

Key words: Fuzheng Yiliu granule; RBC immune function; CD44; nm23; Esophageal carcinoma; Metastasis; Randomized controlled clinical trial Zhao JX, Li XF, Wang XX. Effects of body-resistance strengthening and tumor-suppressing granules on immune adhesion function of red blood cells and expression of metastasis protein CD44 in tumor cells of patients with esophageal carcinoma. World J Gastroenterol 2007; 13(32): 4360-4364

http://www.wjgnet.com/1007-9327/13/4360.asp

INTRODUCTION Fuzheng-Yiliu granules (body-resistance strengthening and tumor-suppressing granules) were made from Gansuproduced herbals including Radix hedysari, radix Radix angelicae sinesis, Rhizoma zekoariae and Radix patriniae at a ratio of 3:1:1:3 by boiling, concentrating and adding an excipient. According to our clinical observations, the

Zhao JX et al . Fuzheng-Yiliu and metastasis

combination of these four herbals at the ratios mentioned may suppress tumor cell growth and concomitantly enhance host immune function. Our previous study of Fuzheng Yiliu granules in an animal model indicated that Fuzheng Yiliu treatment inhibits S180 tumor cell growth in mice associated with suppression of telomerase activity and cell transition from G0/G1 to S phase[1]. Our in vivo studies using tumor tissues derived from cancer patients treated with Fuzheng Yiliu granules have confirmed its suppressive role in cell proliferation and transition from G0/G1 to S phase, further demonstrating its ability to upregulate nuclear transcription factor NF k B and apoptosis in breast and esophageal-gastric carcinomas[2,3]. Very recently, we showed that Fuzheng Yiliu granules can lower the expression of metastasis protein CD46 in esophageal carcinoma[4]. In this report, we describe the effect of Fuzheng Yiliu treatment on the immune adhesion function of red blood cells including RBC-C3bRR and RBC-ICRR as well as expression changes of the metastasis protein CD46 and the metastasis inhibition factor nm23 in tumor tissues.

MATERIALS AND METHODS Patients In this study, 63 in-hospital patients enrolled from Wuwei Oncology Hospital, Gansu Province, included patients with esophageal carcinoma diagnosed by esophagealgastric endoscope and pathology on biopsy, patients who had no previous radiotherapy, and patients who received the complete treatment program. This study was approved by the Institutional Review Board of Wuwei Oncology Hospital. Random groups Sixty-three patients were divided into treatment and control groups using the randomization table. Patients (20 males and 10 females, ranging in age from 45 to 76 years with a mean age of 58.6 ± 7.9 years) in the treatment group were treated with both radiotherapy and Fuzheng Yiliu granules. Patients in the control group (22 males and 11 females, ranging in age from 47 to 70 years with a mean age of 57.3 ± 6.2 years) were treated with radiotherapy alone. These two groups were comparable and had no statistical differences (P > 0.05) in the case number, sex or age. Clinical treatment Patients in the control group were treated with the standard Co60 radiation at a dose of 1.8-2.0 Gy in 3 or 4 fields, 3 cm above and below the tumor lesions, 5 times a week for three weeks (total dose of 50-70 Gy). Patients in the treatment group were also given oral of Fuzheng Yiliu granules, 5.0 g each time, 3 times a day for 21 d. Fuzheng Yiliu granules were developed by the Institute of Combined Western and Chinese Traditional Medicine, Lanzhou University Medical School and manufactured by Lanzhou Foci Pharmacy Company. Five grams (5.0 g) of granules is equivalent to 45 g of original herbs.

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Tumor tissue and blood samples Upon admission to Wuwei Oncology Hospital, patients were subjected to electron esophago-gastroendoscopy for diagnosis and biopsies of 3-4 pieces of tumor tissue. After radiotherapy for both groups, in addition to oral Fuzheng Yiliu granules in the treatment group, the patients were again subjected to electron esophago-gastro endoscopy for diagnosis and biopsies of 3-4 pieces of tumor tissue. Tissues were formalin-fixed and paraffin-embedded for sectioning. Four microns of continuous sections was used for HE staining, pathological diagnosis and immunohistochemical staining. Two milliliters of blood samples drawn into anti-coagulation tubes from each patient before and after 21 d treatment was used to test immune function of red blood cells. RBC-C3bRR and RBC-ICRR Red cell immune adhesion function, RBC-C3bRR and RBC-ICRR, were detected as previously described [5,6]. Yeast cells were obtained from the Blood and Immune Laboratory of Shanghai Hospital, the Second Military Medical University). If one red cell adhered to more than 2 yeast cells, it was counted as one rosette. The percentage of rosettes in 200 red cells was calculated. Immunohistochemistry Antibodies CD44v6 and nm23H-1 and related reagents were purchased from Boshide Company (Wuhan, China). Immunohistochemistry was performed by the avidinbiotin peroxidase complex (ABC) method (Boshide, Wuhan). Briefly, slides were deparaffinized, and rehydrated through xylene and alcohol in Coplin jars. Endogenous peroxidase was blocked with 3% H 2O 2 in phosphatebuffered saline (PBS) for 10 min, and tissues were washed 3 times in ddH2O at room temperature. Microwave oven heat-induced epitope retrieval (HIER) was performed on sections used for CD44v6 antibody, and washed in PBS buffer. Tissue sections for nm23H-1 staining were treated with enzyme buffer from the kit and then washed with ddH2O. The slides were incubated with normal goat serum for 20 min followed by primary antibody (rabbit monoclonal antibody CD44v6 or nm23H-1 with dilutions). Incubation was performed overnight at 4℃. After washed three times, slides were incubated with biotinylated goat anti-rabbit IgG at 31℃ for 20 min. After washed three times, the slides were incubated in SABC solution. Slides were washed 4 times (5 min each time) and then developed with liquid DAB at 31℃ for 20 min, washed twice with H 2 O, and finally counterstained lightly with Mayer’s hematoxyline for 5 s, dehydrated, cleared, and mounted with resinous mounting medium. CD44v6 stained brownyellowish granules mainly on the cell membrane with some cytoplasmic staining. Nm23H-1 stained brown-yellowish granules in cytoplasm. Signal intensity and distribution were [7-9] scored blindly by pathologists as previously described [7-9]: distribution score (DS) was graded as 0: absent; 1: < 10%; 2: 10%-50%; 3: 51%-90%; or 4: > 90%. The intensity score (IS) was graded as IS0: no signal; IS1: weak; IS2: medium; or IS3: strong. The combined total score was determined as a total score (TS) = distribution

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Table 1 Effect of Fuzheng Yiliu granules on RBC immune adhesion function (mean ± SD, %)

b

Group

n

Treatment Control

30 33

RBC-C3bRR Before After treatment treatment 7.78 ± 1.59 10.03 ± 2.01b 7.18 ± 1.29 7.46 ± 1.12

RBC-ICRR Before After treatment treatment 37.68 ± 2.51 22.55 ± 1.65b 37.28 ± 2.41 24.69 ± 1.91

P < 0.01 vs control group after treatment.

Table 2 Effect of Fuzheng Yiliu granules on CD44v6 and NM23H-1 expression (%) Group

n

Treatment

30

Control

b

Treatment

Before After Before 33 After

CD44v6 Positive Negative 21 (70.00) 9 (30.00) 12 (40.00)b 18 (60.00) 23 (69.70) 10 (30.30) 23 (69.70) 10 (30.30)

NM23H-1 Positive Negative 4 (13.33) 26 (86.67) 6 (20.00) 24 (80.00) 6 (18.18) 27 (81.82) 7 (21.21) 26 (78.79)

P < 0.01 vs control group after treatment.

(DS) + intensity (IS) (TS0: sum 0; TS1: sum 1 to 3; TS2: sum 4 to 5; TS3: sum 6 to 7). TS0 and TS1 were considered negative. Statistical analysis Binomial distributions were used to compute P-values for positive and negative immunohistochemical staining of anti-CD44 or anti-nm23 antibodies on the tissue sections. SPSS 8.0, χ2 and t-test were used to test the difference in P-values. P < 0.05 or P < 0.01 was considered statistically significant or very statistically significant, respectively.

RESULTS The results of the immune adherent function of red blood cells before and after radiotherapy for patients with esophageal carcinomas with or without oral Fuzheng Yiliu granules are listed in Table 1. Prior to treatment, RBCC3bRR and RBC-ICRR had no significant differences (P > 0.05). After 21 d of treatment, RBC-C3bRR increased from 7.78% to 10.03% and 7.18 to 7.46% in the treatment and control groups, respectively (P < 0.01). RBC-ICRR decreased significantly from 37.68% to 22.66% and 37.28% to 24.69% in the treatment and control groups, respectively (P < 0.01). The immunohistochemistry results of metastasis protein CD44 and metastasis inhibition factor nm23 in esophageal carcinoma tumor cells of patients before and after radiotherapy, with or without oral FuzhengYiliu granules are listed in Table 2. Prior to treatment, the difference in cases positively stained with CD44v6 or nm223H-1 antibodies between the treatment and control groups was not statistically significant (P > 0.05). The number of cases positively stained with CD44v6 decreased from 21 to 12 in the treatment group, whereas there was no change in the control group (P < 0.05). However, the

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number of cases stained with nm23H-1 increased from 4 to 6 in the treatment group and 6 to 7 in the control group (P > 0.05).

Discussion Esophageal carcinoma occurs frequently in adult and elderly populations. According to traditional Chinese medicine, malignancy is referred to as dysphagia syndrome and its development and progression may be due to aging and deficiency in Qi and Yin or in Qi and blood, or stagnation of phlegm-dampness, heat-toxin accumulation, consumption of body fluid and blood resulted from unhealthy habit of food-intake and emotional upsets. Patients treated with radiotherapy often have invasive and metastatic tumors that may cause difficulty in swallowing or instant vomiting upon food intake, resulting in lack of nutrients to support physiological metabolism. Constant nutrient deficency leads to catabolism of body materials, further decreasing the levels of Yang-Qi and healthy Qi, which in turn make stagnation of phlegm-dampness and heat-toxin accumulation more severe. In Fuzheng Yiliu granules, Radix hedysai enhances Qi, radix angelicae sinesis boosts blood production, Rhizoma zekoariae increases blood circulation to transport stasis, Radix patriniae promotes diuresis to remove damp and toxic metabolites from the body. These four herbs in combination augment healthy Qi and reduce pathogenic factors. Our study indicates that the presence of abnormal immune adhesion function of red blood cells in patients with esophageal carcinoma, could decrease RBC-C3bRR and increase RBC-ICRR [10] . RBC and WBC counts usually begin to decrease three weeks after radiotherapy, preventing patients from continuous treatment. It was reported that the rosette of tumor cells can adhere to RBC [5] , providing morphological support for the hypothesis that erythrocytes might play a role in prevention of tumor cells from metastasis. Gan and Zhang[11] reported that increased RBC-C3bRR and decreased RBC-ICRR play an important role in tumor cell metastasis. These studies strongly suggest that improvement in immune adhesion function of RBC can suppress tumor cell metastasis[12]. Radix angelicae sinesis and Rhizoma zekoariae increase blood microcirculation, modulate immune function, inhibit or kill tumor cells, while Radix patriniae enhances immune function and inhibits S180 tumor cell growth[13]. All these data and the results of our studies on animal model and clinical patients[1-4], demonstrate that Fuzheng Yiliu granules enhance immunity, suppress tumor cell proliferation and induce apoptosis of malignant cells. CD44v6 is one of the alternative spliced variants of CD44[14]. CD44 is an integral cell membrane glycoprotein that may play a role as cell surface adhesion molecules in lymphocyte migration to lymph nodes[15]. Matsumura and Tarin[16] first report that splice variants of CD44 may be associated with metastases and can be used in early diagnosis of cancer patients. Mayer et al [17] found that expression of CD44 is associated with distant metastases, recurrence and increased mortality of gastric cancer. In the

Zhao JX et al . Fuzheng-Yiliu and metastasis

field of esophageal carcinoma, many investigators reported that CD44v6 over-expression is significantly associated with tumor invasion, distant metastasis, and poor prognosis[18-20]. Schmits et al[21] showed that mice deficient in all known variants of CD44 develop granuloma in response to Cryotosporidium parvum infection. SV40induced tumorigenicity of CD44-deficient fibroblasts can be suppressed by reintroduction of CD44 expression[12]. These results suggest that over-expression of CD44 may be a consequence, rather than a cause, of malignant tumor progression. Further studies are needed to elucidate the molecular mechanisms underlying pharmacological function of Fuzheng Yiliu granules on CD44v6-positive cells. NM23H-1 belongs to the NM23 gene family that has been shown to play a critical role in cellular proliferation, embryonic development, differentiation, oncogenesis, and tumor metastasis[22]. Expression of NM23H-1 inversely correlates with metastatic potential and does not correlate to host immune response [23]. MacDonald et al [24] found that specific mutations in the human NM23 gene reduce the ability of the gene product to inhibit cell motility. Granzyme A (GZMA) induces a caspase-independent cell death pathway characterized by single-stranded DNA nicks and other features of apoptosis. Fan et al[25] showed that NM23H1 is a GZMA-activated DNase that interacts with its specific inhibitor SET. After GZMA loading or cytotoxic T lymphocyte (CTL) attack, SET and NM23H1 translocate to nuclei and SET is degraded by GZMA cleavage, allowing NM23H1 to nick chromosomal DNA. Thus, loss of NM23H1 expression in tumors might make them resistant to immune surveillance by CTL and nature killer cells, since target cells with silenced NM23H1 are less sensitive to GZMA-induced apoptosis. The role of NM23H-1 in apoptosis is consistent with clinical observations that reduced expression of NM23H1 is associated with metastatic esophageal carcinoma[26,27] and poor prognosis[28, 29]. NM23H-1-positive cases increased from 4 to 6 in the treatment group, and from 7 in the control group (Table 1, P > 0.05), indicating that Fuzheng Yiliu granules have no effect on NM23H-1 expression. An alternative explanation is that the current sample size was too small to make a valid statistical analysis.

COMMENTS Background

Many patients are diagnosed with esophageal carcinoma at intermediate or advanced stage, making surgical treatment impossible. Late diagnosis may also minimize other treatments such as radiotherapy. Radiotherapy for esophageal carcinoma is limited due to its side effects. Recently, we have used a Chinese herb to improve the efficacy, reduce the side effects of radiotherapy for esophageal carcinoma, and improve the patients’ living quality and prognosis. However, the mechanism remains unknown.

Research frontiers

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Applications

In order to keep the successive efficacy of Fuzheng Yiliu granules, these granules must be used for 21 d or more.

Terminology

Immune adhesion function of red blood cells (RBCs): RBCs are the most important eigen immunocell that has been identified. RBCs function in adhesion, concentration, killing antigen and cleaning immune complex in blood circulation, etc. Through the above functions, RBCs join the regulating immune functions of human body.

Peer review

This is a well-written manuscript describing the research on the anti-tumor effect of the herbal medicine. Fuzheng Yiliu granules enhance the immune adhesion function of RBCs and reduce the number of CD44+-cells in esophageal carcinoma, suggesting a potential role of these Chinese herbals in suppressing invasion and metastasis of malignant cells.

REFERENCES 1 2

3

4 5 6 7

8

9

10 11 12 13 14

Chinese herbs used in anti-metastasis treatment of carcinoma are currently a hot spot in the area of natural anti-cancer agents.

Innovations and breakthroughs

The basic and prophase clinical trial confirmed that Fuzheng Yiliu granules can improve treatment efficacy for esophageal carcinoma. This article explores the effects of these herbs on esophageal carcinoma at cellular and molecular levels.

15 16

Dai EL, Zhao JX, Zhu YZ. Experimental study on effect of fuzheng yiliu decoction on tumor cell cycle and telomerease. Zhongguo Zhongxiyi Jiehe Zazhi 2001; 21: 760-762 Zhao JX, Lian P, Li YT. Effect of fuzheng yiliu granule on nuclear transcriptional factor-kappa B and cell cycle in patients with breast carcinoma. Zhongguo Zhongxiyi Jiehe Zazhi 2003; 23: 421-422 Zhao JX, Qu Y, Chen XZ. Clinical observation on effect of fuzheng yiliu granule on cell cycle and nuclear transcription factor-kappa B in tissue of esophageal-gastric carcinoma. Zhongguo Zhongxiyi Jiehe Zazhi 2003; 23: 908-910 Zhao JX, Li XF. Effects of Fuzheng Yiliu Granule on expression of CD44v6 and nm23-H1 in esophageal carcinoma treated with radiotherapy. Zhongxiyi Jiehe Xuebao 2004; 2: 262-264 Guo F, Qian H, Zhang LZ. Modern Erythrocyte Immunology. Shanghai: 2nd Military Medical University Publishing Press, 2002: 1-44 Guo F, Qian SZ. Preliminary study on the immune function of red cells in patients with pregnancy-induced hypertension. Zhonghua Fuchanke Zazhi 1989; 24: 215-216, 252 Garcia RL, Coltrera MD, Gown AM. Analysis of proliferative grade using anti-PCNA/cyclin monoclonal antibodies in fixed, embedded tissues. Comparison with flow cytometric analysis. Am J Pathol 1989; 134: 733-739 Gillett C, Fantl V, Smith R, Fisher C, Bartek J, Dickson C, Barnes D, Peters G. Amplification and overexpression of cyclin D1 in breast cancer detected by immunohistochemical staining. Cancer Res 1994; 54: 1812-1817 Beasley MB, Lantuejoul S, Abbondanzo S, Chu WS, Hasleton PS, Travis WD, Brambilla E. The P16/cyclin D1/Rb pathway in neuroendocrine tumors of the lung. Hum Pathol 2003; 34: 136-142 Liu LH, Qi LP. Study on change of immune function of lymphocyte and erythrocyte and their relationship. Zhongguo Zhongliu Linchuang 2003; 30: 84-87 Gan RL, Zhang YS. Analysis of erythrocyte immue function in tumor cell metastasis. Zhongguo Mianyixue Zazhi 1997; 13: 484-485 Yang YG. Erythrocyte immune function and tumor metastasis. Zhongguo Zhongliu Linchuang 2003: 30: 68-70 Tian DH. Practical dictionary of traditional Chinese herbs. Beijing: People’s Medical Publishing House, 2002: 1523-1525 Screaton GR, Bell MV, Jackson DG, Cornelis FB, Gerth U, Bell JI. Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons. Proc Natl Acad Sci USA 1992; 89: 12160-12164 Aruffo A, Stamenkovic I, Melnick M, Underhill CB, Seed B. CD44 is the principal cell surface receptor for hyaluronate. Cell 1990; 61: 1303-1313 Matsumura Y, Tarin D. Significance of CD44 gene products for cancer diagnosis and disease evaluation. Lancet 1992; 340: www.wjgnet.com

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1053-1058 Mayer B, Jauch KW, Gunthert U, Figdor CG, Schildberg FW, Funke I, Johnson JP. De-novo expression of CD44 and survival in gastric cancer. Lancet 1993; 342: 1019-1022 18 Chen J, Zhang ZY, Zhu JQ. Clinical significance of CD44v6 expression in esophageal carcinoma. Shijie Huaren Xiaohua Zazhi 1998; B: 534-535 19 Gu HP, Ni CY, Zhou CL. RNA and protein expression of CD44v6 in esophageal carcinoma. Shijie Huaren Xiaohua Zazhi 2001; 9: 1080-1081 20 Nozoe T, Kohnoe S, Ezaki T, Kabashima A, Maehara Y. Significance of immunohistochemical over-expression of CD44v6 as an indicator of malignant potential in esophageal squamous cell carcinoma. J Cancer Res Clin Oncol 2004; 130: 334-338 21 Schmits R, Filmus J, Gerwin N, Senaldi G, Kiefer F, Kundig T, Wakeham A, Shahinian A, Catzavelos C, Rak J, Furlonger C, Zakarian A, Simard JJ, Ohashi PS, Paige CJ, GutierrezRamos JC, Mak TW. CD44 regulates hematopoietic progenitor distribution, granuloma formation, and tumorigenicity. Blood 1997; 90: 2217-2233 22 Lacombe ML, Milon L, Munier A, Mehus JG, Lambeth DO. The human Nm23/nucleoside diphosphate kinases. J Bioenerg Biomembr 2000; 32: 247-258 23 Steeg PS, Bevilacqua G, Kopper L, Thorgeirsson UP, 17

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Talmadge JE, Liotta LA, Sobel ME. Evidence for a novel gene associated with low tumor metastatic potential. J Natl Cancer Inst 1988; 80: 200-204 24 MacDonald NJ, Freije JM, Stracke ML, Manrow RE, Steeg PS. Site-directed mutagenesis of nm23-H1. Mutation of proline 96 or serine 120 abrogates its motility inhibitory activity upon transfection into human breast carcinoma cells. J Biol Chem 1996; 271: 25107-25116 25 Fan Z, Beresford PJ, Oh DY, Zhang D, Lieberman J. Tumor suppressor NM23-H1 is a granzyme A-activated DNase during CTL-mediated apoptosis, and the nucleosome assembly protein SET is its inhibitor. Cell 2003; 112: 659-672 26 Zou DH, Zhang BJ, Zhao L. Association of NM23 expression in primary and lymph note metastasis of esophageal carcinoma. Tumor Prevention & Cure 2002; 7: 456-468 27 Wang XW, Fang JP. Expression of NM23H-1 and CD44v6 in primary and lymph note metastasis of esophageal carcinoma. Practical Tumor J 2002; 17: 10-12 28 Iizuka N, Tangoku A, Hayashi H, Yosino S, Abe T, Morioka T, Oka M. The association between nm23-H1 expression and survival in patients with esophageal squamous cell carcinoma. Cancer Lett 1999; 138: 139-144 29 Wang LS, Chow KC, Lien YC, Kuo KT, Li WY. Prognostic significance of nm23-H1 expression in esophageal squamous cell carcinoma. Eur J Cardiothorac Surg 2004; 26: 419-424 S- Editor Zhu LH L- Editor Wang XL

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E- Editor Yin DH

World J Gastroenterol 2007 August 28; 13(32): 4365-4371 World Journal of Gastroenterology ISSN 1007-9327 © 2007 WJG. All rights reserved.

Online Submissions: wjg.wjgnet.com www.wjgnet.com [email protected]

RAPID COMMUNICATION

Change in expression of apoptosis genes after hyperthermia, chemotherapy and radiotherapy in human colon cancer transplanted into nude mice Han Liang, Hong-Jie Zhan, Bao-Gui Wang, Yuan Pan, Xi-Shan Hao Han Liang, Hong-Jie Zhan, Bao-Gui Wang, Yan Pan, Xi-Shan Hao, Department of Gastrointestinal Oncological Surgery, Tianjin Cancer Hospital, Tianjin Medical University, Key Laboratory of Cancer Prevention and Therapy, Tianjin 300060, China Supported by the Clinical Research Foundation of Tianjin Medical University, No. 2002KY18 Correspondence to: Han Liang, Professor, Department of Gastrointestinal Oncological Surgery, Tianjin Cancer Hospital, Tianjin Medical University, Tibyanbei Hexi District, Tianjin 300060, China. [email protected] Telephone: +86-22-23340123 Fax: +86-22-23359984 Received: 2007-04-19 Accepted: 2007-05-22

CONCLUSION: Hyperthermia enhances the effect of radio- and chemotherapy on tumors by changing the expression of apoptosis genes, such as p53 , Bcl-2 and Bax . Key words: Hyperthermia; Apoptosis; p53 ; Bcl-2 ; Bax ; Nude mice; Colon cancer; Cell line Liang H, Zhan HJ, Wang BG, Pan Y, Hao XS. Change in expression of apoptosis genes after hyperthermia, chemotherapy and radiotherapy in human colon cancer transplanted into nude mice. World J Gastroenterol 2007;

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Abstract

http://www.wjgnet.com/1007-9327/13/4365.asp

AIM: To investigate the change in expression of

p53 , Bcl-2 , and Bax genes in human colon cancer

cells transplanted into nude mice after hyperthermia, chemotherapy, radiotherapy, thermochemotherapy, thermoradiotherapy and thermochemoradiotherapy.

METHODS: Human colon cancer cell line (HT29) was transplanted into the hind limbs of nude mice. Under laboratory simulated conditions of hyperthermia (43℃, 60 min), the actual radiation doses and doses of mitomycin C (MMC) were calculated in reference to the clinical radiotherapy for human rectal cancer and chemotherapy prescription for colon cancer. The mice were divided into 6 groups according to the treatment approaches: hyperthermia, chemotherapy, radiotherapy, thermochemotherapy, thermoradiotherapy, and thermochemoradiotherapy. The mice were sacrificed at different time points and the tumor tissue was taken for further procedures. The morphologic changes in membrane, cytoplasm and nuclei of tumor cells of p53 , Bcl-2 , and Bax after treatment, were observed by immunohistochemistry staining. RESULTS: All of the six treatment modalities downregulated the expression of p53 , Bcl-2 and up-regulated the expression of Bax at different levels. The combined therapy of hyperthermia, with chemotherapy, and/or irradiation showed a greater effect on down-regulating the expression of p53 (0.208 ± 0.009 vs 0.155 ± 0.0115, P < 0.01) and Bcl-2 (0.086 ± 0.010 vs 0.026 ± 0.0170, P < 0.01) and up-regulating Bax expression (0.091 ± 0.0013 vs 0.207 ± 0.027, P < 0.01) compared with any single therapy.

INTRODUCTION Tumor regression after a high fever due to erysipels was first reported in 1866 by the German physician Busch. In 1910 Müller described the potential of hyperthermia as an adjuvant to radiotherapy[1]. In the past two decades there has been a great interest in application of hyperthermia in conjunction with irradiation or/and chemotherapy in cancer treatment. This is associated with a better understanding of several biological parameters such as radiosensitization, chemosensitization, direct cytoxicity, ther motolerance, and stepdown heating, as well as complete changes in micromilieu, especially involving the microvasculature[2-6]. Hyperthermia has become the fifth method of therapy after surgery, chemotherapy, radiation and biological therapy, and plays an important role in multidiscipline therapy for cancer. Hyperthermia is considered a very hopeful anticancer method in the near future. For the first time we observed that hyperthermia at 43℃ for 60 min upregulated E-cadherin and γ-catenin expressions but downregulated β-catenin expression on colon carcinoma cells in vitro. α -catenin expression is not affected by hyperthermia[3]. However, the pathways between apoptosis and hyperthermia (which is one of the inducers) remain unclear. The relative amount of Bcl-2 and Bax proteins determines cell survival or death following an apoptotic stimulus. To clarify the molecular mechanism of hyperthermic cell destruction, radiosensitivity and interaction of heat with chemotherapeutic agents, we designed an experiment to identify the change www.wjgnet.com

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in expression of p53, Bax and Bcl-2 proteins after hyperthermia, chemotherapy, radiotherapy and combined therapies in human colon cancer transplanted into nude mice.

MATERIALS AND METHODS Reagents Human colon adenocarcinoma cell line HT29 was kindly provided by Center Laboratory of Tianjin Medical University Cancer Hospital. One hundred and forty-four 4-5 wk-old BLAB/C nu/nu mice of both sexes, weighing 18-20 g, were purchased from the Animal Center of Chinese Academy of Medical Sciences. The mice were fed with standard maintenance diet and water throughout the experiment period. All animals received care treatment in compliance with the guidelines of China Ministry of Public Health. Reagents and instruments Mitromycin-C (MMC) was produced by Kyowa Hacko Kog yo Co. Ltd. Human anti-p53 gene polycolonal antibodies were purchased from Beijing Zhongshan Biotechnolog y, Inc. Polycolonal human antibody against Bcl-2 and Bax gene, ultrasensitiveTMS-P box for immunohistochemical staining and DAB box were the products of Fujian Maixing Biotechnology Development Inc. Electroheat hemothermia water bath trunk (Shanghai No 7 Medical Equipment Co. Lit., HHW21. Cu 600), 30 self-made wooden shelves for fixing nude mice, WMY-01 digital thermometer were the products of Shanghai Medical Instrument Co. Lit. SL-7510 accelerator, medical adhesive tape, Leitz-1212 paraffin section machine, Olympus CH-2 optical microscope and Bei Hang CMIAS image analysis system were provided by Pathological Department of Tianjin Cancer Hospital. Xenografts of HT29 cells in nude mice HT29 cells were incubated in vitro to logarithmic growth period. The cells were separated and counted. A total of 1 × 107/mL HT29 cells were subcutaneously xenografted in bilateral groins of nude mice. After 22 d, the mice were sacrificed when tumors reached about 9 mm × 10 mm. The mass of tumors was cut into 1 mm × 1 mm × 1 mm pieces, which were then subcutaneously xenografted in bilateral hind limbs of all 144 nude mice. When the tumors grew about 0.8-1.0 cm in diameter, laboratory study was undertaken. Treatment of nude mice with xenografts of HT29 The mice were randomly divided into 6 groups (24 mice in each group) and treated as follows. The mice in group A (hyperther mia) were fixed on a special frame, their bilateral hind limbs were immersed in 43℃ water for 60 min. The mice in group B (chemotherapy) were given MMC through local injection into the tumor at a dose of 1.4 mg/kg. Briefly, 5 mL NS was injected into an ampoule containing 2 mg MMC. MMC was dissolved and the dose for each mouse was calculated according to the formula: X = 5 mgY (1.4/1000) [X: mL www.wjgnet.com

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of MMC solution for injection; Y: weigh of mice (g)]. The mice in group C (themochemotherapy) administrated MMC, hyperthermia was performed after chemotherapy. SL-7510 accelerator with 8 Mev electron beam was used in mice of group D (radiation) with a 1.0 cm-thick plexiglass placed on the surface. Radiation was performed once at 10GY. The mice in group E (themoradotherapy) underwent radiation and then hyperthermia. The mice in group F (themochemoradiotherapy) received radiation followed by chemotherapy and hyperthermia. Three mice from each group were sacrificed at 8 different time points (before treatment, and 2, 4, 8, 12, 24, 48 and 72 h after treatment). The tumor of the hind limbs was resected, fixed with 10% formalin solution, embedded in paraffin wax blocks and cut into 4-µm thick sections for immunohistochemical analysis. Immunohistochemical staining for expression of p53, Bcl-2 and Bax genes in tumor tissue Expression of p53, Bcl-2 and Bax genes in harvested tumor was determined immunohistochemically using the S-P method. Unless otherwise stated, all steps were carried at room temperature. Briefly, after dewaxed and rehydrated, slides were washed for 5 min, put into distilled water for another 1 min and then dried in a microwaven (95℃-98℃) in citric acid buffer (pH 6.0) for 15-20 min to retrieve antigens. The sections were washed three times with phosphate-buffered saline (PBS) for 5 min. Normal goat serum was added to the slides for 20 min. Rabbit anti-human polyclonal antibody against Bcl-2, p53 and Bax was applied to sections and incubated overnight at 4℃. After incubated in PBS for 5 min, second antibody (biotin-conjugated) was added to the sections at 37℃ for 30 min. The enzyme conjugated HRP-streptavidin (third antibody) at 37℃ for 30 min. Between each step, the sections were washed three times with PBS for 5 min. Sites of immunoreaction were visualized with 3, 3’-diaminobenzidine (DAB) for 2-20 min under microscope. Then the sections were washed with tap water for 5 min, soaked in distilled water for 2 min, counterstained with Mayer’s haematoxylin, dehydrated in alcohol, and mounted on Canada balsam. Evaluation criteria for immuohistochemical staining Immunostaining specific for Bcl-2 and Bax gene was membranous and cytoplasmic, p53 gene was nucleolar. These genes were stained yellow or brown under optical microscope. Bei-Hang CMIAS pathological image analysis system was adopted. The brightest area of image was calibrated both for size and optical density; 5 high power fields of each section were selected for image collection. The target area of section was fixed and color division was carried out. The results of division were analyzed statistically and an average optical density was obtained. Statistical analysis Statistical analysis was perfor med by SPSS11.5 for Windows (SPSS, Chicago IL). Comparison of average optical density for Bcl-2, Bax and p53 genes in different groups was made by mono-factor variance analysis. SNQ test was used in comparison of the two groups.

Liang H et al . Change in apoptosis genes after hyperthermia, chemotherapy and radiotherapy

Mean of p53

0.35

A B C D E F

0.30

0.25

0.20

0.15 0

2

4

8

12

24

48

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Table 1 Average optical density of p53 , Bcl-2 and Bax in different groups 48 h after treatment (mean ± SD) Group A B C D E F F

n

p53

Bcl-2

6 6 6 6 6 6

0.208 ± 0.009 0.223 ± 0.0133 0.255 ± 0.0111 0.197 ± 0.0157 0.205 ± 0.100 0.155 ± 0.0115 40.810b

0.086 ± 0.010 0.056 ± 0.0064 0.064 ± 0.0048 0.058 ± 0.0095 0.054 ± 0.0090 0.026 ± 0.0170 21.466b

Bax 0.091 ± 0.0013 0.135 ± 0.0095 0.128 ± 0.0144 0.151 ± 0.011 0.150 ± 0.028 0.207 ± 0.027 23.912b

72 h

Figure 1 Expressions of p53 in different groups at different time points after hyperthermia (A), chemotherapy (B), themochemotherapy (C), radiation (D), themoradiotherapy (E), and themochemoradiotherapy (F).

Group A: hyperthermia; group B: chemotherapy; group C: themochermotherapy; group D: radiation; group E: themoradiotherapy; group F: thempchempradiotherapy. bP < 0.01 in groups A-F. Comparison of average optical density of Bcl-2, Bax and p53 genes in different groups was made by mono-factor variance analysis. SNQ test was used to compare the two groups.

A

B

Figure 2 Expressions of p53 before (A) and after thermochemoradiotherapy (B) (SP, × 200).

RESULTS Change in p53 gene expression Down regulation of p53 expression was found in all the six groups for treatment. The reduction of p53 expression in group F was the highest and occurred at first. In contrast, the reduction of p53 expression in group D was the lowest and occurred at last. There was no significant difference in the reduction of p53 expression in groups A, C and E. The extent of reduction in the above mentioned groups was significant in comparison to that in groups B and D. The extent of reduction of p53 expression was greater in group B than in group D. Dynamic analysis showing reduction of p53 expression was begun immediately in group F after the treatment. The lowest level of p53 expression was found 48 h after the treatment. The optical density was reduced 50% in comparison to that before treatment, and then elevated again 48 h after treatment. p53 expression was down regulated gradually in groups A, C and E 2 h

after treatment, and elevated again in group C 48 h after treatment. p53 expression was down regulated gradually in groups B and D, 4 and 12 h following treatment (Figures 1 and 2). The average optical densities of p53 in different groups 48 h after treatment were analyzed by SNQ test (Table 1). There was no statistical difference between groups A and B, groups A and C, groups A and E (P = 0.425, P = 0.129, P = 0.279), though statistical differences were found between groups D and E (P = 0.014, P = 0.000). There was no statistical difference between groups B and A, groups B and E (P = 0.129, P = 0.279), but between groups B and D, groups B and F (P = 0.014, P = 0.000). There were statistical differences between groups D and C, groups D and E, groups D and F (P = 0.001, P = 0.000, P = 0.000) while no statistical difference occurred between groups C and E (P = 0.97). On the other hand, statistical difference was identified between groups C and F (P = 0.001). These results indicated that p53 expression was significantly down regulated in group F in comparison to other groups and the combined therapy had a greater ability to down regulate p53 expression than any single therapy. Change in Bcl-2 gene expression Down regulation of Bcl-2 expression was demonstrated in six groups. The lowest level of Bcl-2 expression was first observed in group F. Hyperthermia by itself (in group A) could not greatly down regulate Bcl-2 expression. Down regulation of Bcl-2 could be enhanced when hyperthermia was combined with chemotherapy (in group C) or radiation (in group E), but obvious effect of down regulation was found in group E compared to group C. Dynamic analysis indicated that down regulation of Bcl-2 expression was observed in group F 2 h after treatment and the greatest reduction occurred 48 h after treatment and then elevated. In group A, Bcl-2 expression was down regulated temporarily 4 h after hyperthermia, then began to increase at 8 h and returned to the original level at 12 h. Down regulation of Bcl-2 gene was observed again at 24 h following hyperthermia. There was no significant difference at various time

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0.12

A B C D E F

Mean of Bcl-2

0.10 0.08 0.06

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0.25

A B C D E F

0.20 Mean of Bax

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0.15 0.10

0.04 0.05 0.02

0

2

4

8

12

24

48

Figure 3 Expression of Bcl-2 in different groups at the different time points after hyperthermia (A), chemotherapy (B), themochemotherapy (C), radiation (D), themoradiotherapy (E), and themochemoradiotherapy (F).

A

B

Figure 4 Expression of Bcl-2 before (A) and after thermochemoradiotherapy (B) (SP, × 400).

points. Down regulation of Bcl-2 gene was identified 8 h after chemotherapy (in group B) and Bcl-2 expression was down regulated in group D after an additional 4 h. Bcl-2 expression increased at 48 h after chemotherapy and kept stable at 48 h following radiation (in group D). Down regulation of Bcl-2 gene was demonstrated 4 h after themoradiation (in group E) and Bcl-2 decreased in group C after an additional 4 h. Bcl-2 expression increased at 48 h following treatment in groups C and E (Figures 3 and 4). The average optical densities of Bcl-2 in different groups 48 h after treatment were analyzed by SNQ test (Table 1). Statistical differences were found between groups A and B, groups A and D, groups A and C, groups A and E, group A and F (P = 0.03, P = 0.021, P = 0.008, P = 0.003, P = 0.001). However, there were no statistical differences between groups B and D, groups B and C, groups B and E, groups B and F (P = 0.339, P = 1.000, P = 1.000, P = 0.054). No statistical differences existed between groups D and C, groups D and E (P = 0.905, P = 0.356). On the other hand, statistical differences www.wjgnet.com

0

2

4

8

12

24

48

72 h

72 h

Figure 5 Expression of Bax in different groups at the different time points after hyperthermia (A), chemotherapy (B), themochemotherapy (C), radiation (D), themoradiotherapy (E), and themochemoradiotherapy (F).

were shown between groups D and F (P = 0.040). There were no statistical differences between groups C and E (P = 0.999) though statistical differences were indicated between group C and F (P = 0.040). Finally, there were no statistical difference between groups E and F (P = 0.078). The results indicated that Bcl-2 expression was significantly down regulated in groups E and F in comparison to other groups and the combined therapy demonstrated its greater ability to down regulate Bcl-2 expression than any single therapy. Change in Bax gene expression Up regulation of Bax expression was observed in six groups. The highest Bax expression was first found in group F. Once again hyperthermia by itself could not obviously up regulate Bax expression which occurred at last. Chemotherapy increased Bax expression (in group B) followed by radiation (in group D). Bax expression increased more significantly in group E than in group C. Dynamic analysis indicated that Bax expression was up-regulated gradually in group F 2 h after treatment. The highest level was observed 48 h after treatment and then decreased. Bax expression was up regulated 24 h after hyperthermia treatment (in group A), 4 h after themoradiation (in group E), 8 h after themochemotherapy (in group C), 8 h after chemotherapy (in group B), and 12 h after radiation (in group D), respectively (Figures 5 and 6). The average optical densities of Bax in different groups 48 h after treatment were analyzed by SNQ test (Table 1). Statistical differences were shown between groups A and B, groups A and D, groups A and C, groups A and E, groups A and F (P = 0.01, P = 0.012, P = 0.000, P = 0.029, P = 0.000). However, there were no statistical differences between groups B and D, groups B and C, groups B and E (P = 0.988, P = 0.242, P = 0.952). On the other hand, statistical differences existed between groups B and F (P = 0.008). No statistical differences were observed between groups D and C, groups D and E (P = 0.134, P = 0.767), though statistical difference occurred between groups D and F (P = 0.004). There were no statistical differences between groups C and E (P = 1.000), but there was between groups C and F (P = 0.028). Finally, there was no statistical difference between groups E and F (P = 0.059). The results indicated that Bax expression was

Liang H et al . Change in apoptosis genes after hyperthermia, chemotherapy and radiotherapy

A

B

Figure 6 Expression of Bax before (A) and after (B) thermochemoradiotherapy (SP, × 400).

significantly up regulated in groups E and F compared to other groups and the combined therapy had a greater ability to up regulate Bax expression than any single therapy.

DISCUSSION p53 seems to play an important role in mediating DNA damage induced by various drugs because mutations in p53 are associated with decreased susceptibility to chemotherapeutic agents[8], and there is evidence that p53 expression is also related to multidrug resistance (MDR)[9]. It was reported that human non-small-cell lung cancer cell lines with mutant p53 have become more sensitive to cisplatin following transduction of wild-type p53[10]. Finally, p53 molecule has been fused to chemotherapeutic prodrugs to enhance its chemosensitizing properties. It was originally shown that thymocytes readily undergo apoptosis following exposure to γ -irradiation, but loss of wild-type p53 dramatically reduces the number of apoptosis cells[11]. Therefore, efforts to combine p53 gene therapy with radiotherapy have focused on enhancing the radiosensitivity of cancer cells lacking functional p53 to render them [12]. In our experiment, mutant p53 was detected by immunohistochemical staining. We found that themochemotherapy and themoradiation could significantly down regulate mutant p53 expression. Down regulation of mutant p53 enhances radio- and chemosensitivity indirectly with the similar therapeutic effect produced by p53 gene therapy combined with chemotherapy and radiation. Furthermore, hyperthermia has a greater ability to down regulate mutant p53 expression than radiotherapy or chemotherapy. Combined hyperthermia with chemotherapy and radiation can achieve the best result. A recent report

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also indicates that heat-induced growth inhibition of transplanted neck squamous cell carcinoma (HNSCC) may be correlated with the induction of p53-dependent Baxmediated apoptosis. Thus, p53 status appears to be one of the useful parameters for predictive assays in hyperthermic cancer therapy[13]. Bcl-2 family is a huge family comprising various members, which are key regulators of apoptosis, tissue homeostasis, and protection against pathogens [14] . High levels and aberrant patterns of Bcl-2 expression have been reported in colorectal, lung, gastric, renal and other cancers[15]. Elevation of Bcl-2 protein expression contributes not only to the development of cancer, but also to resistance against a wide variety of anti-cancer agents[16]. The protection afforded by Bcl-2 against chemotherapeutic agents defines a novel type of drug resistance in which Bcl-2 over-expression does not prevent drug entry and accumulation in tumor cells or the interaction of drugs with their primary molecular targets. Bcl-2 appears to block the transmission of signals originating from cellular damage to molecular effectors of apoptosiss[15]. This allows cells to survive in the presence of other lethal damage, repairing drug-induced damage after withdrawal of the drugs. Via this mechanism, Bcl-2 essentially converts anti-cancer drugs from cytotoxic to cytostatic, thus increasing the chances of acquiring genetic alterations and favoring the development of a more malignant phenotype. Bcl-2 protein expression is negatively correlated with the radiosensitivity of nasopharyhgeal carcinoma[17]. Our study suggested that hyperthermia combined with chemotherapy could enhance the ability of Bcl-2 to down regulate its expression, although hyperthermia itself could not change the expression of Bcl-2 gene significantly. This may be one of the mechanisms of hyperthermia underlying the enhancement of radiosensitization and cytotoxicity of many chemotherapeutic agents. In contradiction to the Bcl-2 gene, Bax enhances cell death. It was reported that when expression or mutation of Bax gene is low in tumor tissue, cells resist apoptosis and have no response to radiation and chemotherap[18]. On the other hand, over-expression of Bax in human cancer cell lines has been reported to induce apoptosis independently of endogeneous p53 status and to enhance the chemosensitivity or radiosensitivity of selected tumor lines[18]. Research in cell line of childhood acute lymphoblastic leukemia indicates that down regulation of Bcl-2 expression and up regulation of Bax expression by radiation tend to be correlated with increased radiosensitivity[19]. In the present study, expression of Bcl-2 gene was relatively high and expression of Bax gene was low, the ratio of Bcl-2 and Bax was high. However, the expression of Bax was up-regulated significantly after treatment. Bcl-2 gene expression was down regulated in groups B-F. Down regulation of Bcl-2 expression in group F was more obvious than that in groups E and C as well as in groups D and B. Although hyperthermia itself did not change Bcl-2 gene expression, hyperthermia in combination with radiation and chemotherapy increased down regulation of Bcl-2 gene expression significantly, suggesting that hyperthermia can enhance radio- and chemosensitivity. Anti-cancer therapy itself can influence the expression www.wjgnet.com

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of apoptosis-associated genes. Radiation and chemotherapy can change the level of apoptosis[5]. A recent report indicates that apoptosis induced by abating HSP70 expression may enhance the sensitivity of human bladder cancer cells to MMC[20]. In vitro study with malignant histiofibrocytoma cell line has illustrated that the expression of Bax is up-regulated in protein 30 min after 43℃ hyperthermia, accompanying the change in mRNA[6]. In the present study, the extent of up regulation of Bax expression in themoradiation group 24 h after treatment was greater than that in the hyperthermia and radiation treatment groups. This phenomenon was not found in the thermochemotherapy group. In conclusion, hyperthermia in combination with radio-and chemotherapy can achieve a better effect and enhance changes in apoptosis-related genes though its definite mechanism is still unknown. Further research is required to determine the precise effect of hyperthermia in combination with other therapies.

Background

In the past two decades there has been a great interest in application of hyperthermia in conjunction with radiotherapy or/and chemotherapy in cancer treatment. This is associated with a better understanding of several biological parameters such as radiosensitization, chemoxicity, thermotolerance, as well as complete changes in micromilieu, especially involving the microvasculature. However, the pathways between apoptosis and hyperthermia remain unclear.

Research frontiers

We observed that hyperthermia at 43℃ for 60 min up regulated E-cadherin and γ-catenin expression but down regulated β-catenin expression. Anti-cancer therapy itself can influence the expression of apoptosis-associated genes. Radiation and chemotherapy can change the expression of apoptosis.

The study investigated the expression of three apoptosis-related genes (Bcl-2, Bax, p53) in human colon cancer transplanted into nude mice after hyperthermia, chemotherapy and radiotherapy. The authors tried to set up an in vivo tumor model, in which the effect of various treatment modalities and their combination could be investigated. The authors used a histology based model to study the protein expression related to the above mentioned three genes.

REFERENCES 1

3

4 5 6 7

8 9

10

Innovations and breakthroughs

The results of this study show that hyperthermia combined with chemotherapy or/and radiation can change the expression of apoptosis genes such as p53, Bcl-2 and Bax significantly when combined with chemotherapy or/and radiation compared to hyperthermia itself. To our knowledge, no similar studies are available at present.

Applications

According to the results of this study, the molecular mechanism of hyperthermic radiosensitivity and chemosensitivity can be understood. In clinical practice, hyperthermia in combination with radiation and chemotherapy can achieve a better effect.

Terminology

(1) Hyperthermia: A therapeutic method by increasing tissue temperature (range 40-43℃), potentially induces tumor cell death by a spectrum of molecular, metabolic, cellular, and tumor tissue changes. (2) p53 gene: It is a tumor suppressor gene, which is mutated or deleted in over half of all human malignancies. Therefore, one strategy in cancer therapy focuses on the replacement or over-expression of tumor suppressor genes. (3) Bcl-2: It is a huge family composed of various members, which are key regulators of apoptosis. High

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Peer review

ACKNOWLEDGMENTS

COMMENTS

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levels and aberrant patterns of Bcl-2 expression have been reported in a wide variety of human caners. (4) Bax: It is a pro-apoptosis member of the Bcl-2 family, thought to induce apoptosis. Loss of Bax in genetically engineered mice results in increased tumor incidence, suggesting that Bax may play a role in suppressing tumor growth in vivo.

2

The authors thank Professor Riu-Fang Niu, Professor Yun Niu and Ms Yu-Rong Shi at Center Laboratory of Tianjin Cancer Hospital, Tianjin Medical University for their technical supports. The authors also thank Dr. Wienert, Andreas for his kindly review of the English manuscript.

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14 15 16

Lin SY, Li RY, Mao HS, Zhang SW. New developmental therapies, Hyperthermia. In: Oncology (in Chinese). Zhang TZ, Xu GW, editors. Tianjin and Shenyang: Tianjin Science & Technology Press and Liao Ning Science & Technology Press, 2005: 849-880 Song CW, Park HJ, Lee CK, Griffin R. Implications of increased tumor blood flow and oxygenation caused by mild temperature hyperthermia in tumor treatment. Int J Hyperthermia 2005; 21: 761-767 Xia T, Sun Q, Shi X, Fan N, Hiraoka M. Relationship between thermal parameters and tumor response in hyperthermia combined with radiation therapy. Int J Clin Oncol 2001; 6: 138-142 Engin K, Leeper DB, Tupchong L, Waterman FM. Thermoradiotherapy in the management of superficial malignant tumors. Clin Cancer Res 1995; 1: 139-145 Liang H, Hao XS. The biological mechanisms of hyperthermia. Guowai Yixue (Oncological Section) 2001; 28: 438-441 Zhan HJ, Liang H. The status of research for hyperthermia. Guowai Yixue (Oncological Section) 2005; 32: 35-38 Liang H, Li JW, Shi YR, Niu RF, Wang P, Hao XS. Change in E-cadherin, alpha-, beta- and gamma-catenin expression after hyperthermia of a human colon carcinoma cell line in vitro. Zhonghua Yixue Zazhi 2004; 84: 1299-1303 Lowe SW, Ruley HE, Jacks T, Housman DE. p53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell 1993; 74: 957-967 Matsuhashi N, Saio M, Matsuo A, Sugiyama Y, Saji S. The evaluation of gastric cancer sensitivity to 5-FU/CDDP in terms of induction of apoptosis: time- and p53 expressiondependency of anti-cancer drugs. Oncol Rep 2005; 14: 609-615 Fujiwara T, Cai DW, Georges RN, Mukhopadhyay T, Grimm EA, Roth JA. Therapeutic effect of a retroviral wild-type p53 expression vector in an orthotopic lung cancer model. J Natl Cancer Inst 1994; 86: 1458-1462 Lowe SW, Schmitt EM, Smith SW, Osborne BA, Jacks T. p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature 1993; 362: 847-849 Badie B, Goh CS, Klaver J, Herweijer H, Boothman DA. Combined radiation and p53 gene therapy of malignant glioma cells. Cancer Gene Ther 1999; 6: 155-162 Tamamoto T, Yoshimura H, Takahashi A, Asakawa I, Ota I, Nakagawa H, Ohnishi K, Ohishi H, Ohnishi T. Heat-induced growth inhibition and apoptosis in transplanted human head and neck squamous cell carcinomas with different status of p53. Int J Hyperthermia 2003; 19: 590-597 Lanave C, Santamaria M, Saccone C. Comparative genomics: the evolutionary history of the Bcl-2 family. Gene 2004; 333: 71-79 Reed JC. Regulation of apoptosis by bcl-2 family proteins and its role in cancer and chemoresistance. Curr Opin Oncol 1995; 7: 541-546 Fisher TC, Milner AE, Gregory CD, Jackman AL, Aherne GW, Hartley JA, Dive C, Hickman JA. bcl-2 modulation of apoptosis induced by anticancer drugs: resistance to thymidylate stress is

Liang H et al . Change in apoptosis genes after hyperthermia, chemotherapy and radiotherapy independent of classical resistance pathways. Cancer Res 1993; 53: 3321-3326 17 Liu MC, Gelmann EP. P53 gene mutations: case study of a clinical marker for solid tumors. Semin Oncol 2002; 29: 246-257 18 Komatsu K, Suzuki S, Shimosegawa T, Miyazaki JI, Toyota T. Cre-loxP-mediated bax gene activation reduces growth rate and increases sensitivity to chemotherapeutic agents in human gastric cancer cells. Cancer Gene Ther 2000; 7: 885-892

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19 Findley HW, Gu L, Yeager AM, Zhou M. Expression and regulation of Bcl-2, Bcl-xl, and Bax correlate with p53 status and sensitivity to apoptosis in childhood acute lymphoblastic leukemia. Blood 1997; 89: 2986-2993 20 He LF, Guan KP, Ye HY, Ren L, Yan Z, Wang SW, Hou SK. Heat shock protein 70 expression in relation to apoptosis in primary bladder transitional cell carcinoma. Chin Med J (Engl) 2005; 118: 2093-2096 S- Editor Zhu LH L- Editor Wang XL

E- Editor Liu Y

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World J Gastroenterol 2007 August 28; 13(32): 4372-4378 World Journal of Gastroenterology ISSN 1007-9327 © 2007 WJG . All rights reserved.

RAPID COMMUNICATION

A meta-analysis of the yield of capsule endoscopy compared to double-balloon enteroscopy in patients with small bowel diseases Xiang Chen, Zhi-Hua Ran, Jin-Lu Tong Xiang Chen, Zhi-Hua Ran, Jin-Lu Tong, Department of gastroenterology, Renji Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai Institute of Digestive Disease, Shanghai 200001, China Correspondence to: Zhi-Hua Ran, Professor, Department of gastroenterology, Renji Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai Institute of Digestive Disease, Shanghai 200001, China. [email protected] Telephone: +86-21-63260930 Fax: +86-21-63266027 Received: 2007-03-25 Accepted: 2007-04-16

Abstract AIM: To compare the diagnostic yield of capsule endoscopy (CE) with that of double-balloon enteroscopy (DBE). METHODS: Pubmed, Embase, Elsevier ScienceDirect, the China Academic Journals Full-text Database, and Cochrane Controlled Trials Register were searched for the trials comparing the yield of CE with that of DBE. Outcome measure was odds ratio (OR) of the yield. Fixed or random model method was used for data analysis. RESULTS: Eight studies (n = 277) which prospectively compared the yield of CE and DBE were collected. The results of meta-analysis indicated that there was no difference between the yield of CE and DBE [170/277 vs 156/277, OR 1.21 (95% CI: 0.64-2.29)]. Based on sub analysis, the yield of CE was significantly higher than that of double-balloon enteroscopy without combination of oral and anal insertion approaches [137/219 vs 110/219, OR 1.67 (95% CI: 1.14-2.44), P < 0.01), but not superior to the yield of DBE with combination of the two insertion approaches [26/48 vs 37/48, OR 0.33 (95% CI: 0.05-2.21), P > 0.05)]. A focused meta-analysis of the fully published articles concerning obscure GI bleeding was also performed and showed similar results wherein the yield of CE was significantly higher than that of DBE without combination of oral and anal insertion approaches [118/191 vs 96/191, fixed model: OR 1.61 (95% CI: 1.07-2.43), P < 0.05)] and the yield of CE was significantly lower than that of DBE by oral and anal combinatory approaches [11/24 vs 21/24, fixed model: OR 0.12 (95% CI: 0.03-0.52), P < 0.01)]. CONCLUSION: With combination of oral and anal approaches, the yield of DBE might be at least as high as

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that of CE. Decisions made regarding the initial approach should depend on patient’s physical status, technology availability, patient’s preferences, and potential for therapeutic endoscopy. © 2007 WJG . All rights reserved.

Key words: Capsule endoscopy; Double balloon enteroscopy; Yield; Meta-analysis Chen X, Ran ZH, Tong JL. A meta-analysis of the yield of capsule endoscopy compared to double-balloon enteroscopy in patients with small bowel diseases. World J Gastroenterol 2007; 13(32): 4372-4378 http://www.wjgnet.com/1007-9327/13/4372.asp

INTRODUCTION Small intestine is the middle part of digestive tract, due to its anatomic location, structural characteristics, and physiological functions, it is difficult to inspect via gastroscopy and colonoscopy. Push endoscopy is limited in that it only allows inspection of the proximal small intestine for variable distances. Intraoperative endoscopy has not met with widespread acceptance for the requirement of open laparotomy with surgically assisted passage of the endoscope through the intestine, although it has been considered the most reliable procedure for several years[1]. The diagnostic yield and accuracy of other modalities such as barium study, DSA, radionuclide and computerized tomography, magnetic resonance imaging is poor. All these make the management of small bowel disease exceptionally difficult. The techniques of capsule endoscopy (CE) and double-balloon enteroscopy (DBE) are regarded as excellent tools for the diagnosis and treatment of small-bowel disease, as well as for complementary procedures. Considerable research has demonstrated that they were more effective than other diagnostic modalities[2,3]. It’s confusing, however, when a choice should be made between the two advanced tools. Gay et al[4] concluded that indication and route for DBE could be determined according to the outcome of CE. The protocol has been widely accepted in developed countries and it seems effective, but it means that patients should undergo and pay for both of these two expensive items. It’s not clear whether the above protocol

Chen X et al . Capsule endoscopy vs double-balloon enteroscopy

was a good choice judging from the cost benefit. The aim of the present study was to assess the diagnostic efficacy of CE and DBE and to determine the optimal diagnostic procedure for patients with small bowel disease.

MATERIALS AND METHODS Participants Patients with symptoms indicating organic small bowel disease such as obscure GI bleeding, abdominal pain (functional GI diseases should be excluded), diarrhea etc., had been enrolled in the included studies, they had undergone gastroscopy, colonoscopy and other diagnostic modalities without positive findings. Search strategy The electronic databases: Pubmed (1966 to February, 2007), Embase (1980 to February, 2007), Elsevier ScienceDirect (1995 to February, 2007), the China Academic Journals Full-text Database (1979 to February, 2007), and Cochrane Controlled Trials Register (Issue 1, 2007) were used for systematic literature searches. We employed the text words: capsule endoscopy, video capsule endoscopy combining with double-balloon endoscopy, double-balloon enteroscopy, push-and-pull enteroscopy, as search terms. The search of abstracts presented at the proceedings of Digestive Disease Week (USA) and the World Congress of Gastroenterology was also performed. Description of included literature Literature was searched and prospective studies were collected. When two or more publications from one institution appeared to review the same patients, only the most recent study results were included. CE and DBE should be performed on each patient successively in included studies, so the diagnostic yields of two tests are able to be compared under equal conditions. All papers should be examined independently for eligibility by two reviewers. Disagreements were resolved by consulting a third reviewer. Statistical analysis Data were entered into the Cochrane Collaboration review manager software RevMan 4.2.8 (The Cochrane Collaboration, Oxford, U.K.). The odds ratios of diagnostic yields of the two tests were examined as a measure of the outcome. Heterogeneity among the studies was assessed by chi-square test. Statistical significance for the test of heterogeneity was set at 0.10. If significant heterogeneity exists, it would be inappropriate to combine the data for further analysis using a fixed-effects model, alternatively, the random model was used for calculations, then sub analyses for the meta-analysis was planned according to the cause of heterogeneity.

RESULTS One hundred and sixty-three pieces of literature were initially identified using the search strategy described. One hundred and fifty-three articles were excluded after preliminary review for not having comparative studies, leaving

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Potentially relevant trials identified from search strategy. (n = 163)

One hundred and fifty-three articles were excluded after preliminary review of the titles.

Literatures retrieved for detailed evaluation. (n = 10) Two articles were excluded for no prospective trials. (n = 2)

Eight trials met inclusion criteria: 5 full-text articles and 3 abstracts. (n = 8)

Figure 1 Trial search flow for meta-analysis.

10 for detailed evaluation by two independent reviewers. Among these 10 potentially appropriate studies, an article retrospectively studied the yield of CE and DBE[5] and a review[6] were excluded. Finally, eight pieces of literature with 277 subjects met inclusion criteria, to include 5 fully published articles and 3 abstracts (Figure 1). Description of the studies Most patients suffered obscure GI bleeding. Besides, two patients with diarrhea and one patient with unclear weight loss were included in Damian’s study[7]. Three patients in Wi’s study[8] complained of chronic abdominal pain, one of them was excluded from statistics because his final diagnosis was functional GI disease. Nine patients with polyposis in Matsumots’s study[9] were excluded from data analysis because their diagnosis had been made in advance and multi-segments of GI tract were involved. In Nakamura’s study[10], two patients did not undergo DBE for severe cardiopulmonary impairment, one patient with anal bleeding was identified as having colon cancer before DBE, one patient suddenly declined DBE just before the examination, so the data of the 4 patients were deleted from statistical analysis. All patients of the other 7 studies underwent both CE and DBE. Double blind method was used in five studies: physicians evaluating CE and DBE should be blinded to the results of the other method. Matsumoto et al[9] did not mention this aspect in their paper. In Mehdizadeh’s[11,12] study, CE was performed prior to DBE. In Hadithi’s trial[13], the endoscopists were aware of the identity and clinical presentations of the patients, and of the results of the VCE at the time of performing DBE, but the outcome of his study indicated that the detection rate of CE was superior to DBE. Lesions causing GI bleeding were classified and counted in six studies. Capsule retention occurred in three patients. No DBE related adverse events were reported (Table 1). Among 277 subjects of the 8 studies, one hundred and seventy patients produced findings via CE and 156 by DBE. Homogeneity test of each OR displayed that heterogeneity did exist, the random model was used for calculations. The summary OR was 1.21 (95% CI: 0.64-2.29). There was no significant difference between the yield of CE and DBE (Figure 2).

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Table 1 Description of the included studies (CE/DBE) Researcher (yr)

Case number enrolled in study

Kameda N[14] (2006)1 Hadithi M (2006) Nakamura M (2006) Matsumoto T (2005) Wi JH (2006)1 Damian U (2006)1 Mehdizadeh S (2006) Zhong J[15] (2004)

Mean age

24 35 32 13 11 28 188 24

62 63 59 48 44 60 58 39

Male patients number 9 22 21 7 5 Not reported 85 16

Case number enrolled in analysis

Positive findings

24 35 28 13 10 28 115 24

15/16 28/21 17/12 10/6 7/9 19/14 63/57 11/21

Complication 1/0 0 0 0 0 0 0 2/0

Double blind method Yes No Yes Yes Yes Not reported No Yes

1

Abstracts from national meetings.

Study or sub-category Damian U Hadithi M Kameda N Matsumoto Mehdizadeh S Nakamura M Zhong J Wi JH

CE

DBE

n /N

n /N

19/28 28/35 15/24 10/13 63/115 17/28 11/24 7/10

OR (random) 95% CI

Weight %

14/28 21/35 16/24 6/13 57/115 12/28 21/24 9/10

277 Total (95% CI) 277 Total events: 170 (CE), 156 (DBE) 2 2 Test for heterogeneity: Chi = 17.40, df = 7 (P = 0.02), I = 59.8% Test for overall effect: Z = 0.58 (P = 0.56)

OR (random) 95% CI

13.94 14.11 12.95 8.87 20.19 14.14 10.59 5.21

2.11 2.67 0.83 3.89 1.23 2.06 0.12 0.26

[0.71, 6.25] [0.92, 7.77] [0.25, 2.72] [0.72, 21.06] [0.73, 2.07] [0.71, 5.98] [0.03, 0.52] [0.02, 3.06]

100.00

1.21 [0.64, 2.29]

0.1 0.2 0.5 1  2  5  10 Favours DBE Favours CE

Figure 2 Comparison of the yield of CE and DBE (All studies).The existence of heterogeneity was confirmed (chi2 test, P = 0.02), random model was used. The summary OR was 1.21 (95% CI = 0.64-2.29). There was no significant difference between the yield of CE and DBE (P = 0.56).

Sub analysis Homogeneity test displayed that there was heterogeneity in the meta-analysis. After reviewing each study intensively, we discovered that there were two insertion approaches applied in DBE procedures: oral and anal approaches. In some studies, two insertion approaches were combined: if no lesions were identified by one approach, the other one was performed, or all patients underwent both of the two approaches; in the other studies, most patients underwent just single DBE procedure, regardless of whether lesions were found or not. Single insertion approach could hardly cover the whole GI tract, so the detection rate it yielded was inevitably lower compared to the combination of both approaches. Therefore, we performed sub analysis according to the DBE insertion approaches used in each study. The method that DBE should be performed in each patient with combination of two insertion approaches was not used in 5 studies including 219 subjects, while the combination method was used in 2 studies including 48 subjects. Wi et al did not mention the DBE insertion approaches used in his abstract, therefore the data was given up in further sub analyses. The result of sub analysis indicated that the yield of CE was significantly higher than that of DBE without the combination of the two insertion approaches [137/219 vs 110/219, fixed model: OR 1.67 (95% CI: 1.14, 2.44), P < 0.01)] (Figure 3A); while the www.wjgnet.com

detection rate of DBE with combinatory oral and anal routes was higher, though not significantly, than that of CE, [26/48 vs 37/48, random model: OR 0.33 (95% CI: 0.05, 2.21), P > 0.05)] (Figure 3B). Unfortunately, the sample size in this study was not large enough to lead to an absolute conclusion, we expect that this difference will tell us more if the we enlarge the sample size. Meta-analysis focused on fully published literature on obscure GI bleeding Since meta-analyses are rigorous exercises that need to be subject to peer review, it seems that the data of abstracts from national meetings may not be reliable; abdominal pain and diarrhea will have incredibly low yields on DBE and CE and most will be functional, therefore, we focused the analysis on the fully published papers concerning obscure bleeding. Focused analysis also revealed the significantly higher yield of CE compared to DBE when the combination of two approaches was not used [118/191 vs 96/191, fixed model: OR 1.61 (95% CI: 1.07, 2.43), P < 0.05)] (Figure 4A); and the yield of CE was significantly lower than that of DBE when combinatory insertion approaches were used [11/24 vs 21/24, fixed model: OR 0.12 (95% CI: 0.03-0.52), P < 0.01)], just one study was included however (Figure 4B).

Chen X et al . Capsule endoscopy vs double-balloon enteroscopy

A

Study or sub-category Damian U Hadithi M Matsumoto T Mehdizadeh S Nakamura M

CE

DBE

n /N

n /N

19/28 28/35 10/13 63/115 17/28

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OR (fixed) 95% CI

Weight %

14/28 21/35 6/13 57/115 12/28

Total (95% CI) 219 219 Total events: 137 (CE), 110 (DBE) 2 2 Test for heterogeneity: Chi = 3.34, df = 4 (P = 0.50), I = 0% Test for overall effect: Z = 2.61 (P = 0.009) 0.1 0.2 0.5 1  2 Favours DBE

B

Study or sub-category

CE

DBE

n /N

n /N

OR (fixed) 95% CI

11.09 10.35 3.41 63.53 11.62

2.11 2.67 3.89 1.23 2.06

100.00

1.67 [1.14, 2.44]

[0.71, 6.25] [0.92, 7.77] [0.72, 21.06] [0.73, 2.07] [0.71, 5.98]

O-E 0.00 0.00 0.00 0.00 0.00

Variance 0.31 0.30 0.74 0.07 0.30

 5  10 Favours CE

OR (random) 95% CI

Weight %

01 Twe insertion routes of DBE were combined 16/24 Kameda N 15/24 21/24 Zhong J 11/24 48 48 Subtotal (95% CI) Total events: 26 (CE), 37 (DBE) 2 2 Test for heterogeneity: Chi = 4.09, df = 1 (P = 0.04), I = 75.5% Test for overall effect: Z = 1.14 (P = 0.25)

52.46 47.54 100.00

0.83 [0.25, 2.72] 0.12 [0.03, 0.52] 0.33 [0.05, 2.21]

O-E

0.00 0.00

Variance

0.37 0.55

Total (95% CI) 48 48 Total events: 26 (CE), 37 (DBE) 2 2 Test for heterogeneity: Chi = 4.09, df = 1 (P = 0.04), I = 75.5% Test for overall effect: Z = 1.14 (P = 0.25)

100.00

0.1 0.2 0.5 1  2 Favours DBE

OR (random) 95% CI

0.33 [0.05, 2.21]

 5  10 Favours CE

Figure 3 A: Comparison of the yields of CE and DBE without combination of the two insertion approaches. The yield of CE was significantly higher than that of DBE when the combination of two approaches was not used (fixed model, P = 0.009); B: Comparison of the yields of CE and DBE with the two insertion approaches combined. The yield of DBE with combinatory oral and anal routes was moderately higher than that of CE (random model, P = 0.25).

Comparison of counting Statistics of various lesions identified by the two endoscopies Lesions detected in 239 patients with obscure GI bleeding were gathered from 6 studies and counting statistics were calculated. There was no significant difference in detection rate of various lesions listed below between the two endoscopies. (SPSS11.0, chi-square test, The P values less than 0.05 were considered to be statistically significant, (Table 2).

DISCUSSION CE and DBE are both advanced inspection items. They have common indications and quite different features. CE can cover the whole GI tract; the procedure requires no sedation[16] and is better tolerated. Its major limitations are the inability to obtain a biopsy, precisely localize a lesion, or perform therapeutic endoscopy. Additionally, it may provide false-positive and false-negative findings due to its incontrollable movement and low-resolution pictures it takes. It was recently reported that CE missed an advanced small-intestinal cancer that was later diagnosed with push enteroscopy[17], suggesting that CE is not an exclusive procedure but should instead be complementary to other diagnostic tools in the assessment of small intestinal pathology. On the contrary, DBE has more advantages in that it is much better adjusted because the movement of

the scope can be handled according to the viewing angle and observation time needed; it can provide high-quality pictures, biopsy availability and therapeutic endoscopy[18,19]. DBE can be considered the gold standard if the whole small intestine were inspected. It appears to be equally as effective for the management of small-bowel lesions compared with intraoperative enteroscopy, and is associated with fewer complications[20,21]. Alternatively, it is not able to visualize the whole GI tract unless a combination of oral and anal insertion approaches is performed. The procedure is invasive and not as well tolerated as CE, requiring additional staff, typically two physicians or an additional nursing assistant. The meta-analysis indicated that the yield of CE was higher compared to DBE with a single insertion approach, but might be lower than that of DBE with a combination of oral and anal approaches. Currently, most patients will have DBE that is capsule-directed, and therefore physicians should be able to find the majority of lesions that are causing bleeding, taking into account that the course is less invasive and more likely to be accepted. Though CE is a highly sensitive modality for the diagnosis of small bowel disease, it cannot always provide a right instruction for DBE due to the potential false-positive and false-negative findings produced. In Mehdizadeh’s series, CE found a potential bleeding source in 63 patients, but 22 (34.9%) www.wjgnet.com

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Study or sub-category Matsumoto T Hadithi M Nakamura M Mehdizadeh S

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CE

DBE

n /N

n /N

10/13 28/35 17/28 63/115

World J Gastroenterol OR (fixed) 95% CI

Weight %

6/13 21/35 12/28 57/115

Total (95% CI) 191 191 Total events: 118 (CE), 96 (DBE) 2 2 Test for heterogeneity: Chi = 3.13, df = 3 (P = 0.37), I = 4.1% Test for overall effect: Z = 2.28 (P = 0.02) 0.1 0.2 0.5 1  2

Study or sub-category

CE

DBE

n /N

n /N

01 Twe insertion routes of DBE were combined 21/24 Zhong J 11/24 Subtotal (95% CI) Total events: 11 (CE), 21 (DBE) 24 Test for heterogeneity: no applicable Test for overall effect: Z = 2.85 (P = 0.004) Total (95% CI) 24 Total events: 11 (CE), 21 (DBE) Test for heterogeneity: no applicable Test for overall effect: Z = 2.85 (P = 0.004)

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OR (fixed) 95% CI

3.84 11.64 13.07 71.45

3.89 2.67 2.06 1.23

100.00

1.67 [1.07, 2.43]

[0.72, 21.06] [0.92, 7.77] [0.71, 5.98] [0.73, 2.07]

O-E

  Number 32 Variance

0.00 0.00 0.00 0.00

0.74 0.30 0.30 0.07

O-E

Variance

0.00

0.55

 5  10

Favours DBE

B

August 28, 2007

Favours CE OR (fixed) 95% CI

Weight %

OR (fixed) 95% CI

100.00 100.00

0.12 [0.03, 0.52] 0.12 [0.03, 0.52]

100.00

0.12 [0.03, 0.52]

24

24

0.1 0.2 0.5 1  2 Favours DBE

 5  10 Favours CE

Figure 4 A: Comparison of the yield of CE and DBE without combination of the two insertion approaches (focused on the fully published papers on obscure GI bleeding). The yield of CE was significantly higher than that of DBE when the combination of two approaches was not used (fixed model, P = 0.02); B: Comparison of the yield of CE and DBE with 2 insertion approaches combined (focused on the fully published papers on obscure GI bleeding). The yield of DBE with combinatory oral and anal routes was significantly higher than that of CE (fixed model, P = 0.004), just one study was included however.

Table 2 Counting statistics of various lesions identified by CE and DBE in patients with obscure GI bleeding

Angiodysplasia or phlebangioma Tumor Polyp Ulcer Erosion Crohn disease Diverticulum Fresh blood and clot or red spot Others Total Patients number

CE

DBE

P value

76 15 9 20 11 4 1 7 3 146 239

62 23 2 20 4 7 5 2 7 132 239

0.189 0.236 0.062 1 0.113 0.544 0.216 0.176 0.339 0.228

of them received negative results in the following DBE procedure. In Hadithi’s series, eight (28.6%) patients had positive findings on CE, whereas these lesions could not be verified by DBE. The inclusion of all lesions detected by CE, even trivial, may partly explain the large number of false positives. The fact that in the majority of cases DBE actually visualized only the jejunum, thereby decreasing its detection rate may provide an alternate explanation. It is clear that a single insertion procedure of DBE could not provide a full-scale evaluation for the multi-segment involving or multi-segment originating diseases like Crohn’s www.wjgnet.com

disease and polyposis. In fact, we cannot determine whether an identified lesion is localized until the whole GI tract was inspected, therefore a second DBE should usually be considered. The procedure of DBE in combination with the two insertion approaches may prove to be more sensitive and the outcomes would be more reliable. At the same time, there will be no need for the course and charge of CE if the diagnosis was made by a single DBE procedure or when a second DBE could not be avoided. This would be more economical in some countries like China (Table 3). Funnel plot (Figure 5) for the analysis of publication bias was performed on the six studies in which the DBE was carried out with single insertion approach, visual inspection of the plot revealed no evidence of publication bias. The number of the fully published papers was too small to analyze the publication bias and more studies on comparison of the yield of DBE with combinatory insertion approaches to CE are needed. In summary, our study displayed that CE and DBE are both effective modalities for diagnosis of small bowel diseases. A capsule-directed DBE procedure might be better tolerated; the procedure of DBE with combination of oral and anal approaches might be more sensitive and the outcome might be more reliable (Figure 6). Choice for initial test should depend on patient’s physical status, available technology, patient’s preferences, and potential for therapeutic endoscopy. On the other hand, more studies are needed

Chen X et al . Capsule endoscopy vs double-balloon enteroscopy

0.4 0.8

Cost per patient evaluated (RMB: Yuan) 8500 3746 12246 15992 7492

F i g u re 5 Funnel plot for the analysis of publication bias on the studies in which the DBE was performed without combination of the two approaches.

0.0 SE (log OR)

Table 3 Cost analysis (according to the charging standard in China, 2006)

CE DBE CE + DBE × 1 CE + DBE × 2 DBE × 2

4377

1.2 1.6

0.1

0.2

0.5  

1

A patient with suspicious small bowel disease who needs small bowel inspection The physical status is poor.

A less invasive modality is preferred. CE No lesion is identified; further ascertainment or therapeutic endoscopy is needed. DBE No lesion is identified1 or multi-segment involving lesion is found

A full-scale small bowel inspection is preferred or an intervention might be required.

CE A diagnosis is made, no need for CE DBE

Second DBE

1

Including that both CE and first DBE are negative, or about 30% of positive results from CE which cannot be verified by first DBE.

to evaluate the yield and accuracy of DBE with combinatory oral and anal insertion approaches compared to CE.

COMMENTS

Research frontiers The existing study concluded that indication and route for DBE could be determined according to the outcome of CE, which has been widely accepted in developed countries.

1 2

3

4 5

Innovations and breakthroughs The meta-analysis evaluated the diagnostic efficacy of CE compared to DBE and systematically summarized the included clinical trials and related literatures.

Applications The outcome of the meta-analysis is of referential value for management of small bowel disease. The choice between the two endoscopies could be made according to their diagnostic efficacy, cost benefit and patient’s tolerability.

6 7

Terminology

The term yield in the meta-analysis means the detection rate of capsule endoscopy or double-balloon enteroscopy, no matter the positive findings are truepositive or false-positive.

8

Peer review

The manuscript describes an interesting meta-analysis that is of outstanding relevance, well conducted, finely written and done according to a rigorous methodology.

Figure 6 Diagnosing flow according to DBE performed based on a capsuledirected strategy or a combinatory oral and anal routes strategy.

A second DBE is optimal, still no need for CE.

REFERENCES

Background Small intestine is the middle part of digestive tract which is hard to be inspected by conventional diagnostic modality. Capsule endoscopy and double-balloon enteroscopy, which are regarded as excellent tools for the diagnosis and treatment of small-bowel disease, have common indications and quite different features. How should we make a choice between the two diagnosis modality?

5 1 OR (Fixed)

No lesion is identified or multi-segment involving lesion is found.

Treatment Second DBE

2

9

Delmotte JS, Gay GJ, Houcke PH, Mesnard Y. Intraoperative endoscopy. Gastrointest Endosc Clin N Am 1999; 9: 61-69 Triester SL, Leighton JA, Leontiadis GI, Fleischer DE, Hara AK, Heigh RI, Shiff AD, Sharma VK. A meta-analysis of the yield of capsule endoscopy compared to other diagnostic modalities in patients with obscure gastrointestinal bleeding. Am J Gastroenterol 2005; 100: 2407-2418 May A, Nachbar L, Schneider M, Ell C. Prospective c o m p a r i s o n o f p u s h e n t e r o s c o p y a n d p u s h - and-pull enteroscopy in patients with suspected small-bowel bleeding. Am J Gastroenterol 2006; 101: 2016-2204 Gay G, Delvaux M, Fassler I. Outcome of capsule endoscopy in determining indication and route for push-and-pull enteroscopy. Endoscopy 2006; 38: 49-58 Masatsugu Shiba, Kazuhide Higuchi, Natsuhiko Kameda, Kaori Kadouchi, Hirohisa Machida, Kazuki Yamamori, Hirotoshi Okazaki, Masaki Hamaguchi, Tomoko Wada, Yoshio Jinnno, Eiji Sasaki, Kenji Watanabe, Kazunari Tominaga, Toshio Watanabe, Yasuhiro Fujiwara, Shirou Nakamura, Nobuhide Oshitani, Tetsuo Aeakawa. Wireless Capsule Endoscopy and Double-Balloon Enteroscopy in Japanese Patients with Obscure Gastrointestinal Bleeding. Gastrointest Endosc 2005; 61, AB182 Gerson LB, Van Dam J. Wireless capsule endoscopy and double-balloon enteroscopy for the diagnosis of obscure gastrointestinal bleeding. Tech Vasc Interv Radiol 2004; 7: 130-135 Damian U, Schilling D, Hartmann D, Weickertb U, Eickhoff A, Jakobs R, Kudis V, Riemann JF.Double- Balloon Enteroscopy (Push and Pull Enteroscopy) of the Small Bowel: Comparison with Video Capsule Endoscopy and Magnetic Resonance Imaging. Gastrointest Endosc 2006; 63: AB174 Wi JH, Kim JO, Eun SH, Kim SY, Jung IS, Ko BM, Cho JY, Lee JS, Lee MS, Shim CS, Kim BS.A Prospective Comparison of Capsule Endoscopy and Double Balloon Enteroscopy in Small Bowel Disease. Gastrointest Endosc 2006; 63: AB175 Matsumoto T, Esaki M, Moriyama T, Nakamura S, Iida M. Comparison of capsule endoscopy and enteroscopy with the www.wjgnet.com

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double-balloon method in patients with obscure bleeding and polyposis. Endoscopy 2005; 37: 827-832 Nakamura M, Niwa Y, Ohmiya N, Miyahara R, Ohashi A, Itoh A, Hirooka Y, Goto H. Preliminary comparison of capsule endoscopy and double-balloon enteroscopy in patients with suspected small-bowel bleeding. Endoscopy 2006; 38: 59-66 Mehdizadeh S, Ross AS, Leighton J, Kamal A, Chen A, Schembre D, Binmoeller K, Kozarek R, Waxman I, Gerson L, Jones B, Semrad C, Lo SK. Double Balloon Enteroscopy (DBE) Compared to Capsule Endoscopy (CE) Among Patients with Obscure Gastrointestinal Bleeding (OGIB): A Multicenter U.S. Experience. Gastrointest Endosc 2006; 63: AB90 Mehdizadeh S, Ross A, Gerson L, Leighton J, Chen A, Schembre D, Chen G, Semrad C, Kamal A, Harrison EM, Binmoeller K, Waxman I, Kozarek R, Lo SK. What is the learning curve associated with double-balloon enteroscopy? Technical details and early experience in 6 U.S. tertiary care centers. Gastrointest Endosc 2006; 64: 740-750 Hadithi M, Heine GD, Jacobs MA, van Bodegraven AA, Mulder CJ. A prospective study comparing video capsule endoscopy with double-balloon enteroscopy in patients with obscure gastrointestinal bleeding. Am J Gastroenterol 2006; 101: 52-57 Kameda N, Higuchi K, Shiba M, Tabuchi M, Sugimori S, Yukawa T, Kadouchi K, Okazaki H, Machida H, Inagawa M, Wada T, Tanigawa T, Yamagami H, Watanabe K, Watanabe T, Tominaga K, Fujiwara Y, Oshitani N, Arakawa T. A Prospective Trial Comparing Wireless Capsule Endoscopy

15

16

17

18 19 20 21

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and Double-Balloon Enteroscopy in Patients with Obscure Gastrointestinal Bleeding. Gastrointest Endosc 2006; 63: AB162 Zhong J, Zhang CL, Ma TL, Jin CR, Wu YL, Jang SH. Comparative study of double-balloon enteroscopy and capsule endoscopy in etiological diagnosis of small intestine bleeding. Zhonghua Xiaohua Zazhi 2004; 24: 741-744 Gerson LB, Van Dam J. Wireless capsule endoscopy and double-balloon enteroscopy for the diagnosis of obscure gastrointestinal bleeding. Tech Vasc Interv Radiol 2004; 7: 130-135 Madisch A, Schimming W, Kinzel F, Schneider R, Aust D, Ockert DM, Laniado M, Ehninger G, Miehlke S. Locally advanced small-bowel adenocarcinoma missed primarily by capsule endoscopy but diagnosed by push enteroscopy. Endoscopy 2003; 35: 861-864 Kita H, Yamamoto H. Double-balloon endoscopy for the diagnosis and treatment of small intestinal disease. Best Pract Res Clin Gastroenterol 2006; 20: 179-194 Yamamoto H, Kita H. Double-balloon endoscopy. Curr Opin Gastroenterol 2005; 21: 573-577 Gerson LB. Double-balloon enteroscopy: the new gold standard for small-bowel imaging? Gastrointest Endosc 2005; 62: 71-75 May A, Nachbar L, Ell C. Double-balloon enteroscopy (pushand-pull enteroscopy) of the small bowel: feasibility and diagnostic and therapeutic yield in patients with suspected small bowel disease. Gastrointest Endosc 2005; 62: 62-70 S- Editor Liu Y L- Editor Li M E- Editor Li JL

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World J Gastroenterol 2007 August 28; 13(32): 4379-4384 World Journal of Gastroenterology ISSN 1007-9327 © 2007 WJG . All rights reserved.

RAPID COMMUNICATION

Safety evaluation of donors for living-donor liver transplantation in Chinese mainland: A single-center report Guo-Qiang Li, Feng Zhang, Xiang-Cheng Li, Bei-Cheng Sun, Feng Cheng, Wen-Gang Ge, Xue-Hao Wang Guo-Qiang Li, Feng Zhang, Xiang-Cheng Li, Bei-Cheng Sun, Feng Cheng, Wen-Gang Ge, Xue-Hao Wang, Liver Transplantation Center, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China Correspondence to: Dr. Xue-Hao Wang, Liver Transplantation Center, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China. [email protected] Telephone: +86-25-83718836-6746 Fax: +86-25-83672106 Received: 2007-02-23 Accepted: 2007-03-26

Abstract AIM: To discuss the safety of donors during living donor liver transplantation (LDLT) and the authors’ experience with 50 cases. METHODS: Between January 1995 and March 2006, 50 patients with end-stage liver disease received LDLT in our department. Donors (at the age of 27-58 years) were healthy and antibody (ABO)-compatible. The protocol of evaluation and selection of donors, choice of surgical methods and strategy applied in the safety evaluation of donors were analyzed. RESULTS: A total of 115 candidate donors were e va l u a t e d f o r L D LT a t o u r c e n t e r. O f t h e s e , 5 0 underwent successful hepatectomy for living donation. The elimination rate for donors was 43.5%. Positive hepatitis serology and ABO incompatibility were the main factors for excluding candidates. All donors recovered uneventfully. The follow-up time ranged from 3 to 135 mo. The incidence of major and minor medical complications was 12.0% and 28.0%, respectively. CONCLUSION: LDLT provides an excellent approach to the problem of donor shortage in China. With a thorough and complete preoperative workup and meticulous intraand postoperative management, LDLT can be performed with minimal donor morbidity. © 2007 WJG . All rights reserved.

Key words: Liver transplantation; Living donor; Safety; Evaluation Li GQ, Zhang F, Li XC, Sun BC, Cheng F, Ge WG, Wang XH. Safety evaluation of donors for living-donor liver transplantation in Chinese mainland: A single-center Report. World J Gastroenterol 2007; 13(32): 4379-4384

http://www.wjgnet.com/1007-9327/13/4379.asp

INTRODUCTION Global shortage of cadaveric organs has been a serious obstacle to the development of liver transplantation (LTX), limiting the application of this life-saving therapy[1]. Living-donor liver transplantation (LDLT), as an innovative surgical technique to expand the available donor pool, has become popular in recent years. It was initially performed in Sao Paulo, Brazil, in 1988[2]. The recipients, from a Brazilian case series, died of medical complications shortly after the procedure. This was soon followed by a report from Strong et al[3] in Australia and Broelsch et al[4] in USA in 1989. Subsequently, the procedure was developed both in Asia and USA. In early 1990s, almost all the recipients were children. Removal of a partial left liver lobe for donation is a more conservative surgical procedure and has been shown to have a low risk for morbidity and mortality. With the experience in pediatric cases and cadaveric splitliver transplantation, LDLT has finally been extended to adult patients using mainly right lobe grafts. The first right hepatic lobe LDLT was reported in 1994[5], which has led to a dramatic increase in adult-to-adult right hepatic lobe LDLT. Despite the advantages of LDLT, the procedure has received criticism for the risk it imposes on healthy persons who will undergo a major operation without any potential health benefit. There have been several cases reported about donor death and significant donor morbidity (as high as 67%) [6-8]. It is clear that the risk of living liver donation is not negligible. The dictum “primum non nocere” (first do no harm) for the donor should remain the central factor for the whole process of LDLT[9]. In response to the shortage of cadaveric organs and a continually growing waiting list, the first case of LDLT in Chinese mainland was initiated at our center in 1995. The recipient, a man with primary liver cancer, received the left liver graft from his wife. Since then LDLT activity in Chinese mainland has been increasing rapidly. Accordingly, there is a greater concern for the safety of donors. However, no systemic reports on the safety of living liver donors are available in China. This study was to analyze the data of donors for LDLT in our center to illuminate the factors affecting the safety of donors. www.wjgnet.com

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MATERIALS AND METHODS From January 1995 to March 2006, 50 patients including 14 adults (aged 18 years or older) underwent LDLT at the First Affiliated Hospital of Nanjing Medical University. The age of donors ranged from 27 to 58 (mean 36.4 ± 12.3) years. Forty-nine donors fulfilled relationship within the third degree of consanguinity with recipients and 1 donor was a volunteer having good relationship with the recipient. Characteristics of donors are listed in Table 1. Evaluation and selection of donors The criteria for donor selection among different centers, and a thorough evaluation process consisting of three to six stages in most centers are described elsewhere[10,11]. The donor evaluation protocol was designed for testing proceeded from simple and noninvasive to more complex and invasive, assuming continued donor willingness and lack of contraindications to donation. Testing assured the donor safety and then evaluated the quality of graft. The minimal age for consideration was 20 and the upper age limit 60. The donor-recipient pair must be blood group identical or compatible. The donor evaluation protocol followed at our center is outlined in Table 2. When a potential recipient came to our center, he and his family members were informed of the need for an early liver transplantation and provided with information, upon their request, regarding living donor transplantation without specific reference to those who might be an appropriate candidate for liver donation. If the family members agreed to receive LDLT, the risks and benefits of the procedure would be explained in general. Initial counseling focused on the evaluation protocol, with emphasis on invasive testing, surgical procedure, and all possible risks of the donor hepatectomy. The donor should make the decision voluntarily, without any coercion. To reduce the pressure on potential donors, informed consent was obtained in the absence of other family members. The donor was given the opportunity to withdraw at any time, with the assurance that an excuse would be provided by the transplant team. Only after informed consent was made, could the evaluation of donors for medical or surgical suitability be commenced. Acute or chronic medical illness was excluded by a detailed history and physical examination, and all donors were screened by laboratory tests including complete blood cell count, liver and renal biochemistry values, and viral serologic studies. Positivity of hepatitis B surface antigen, human immunodeficiency virus antibody, or hepatitis C virus antibody constituted an outright ineligibility of the potential donor. Donors with diabetes mellitus or hypertension under regular control were not rejected. The psychologic status of the potential donor was assessed by a clinical psychologist. High-resolution abdominal ultrasonography (US) was performed to evaluate the quality of liver parenchyma, exclude the presence of tumors, and confirm the patency of blood vessels. Chest radiography and electrocardiography were performed to exclude cardiopulmonary disease. Computed tomography (CT), CT volumetry, multiple detector three-dimensional CT angiography, and three-dimensional magnetic resonance (MR) cholangiography were performed to assess liver volume and identify unsuspected intra-abdominal pathology and www.wjgnet.com

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Table 1 Evaluated donor demographics (n = 115) Evaluated donors Age (yr, range, mean ± SD) Weight (kg, mean ± SD) Sex Relationship to recipient

Evaluated and accepted donors 27-58 (36.4 ± 12.3) 53.7 ± 12.9 M F Mother Father Wife Husband Uncle Sister/brother Volunteer

Evaluated and rejected donors 31-55 (37.4 ± 10.6) 64.4 ± 14.3

13 37 33 12 2 0 1 1 1

23 42 16 28 9 3 5 4 0

Table 2 Current donor evaluation protocol at the First Affiliated Hospital of Nanjing Medical University Basic requirements Step Ⅰ

Step Ⅱ

Step Ⅲ

20–60 yr Relationship: relatives or unrelated volunteers Blood type: identical or compatible Clinical evaluation: initial informed consent, history and physical examination Laboratory: blood group, liver and renal function Serology: HBsAg, HBsAb, HBcAb, anti-HCV, anti-HIV Clinical examination: psychological evaluation Laboratory: hematology, coagulation profile, blood sugar, electrolytes, HLA typing, cross-matching, alpha-1antitrypsin, ferritin, tumor markers (AFP, CEA,CA199, CA50), arterial blood gas,urine and stool analysis, pregnancy test (female) Serology: HBV DNA, RPR, antibody for CMV, EBV, HSV, varicella and rubella viruses Imaging study: Chest radiograph, abdominal ultrasound, ECG Imaging study: CT angiography and volumetry, MR cholangiography

anomalous vasculature incompatible with donation. Liver biopsy was not routinely performed in our center. If there was radiographic evidence of fatty infiltration or parenchymal liver disease, even with normal liver function, echoguided liver biopsy of the segments to be donated was performed. If a questionable result arose from the standard protocol, more testing may have been indicated. After completion of each step, the donors’ statuses were reevaluated and a decision of whether to proceed was made by the transplant team. The Ethics Committee of the First Affiliated Hospital of Nanjing Medical University approved the surgical procedures. Donor evaluation continued in the operating room with intraoperative cholangiography and ultrasonography. These studies were used for planning operative strategy and excluding the presence of prohibitive anatomical variations not detected by the preoperative work-up. Surgical procedures for donors The donors were prepared on the operating table with care to avoid sores at the pressure points. The abdomen was entered through a bilateral subcostal incision with a

Li GQ et al . Safety of donors in living donor liver transplantation

Table 3 Exclusion criteria for 65 potential donors Step

Exclusion

n

Ⅰ

Clinical evaluation Blood type Diabetes Hepatitis B (core Ab +) Hepatitis C Autoimmune hepatitis Psychological evaluation Cardiac evaluation CT or MRI findings CT volumetry (steatosis-GRBW) Second consent

4 27 1 17 2 1 1 2 1 3 6

Ⅱ Ⅲ Ⅳ

GRBW: graft recipient body weight.

vertical midline extension. Harvesting of the left lobe of liver from a living donor was performed as previously described[12,13]. The left hepatic artery was dissected, exposing the left portal vein lying posteriorly. After complete left portal vein dissection, the bile duct or ducts were sharply transected at the edge of the graft. Finally, parenchymal dissection was performed using an ultrasonic dissector along the line marked on the liver surface according to intraoperative ultrasonogram, and all tubular structures were ligated or sutured. For right lobectomy, the surgical technique has been described in detail elsewhere. After mobilization of the right lobe, the hilar plate was lowered and the right bile duct or ducts were divided sharply. All portal vein branches to the caudate process were divided and ligated. The right lobe of liver was then rotated toward the left side for division of right triangular ligament and tiny venous branches between the anterior surface of the inferior vena cava and posterior surface of paracaval portion of the caudate lobe. The right hepatic vein and the right inferior hepatic vein larger than 5 mm were preserved for reimplantation. Transection was done with both electrocautery and an ultrasonic dissector. The stumps of the right hepatic artery, right portal vein, and hepatic veins were closed with continuous nonabsorbable surgical sutures. The grafts were harvested without blood vessel clamping or graft manipulation in order to optimize their viability. Intraoperative ultrasonography was performed to identify the presence of a large inferior hepatic vein (accessory left or right hepatic vein) and to study the junction of the middle hepatic vein with the inferior vena cava. After cholecystectomy, operative cholangiography via cystic duct cannulation was performed to study the bile duct anatomy. In all cases, the graft was immediately transferred to the back-table for flushing of the hepatic artery and portal vein with preservation fluid at 4℃ and prepared for implantation. Postoperative management The donors were cared for in the intensive care unit with attention to adequate tissue oxygenation and perfusion. Antimicrobial medications were prescribed to prevent infection. Parenteral nutrition consisting of a mixture of branched-chain amino acid-enriched solution, dextrose, and medium- and long-chain triglycerides was administered

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in all donors immediately after the hepatectomy to stimulate liver regeneration. Oral nutrition was encouraged once bowel activity returned. Chest physiotherapy and incentive spirometry were routinely given. Early ambulation was advocated. Daily laboratory monitoring continued until liver function normalized and hemoglobin and electrolytes were stable. Follow-up Outcomes related to complications and ongoing symptoms were defined as medical outcomes. A major medical complication was defined as a medical problem requiring surgical or procedural repair, hospitalization, or intravenous therapy. A minor medical complication was defined as a medical problem either resolved spontaneously or requiring oral medical therapy. A specific research assistant was in charge of the whole follow-up. The methods were taken including record table, telephone follow-up and return visit. Statistical analysis Data are expressed as mean ± SD. Statistical analysis was completed by using SPSS 10.0 (SPSS Inc, Chicago, IL).

RESULTS A total of 115 candidate donors were evaluated for LDLT at our center. Of these, 50 underwent successful hepatectomy for living donation. A total of 65 potential donors (56.5%) were excluded at different points of the work-up. Positive hepatitis serology and ABO incompatibility were the main contraindications to donation. After the first step, volunteers withdrew from donation due to effects from family, relatives and society, with society being the main reason for exclusion. Reasons for exclusion of potential donors are listed Table 3. Donor livers were resected from segments Ⅱ, Ⅲ, and (including the middle hepatic veins) in 36 cases, segⅣ ments Ⅱ, Ⅲ, and part of Ⅳ (not including the middle hepatic veins) in 4 cases, segments Ⅴ, Ⅵ, Ⅶ, and Ⅷ (not including the middle hepatic veins) in 9 cases and segments Ⅴ, Ⅵ, Ⅶ, and Ⅷ (including the middle hepatic veins) in 1 case. The mean measured graft volume was 246 ± 57 g in pediatric recipients and 736 ± 189 g in adult recipients. The average graft/recipient weight ratio (GRWR) was (1.21 ± 0.43)%. Two child patients, who received small-graft with a GRWR less than 0.8%, recovered without any serious complications. The mean duration of the operation from skin incision to closure was 7.6 ± 1.2 h and the mean blood loss was 450 ± 130 mL. The mean stay of donors in the intensive care unit (ICU) was 1.2 ± 0.4 d and the mean hospital stay was 9.3 ± 1.8 d. In the immediate postoperative period, all donors exhibited a significant transient elevation of liver enzymes and hyperbilirubinemia on postoperative day. Normalization of serum transaminases and total bilirubin was accomplished by postoperative d 5 to 7. In contrast, prothrombin time exhibited a mild postoperative elevation that declined to normal level within 3 d. During followup, CT showed that the remaining liver in the right-lobe www.wjgnet.com

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Survival Complications Major Biliary leak Pneumonia Initial stage of liver dysfunction Pleural effusions Minor Delay in return to normal bowel function Pain Sore throat Foot paresthesias Persistent short-term memory loss Chronic fatigue Ongoing symptoms Yes No Abdominal discomfort Scar numbness Loss of appetite, nausea Poor appetite Diarrhea Weakness Nausea Difficulty sleeping Sought other physician assistance Yes No

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n (%) 50/50 (100)

DISCUSSION

6/50 (12.0) 2 2 1 1 14/50 (28.0) 5 3 2 2 1 1

The development of LDLT in China experiences three stages: pediatric living donor liver transplantation (PLDLT), adult-to-adult living donor liver transplantation (ALDLT), emergency or high-urgency living donor liver transplantation (ELDLT). To ensure the safety of donors, we identified three basic principles for the selection of donors: independent decision on donation, no contraindication for donation, and avoidance of coercion in the process of donation. These principles were strictly fulfilled with no exceptions. The evaluation process may involve invasive liver biopsy, angiography, and endoscopic retrograde cholangiopancreatography, etc. With advances in radiologic evaluation of the liver, donors can be safely evaluated and liver resection approaches can be planned with new imaging techniques. In our series, CT volumetry, multiple detector, three-dimensional CT angiography, and three-dimensional magnetic resonance (MR) cholangiography were employed to provide an accurate picture of liver vascular anatomy and liver volume measurement for surgery. Kubota et al[14] showed that individuals with normal liver function could tolerate resection of up to 60% of nontumorous liver. The present data, albeit limited, indicate that residual liver volume, accounting for 27% of the total volume, is the lowest limit for supporting survival, provided that the liver itself is not fatty. To allow a safety margin, the residual liver volume, accounting for 30% of the total volume, is probably the lowest limit. Accordingly, CT volumetry should be routinely performed because the volume of the resected graft could be quite variable. Liver biopsy is not performed only upon indication. If there is radiographic evidence of fatty infiltration or parenchymal liver disease, even with normal liver function, echo-guided liver biopsy from the segments to be donated is performed. The amount of steatosis is a matter of concern because fatty liver is probably more vulnerable to injury during ischemia-reperfusion. Liver grafts with a mild degree of fatty change can be used for liver transplantation without adverse effects. However, liver grafts with a moderate or severe degree of fatty change lead to primary graft dysfunction in recipients[15]. Therefore, it is wise to limit the macroscopic fat content to less than 10% and even lower when the planned resection volume exceeds 60%. Our lower and upper age limits were set at 20 and 60 years, respectively. The minimal age of 20 years mainly concerns the issue of informed consent. There are some issues on which the upper age limit should be set. It was reported that liver transplantation with grafts from donors older than 65 years can achieve good results[16]. To lower the risk of donors and recipients, we set the upper age limit at 60 years. Parents are the majority of donors for pediatric patients, but the parents of adult recipients are frequently not candidates for donation due to their advancing age or underlying disease. The “one-child” policy in China

28 (56.0) 22 (44.0) 15 6 3 3 2 1 1 1 12 (42.9) 16 (51.1)

Major and minor complications are defined in Methods. Under ongoing symptoms, the number of symptoms reported is greater than 28.

donors grew as large as (or larger than) the original one after 10 to 14 mo, whereas the recovery time of the leftlobe donors was 6 to 11 mo. Maximum liver growth occurred within the first month after donation, followed by a gradual increase during the first postoperative year. The follow-up time ranged from 3 to 135 mo. Followup was not lost for any one. Up to March 2006, the first living donor in Chinese mainland, who donated her left liver graft to her husband, was followed up over 11 years. She was in good health and returned to work. The mean recovery time of 41 donors who were followed up for more than 6 mo, was 6.0 ± 1.5 mo, the mean time to return to work was 8.0 ± 1.0 mo, and 35 of them returned to normal work even earlier. No reoperation was performed and no death occurred in this series. Six of the 50 (12.0%) patients had major complications. One donor suffered from symptoms at the initial stage of liver dysfunction, which disappeared after conservative management. Two donors had biliary leakage requiring percutaneous drainage and antibiotic treatment. One donor required chest tube placement because of right pleural effusions. Two donors had pneumonia, requiring antibiotics intravenously. Fourteen of the 50 (28.0%) patients had minor complications related to the operation and were managed conservatively. Complications included delayed bowel function, pain, sore throat, foot paresthesias, persistent short-term memory loss and chronic fatigue. Symptoms occurred at the time of survey in 28 patients (56.0%). These symptoms led 12 patients to seek

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medical intervention and resolved after treatment. Medical outcomes are listed in Table 4.

Table 4 Donor medical outcomes Donor medical outcomes

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Li GQ et al. S afety of donors in living donor liver transplantation

limits the availability of siblings as donors. Consequently, genetically unrelated donors may be necessary alternatives to conventional living donation for adults. However, any commercially available human organs must be extremely forbidden. In our series, one man with severe liver failure due to hepatitis B and cirrhosis needed timely liver transplantation. Since his wife and relatives were excluded because of medical or psychological reasons, he finally received the right liver graft from his friend. In our study, positive hepatitis B serology was the main reason for exclusion besides ABO incompatibility. Positive hepatitis B serology occurred in 26.2% of potential donors. Because of the high prevalence of hepatitis B virus infection in China, whether donors with positive hepatitis B core antibody should be ruled out is still controversial. Four anatomic grafts have been described for LDLT[17], including the entire right liver lobe (Couinaud segments ⅤⅧ), the entire left liver lobe (Couinaud segments Ⅱ-Ⅳ), the left lateral segment (Couinaud segments Ⅱ-Ⅲ), and the extended right liver (Couinaud segments Ⅳ-Ⅷ). The type of graft selection depends mostly on the size disparity between the donor and recipient. In general, the left lobe comprising segments Ⅱ-Ⅳ (including or not including the middle hepatic veins) is used for pediatric recipients and segments Ⅴ-Ⅶ (including or not including the middle hepatic veins) for adult recipients. Whether graft resection for LDLT includes the middle hepatic vein is an important issue. Maema et al[18] have reported their experience in living donor left lobectomy with (extended left lobe) or without (standard left lobe) resection of the middle hepatic vein. They believe that venous outflow is essential to both function and regeneration[18]. Inadequate outflow results in decreased segmental portal flow and poor segmental regeneration. Even when no grafts are grossly congested, regeneration is clearly impaired in grafts but not in the middle hepatic vein, suggesting that the right lobes should be used. Sufficient medical data indicate that segments Ⅱ-Ⅲand part of segment Ⅳ (not including the middle hepatic veins) can be removed and would not compromise the donor’s metabolic potential. In the series, LDLT was performed in 4 cases with removal of segments Ⅱ-Ⅲ and part of segment Ⅳ (not including the middle hepatic veins). Although the graft volume met the metabolic demand of the recipient, graft congestion due to poor venous outflow delayed recovery of the recipient postoperatively. As experience and confidence grow, extended left lobectomy (segments Ⅱ-Ⅳ, including the middle hepatic veins) is wildly applied in LDLT in our center. LDLT was performed in 36 cases and extended left lobectomy in 2 cases whose GRWR was 0.97% and 1.06%, respectively. Both donors and recipients recovered uneventfully and were in good health. We concluded by reconstructing middle hepatic vein tributaries or other accessory venous tributaries, graft congestion, apt to thrombosis and graft dystrophia or primary dysfunction due to poor venous outflow can be solved properly. To overcome the barrier of graft size matching for adult recipients, right lobectomy was performed for ALDLT in our study. This method was initially described by Habib and Tanaka[19], who attempted to harvest a left lobe for LDLT when anatomic considerations favored a right-

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lobe hepatectomy. We consider that for right lobe grafts, inclusion or exclusion of the middle hepatic vein depends mostly on the size disparity between the donor and recipient, as well as the adequacy of graft volume, remnant liver volume, and configuration of the hepatic venous anatomy. If the size of the donor and recipient is similar, right lobectomy (Couinaud segments Ⅴ-Ⅷ, not including the middle hepatic veins) is preferred to ALDLT in our center. To avoid graft congestion, the right inferior hepatic vein larger than 5 mm can be reconstructed to secure adequate drainage. If the size disparity between the donor and recipient is conspicuous (the size of the recipient was much bigger than that of the donor), extended right lobectomy (Couinaud segments Ⅳ-Ⅷ, including the middle hepatic veins) can be employed to obtain an adequate graft volume. Utilization of extended right lobe liver grafts (the middle hepatic veins) in ALDLT was introduced by the University of Hong Kong Medical Center in 1996[20]. We first performed extended right lobe-LDLT in 2005. About 60% of the donor liver volume was resected for donation. The donor suffered from symptoms at the initial stage of liver dysfunction, which disappeared after conservative treatment. We consider that it is necessary to leave the donor with a residual liver volume, accounting for at least 30% of the total liver volume, and to use grafts containing minimal fatty to secure the safety of donors. To ensure that the remnant liver regenerates quickly after operation, intraoperative trauma to the remnant liver must be minimal. Thus, we exercised the liver carefully and intermittently rotated the right lobe during mobilization, exerted no Pringle maneuver during liver transection, reconstructed the falciform ligament to prevent left lobe rotation into the right subphrenic cavity, and initiated postoperative parenteral nutrition support to stimulate liver regeneration[21]. Methylene blue was injected into the common bile duct via the cystic duct cannula to detect bile leakage from the right hepatic duct stump and transection surface. In conclusion, LDLT can achieve acceptable survival and solve the issue of donor availability. We believe that with a thorough and complete preoperative workup and meticulous intraoperative and postoperative management, LDLT can be performed.

REFERENCES 1

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Manyalich M, Cabrer C, Valero R, Paredes D, Navarro A, Trias E, Vilarrodona A, Ruiz A, Rodriguez C, Paez G. Transplant procurement management: a model for organ and tissue shortage. Transplant Proc 2003; 35: 2533-2538 Raia S, Nery JR, Mies S. Liver transplantation from live donors. Lancet 1989; 2: 497 Strong RW, Lynch SV, Ong TH, Matsunami H, Koido Y, Balderson GA. Successful liver transplantation from a living donor to her son. N Engl J Med 1990; 322: 1505-1507 Broelsch CE, Emond JC, Whitington PF, Thistlethwaite JR, Baker AL, Lichtor JL. Application of reduced-size liver transplants as split grafts, auxiliary orthotopic grafts, and living related segmental transplants. Ann Surg 1990; 212: 368-375; discussion 375-377 Baltz AC, Trotter JF. Living donor liver transplantation and hepatitis C. Clin Liver Dis 2003; 7: 651-665, viii

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Pomfret EA, Pomposelli JJ, Lewis WD, Gordon FD, Burns DL, Lally A, Raptopoulos V, Jenkins RL. Live donor adult liver transplantation using right lobe grafts: donor evaluation and surgical outcome. Arch Surg 2001; 136: 425-433 Surman OS. The ethics of partial-liver donation. N Engl J Med 7 2002; 346: 1038 Vastag B. Living-donor transplants reexamined: experts cite 8 growing concerns about safety of donors. JAMA 2003; 290: 181-182 Rao AR, Chui AK, Chan HL, Hui AY, Island E, Lau WY. 9 Complications of liver donation for living related liver transplantation. Transplant Proc 2004; 36: 2224-2225 10 Marcos A, Fisher RA, Ham JM, Olzinski AT, Shiffman ML, Sanyal AJ, Luketic VA, Sterling RK, Olbrisch ME, Posner MP. Selection and outcome of living donors for adult to adult right lobe transplantation. Transplantation 2000; 69: 2410-2415 11 Chen YS, Cheng YF, De Villa VH, Wang CC, Lin CC, Huang TL, Jawan B, Chen CL. Evaluation of living liver donors. Transplantation 2003; 75: S16-S19 12 Broelsch CE, Whitington PF, Emond JC, Heffron TG, Thistlethwaite JR, Stevens L, Piper J, Whitington SH, Lichtor JL. Liver transplantation in children from living related donors. Surgical techniques and results. Ann Surg 1991; 214: 428-437; discussion 437-439 13 Emond JC, Heffron TG, Kortz EO, Gonzalez-Vallina R, Contis JC, Black DD, Whitington PF. Improved results of livingrelated liver transplantation with routine application in a pediatric program. Transplantation 1993; 55: 835-840

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Kubota K, Makuuchi M, Kusaka K, Kobayashi T, Miki K, Hasegawa K, Harihara Y, Takayama T. Measurement of liver volume and hepatic functional reserve as a guide to decisionmaking in resectional surgery for hepatic tumors. Hepatology 1997; 26: 1176-1181 15 Angelico M. Donor liver steatosis and graft selection for liver transplantation: a short review. Eur Rev Med Pharmacol Sci 2005; 9: 295-297 16 Lopez-Navidad A, Caballero F. Extended criteria for organ acceptance. Strategies for achieving organ safety and for increasing organ pool. Clin Transplant 2003; 17: 308-324 17 Renz JF, Yersiz H, Farmer DG, Hisatake GM, Ghobrial RM, Busuttil RW. Changing faces of liver transplantation: partialliver grafts for adults. J Hepatobiliary Pancreat Surg 2003; 10: 31-44 18 Maema A, Imamura H, Takayama T, Sano K, Hui AM, Sugawara Y, Makuuchi M. Impaired volume regeneration of split livers with partial venous disruption: a latent problem in partial liver transplantation. Transplantation 2002; 73: 765-769 19 Habib N, Tanaka K. Living-related liver transplantation in adult recipients: a hypothesis. Clin Transplant 1995; 9: 31-34 20 Fan ST, Lo CM, Liu CL, Wang WX, Wong J. Safety and necessity of including the middle hepatic vein in the right lobe graft in adult-to-adult live donor liver transplantation. Ann Surg 2003; 238: 137-148 21 Fan ST, Lo CM, Liu CL, Yong BH, Chan JK, Ng IO. Safety of donors in live donor liver transplantation using right lobe grafts. Arch Surg 2000; 135: 336-340 S- Editor Zhu LH L- Editor Wang XL

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World J Gastroenterol 2007 August 28; 13(32): 4385-4390 World Journal of Gastroenterology ISSN 1007-9327 © 2007 WJG . All rights reserved.

RAPID COMMUNICATION

New precut sphincterotomy for endoscopic retrograde cholangiopancreatography in difficult biliary duct cannulation Deng-Hao Deng, Hong-Mei Zuo, Jia-Feng Wang, Zhi-E Gu, Hong Chen, Yuan Luo, Ming Chen, Wen-Nuo Huang, Lu Wang, Wei Lu Deng-Hao Deng, Hong-Mei Zuo, Jia-Feng Wang, Zhi-E Gu, Hong Chen, Yuan Luo, Ming Chen, Wen-Nuo Huang, Lu Wang, Wei Lu, Department of Gastroenterology, Clinical Medical College of Yangzhou University, No. 98 Nangtong West Road, Yangzhou 225001, Jiangsu Province, China Correspondence to: Deng-Hao Deng, Department of Gastroenterology, Clinical Medical College of Yangzhou University, No.98 Nangtong West Road, Yangzhou 225001, Jiangsu Province, China. [email protected] Telephone: +86-514-7101209 Fax: +86-514-7937406 Received: 2006-12-28 Accepted: 2007-01-25

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group (13.3% vs 9.3%, χ = 0.478, P > 0.05). CONCLUSION: VNTPS procedure and Deng’s precut are highly effective methods to get biliary access during ERCP with DBC. With skillful techniques, it can increase the successful rate for deep cannulation of biliary duct and decrease complications. VNTPS technique, especially Deng’s precut is as effective and safe as EST. This technique can be well performed in hospitals without particular equipments. © 2007 WJG . All rights reserved.

Abstract AIM: To retrospectively investigate the effect and safety of various new type precut sphincterotomy techniques (VNTPST) in endoscopic retrograde cholangiopancreatography (ERCP) due to difficult biliary duct cannulation (DBC). METHODS: A plough-like pull-type sphincterotome (PLPTS) or improved short nose sphincterotome or improved needle knife was applied. VNTPST was carried out in 30 of 280 patients, whose biliary tract could not be exposed well or deep cannulation was difficult to perform during ERCP with traditional methods. Patients were followed up for short-term complications and the therapeutic effect of VNTPS was observed and compared with that of traditional endoscopic sphincterotomy (EST). RESULTS: A total 280 patients underwent ERCP, of which 3 failed in operation because of pathological features in stomch or duodenum, 247 successfully underwent traditional ERCP (89.1%, 247/277), 30 failed (10.8%, 30/277). VNTPS technique succeeded in 24 (80%, 24/30) of 30 cases. The successful rate of deep biliary duct cannulation increased 8.6% (24/277), the total cannulation successful rate following precut was 97.7%. There was a significant difference between the 2 two groups (97.7% vs 89.1%, χ = 17.1, P < 0.01). The incidence of complications was 9.3% (26/277) for traditional ERCP group and 13.3% (4/30) for VNTPS technique group. Guideline tip was broken in pancreatic d u c t ( K P D G P ) o f one pat ie nt , and t he re was no pancreatitis, slight or moderate bleeding postoperatively occurred in 2 patients, 1 patient had bleeding during operation (PDWN). There were no differences between VNTPS technique group and traditional ERCP (TRERCP)

Key words: Endoscopic retrograde cholangiopancreatogr aphy; Difficult deep cannulation; Varied new type precut sphincterotomy techniques; Postoperative complications Deng DH, Zuo HM, Wang JF, Gu ZE, Chen H, Luo Y, Chen M, Huang WN, Wang L, Lu W. New precut sphincterotomy for endoscopic retrograde cholangiopancreatography in difficult biliary duct cannulation. World J Gastroenterol 2007; 13(32): 4385-4390 http://www.wjgnet.com/1007-9327/13/4385.asp

INTRODUCTION McCune reported ERCP in 1968 for the first time[1]. In China, ERCP was imported by Peking Union Hospital in 1974. Dr. Ming-Zhang Chen initially reported endoscopic sphincterotomy (EST), but it has a lower successful rate. With the advances in surgical techniques and equipments, the successful rate for ERCP was 96.1% in 1990s in China[2], which is similar to the international reports[3,4]. Due to various factors, catheter cannot always be inserted into the bile duct successfully, leading to failure in TERCP. Precut was initially reported by Siegel[5], the successful rate for ERCP can go up with the success of precut. It was reported that the successful rate for precut is 77%-91% [6,7]. The application of precut is limitted [8-11] because more complications occur, few reports on its use are available. We retrospectively analyzed the clinical data obtained from 280 cases and evaluated the effect and safety of VNTPST in diagnostic and therapeutic endoscopic retrograde cholangiopancreatography (DTERCP) with DBC. www.wjgnet.com

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MATERIALS AND METHODS Patients Two hundred and eighty patients underwent ERCP in our hospital from April 2004 to September 2006, of them 247 succeeded in TRERCP, 30 (13 males, 17 females, aged 26-88 years, with a mean age of 66 years) failed, and 24 succeeded in percut technique. Among the 30 cases, 7 were diagnosed as Oddi’s sphincer stenosis (OSS) combined with choledocholithiasis, 2 as inlayed choledocholithiasis, 8 as simple constrictive papillitis (one as combined juxtapapillary diverticula), 3 as pancreatic head carcinoma, 2 as duodenal papilla carcinoma, 1 as papillary adenomatoid hyperplasia, 3 as ampullary carcinoma and distal bile duct cancer (DBDC), 2 as common bile duct inflammatory strictures, 2 as upper and middle bile duct carcinoma combined with inflammatory papilla stenosis. GF240 electric duodenal endoscope and PLPTS were from Olympus Company. Modified short nose sphincterotome or needle knife was a plough-like pull-type sphincterotome (PLPTS). An Olympus’s high-frequency electrosurgical unit and several kinds of guide wire were used in all procedures. Procedures Preoperative preparation was the same as the standard ERCP. Patients underwent standard ERCP at first, followed by VNTPST when deep cannulation was difficult. Modified pancreatic sphincter precutting (MPSP): Before precut, the exact direction of incision was determined and some movements were made in the correct direction as previously described (Figure 1 A-C)[12]. Incision was made in 10-12 o’clock point from the papillary orifice. The pure precut currently used was set at index 3.5-4.5, precut was performed by exerting pressure with the wire at the roof of PLPTS. The spin and PLPTS were lifted and the precut length was about 0.5-0.8 cm. The incision was extended in the submucosa until biliary effusion was detected. The precut was successful when catheter or guide wire was inserted into the common bile duct (CBD). General EST was performed with guide wire. A needleknife precut was used if biliary cannulation failed. MPSP was more effective for type “Y” pancreaticobiliary ductal junction or type “ Ⅴ ” without papillary stenosis. The pancreatic sphincter could be inserted with the help of guide wire or scalpel. PSP was not available for type “Ⅱ”. No bleeding and perforation occurred. MPSP was safe and less dangerous, but complications such as pancreatitis occurred. Electrical coagulation and blending current should be limited Precut down with needle (PDWN): When biliary cannulation was unsuccessful due to papillary stenosis, ampullary edema or abnormal ampullary anatomy (Figure 2 A-D). PDWN improved by PLPTS should be applied. To expose the needle (3-5 mm in length), the power source was set at index 3.5-4.5 with the exact direction adjusted. Incision was made in the 11 o’clock direction to the papillary orifice. The electric current index depended on the ampullary size and the edema degree. The incision was extended in www.wjgnet.com

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Figure 1 Different MPSP precut methods (A-C).

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Figure 2 Before precut down with needle knife (A, B) and after precut (C, D).

layers from the papilla duodenal mucosa to the bile duct sphincter or bile seepage, then a catheter assisted with ultra-slipping guide wire was inserted into the bile duct. PDWN was performed repeatedly if biliary cannulation failed. When the bile duct sphincter was exposed or the incision was very deep, and the bile duct orifice could not be found or a catheter could not be inserted, it suggested that the anatomy of common bile duct was abnormal and precut should be stopped. Otherwise biliary duct or duo-

Deng DH et al . Precut sphincterotomy for ERCP

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Figure 3 Before precut up with needle knife (A) and after precut (B).

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Figure 5 Precut with up-removal orifice technique (A, B) and after precut (C).

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The depth of incision by SNKP was easy to control, not resulting in perforation, but it was not so convenient in comparison with a needle knife for duodenal ampulla calculus incarceration. Pancreatic duct guideline precut (PDGP): A short nose knife or needle-knife was inserted into the papilla orifice while keeping the pancreatic duct guideline. The direction and depth could be controlled by pancreatic duct guide wire, which was not convenient for the small aperture endoscope.

Figure 4 Before Deng'S precut (A-C) and after Deng'S precut (D).

denum perforation might occur. Since perforation would occur after EST due to moving up of the incision orifice after precut, EST should not be very long. Precut up with needle (PUWN): PUWN was suitable for the mini papilla with a small orifice (Figure 3 A-B). The pure cutting current was used in the course, with the papilla adjusted to the left side of the visual field, the wire (3 mm) exposed, the needle anchored at the orifice. The bridge or elevator rotating the endoscope in the direction of anticlockwise was lifted while the current was applied for incision. PUWN was performed. The following step was the same as PDWN. This kind of precut might cause pancreatitis and the depth of incision was variable and uncontrolled. PUWN was not performed when the pailla orifice was not detectable. Short nose knife precut (SNKP): SNKP could be performed when papilla orifice was variable. A short nose knife was inserted into the orifice in 11 o’clock direction with pure cutting current until a seepage of bile was detected, then a standard catheter was inserted into CBD.

Mucosal bridge precut (Deng’s precut): When PLPTS was inserted into the bile duct with difficulty, the assistant strained the knife tightly to bend the sphincterotome, with the knife action adjusted in line with bile duct axis, and inserted the PLPTS tip from the papillary orifice, the hard guideline piercing through the ampullary duodenal mucosa, cut the ampullary duodenum side mucosal bridge until a seepage of bile was detectable, then inserted the guideline or the catheter into the duct (Figure 4 A-D). The performance could be followed by NKF if the attempt failed. This method could also be applied in patients with inflammatory papilla stenosis or combined upward papillary diverticula. Up-removal orifice technique (UROT): This technique was applied when bile duct cannulation could not be achieved for the papilla orifice or bending endoscope could not be lifted up to the orifice (Figure 5 A-C). The assistant strained PLPTS tightly and tried to access the orifice with PLPTS tip, then raised the papilla mucosa and cut it for removing the orifice. When the bile duct axis was exposed after UROT, normal standard cannulation was easily performed. Bending or rotating endoscope technique(BERET): When the papilla could not be lifted due to pathological www.wjgnet.com

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chi-square test. P < 0.05 was considered statistically significant.

RESULTS

Figure 6 Before (A) and after precut (B) with bending endoscope technique (B).

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Figure 7 Before (A) and after PDGC (B).

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Figure 8 Before (A) and after comprehensive technique (B).

changes or postoperative adhesions in the duodenum, which may result in standard cannulation failure, deep cannulation could be achieved by bending or rotating endoscope (Figure 6 A and B). Pancreatic duct guideline cannulation (PDGC): When the knife was inserted into the pancreatic duct repeatedly, the guide wire might be kept in the pancreatic duct, then the knife or catheter must be inserted into bile duct through the endoscopic biopsy duct (Figure 7 A and B). Comprehensive technology (CT): When incision could not be made by one single method, other precut procedures such as NKF, could be performed in combination until cannulation was achieved (Figure 8 A and B). Statistical analysis Data were expressed as percent and processed with www.wjgnet.com

A total of 280 consecutive patients underwent ERCP with a standard catheter, of them, 3 gave up the operation because of pathological changes in stomach or in duodenum, 247 succeeded (89.1%, 247/277), 30 failed (10.8%, 30/277). Twenty-four of the 30 cases (80%, 24/30) succeeded in VNTPS technique. The successful rate for deep duct bile cannulation was increased 8.6% (24/277), the total successful cannulation rate following precut reached 97.7% (271/277). There was a significant difference between them (97.7% vs 89.1%, χ2 = 17.1, P < 0.01). Among the successful cases, 2 succeeded in MPSP, 3 in PDWN, 2 in PUWN, 1 in SNKP, 2 in PDGP, 2 in Deng’s precut (one was inflammatory papilla combined with upward papilla diverticula), 3 in UROT, 2 in BERET, 1 in KPDGC. Comprehensive technology achieved success in 6 cases and failed in 6 cases. Among the unsuccessful cases, 1 was diagnosed as OSS combined with cholelocholithiasis, 1 as pancreatic head carcinoma, 2 as duodenal papilla carcinoma, 1 as distal bile carcinoma, 1 as lower segmental stenosis of common bile duct (over 1.5 cm in length). The incidence of complications in traditional ERCP was 9.3% (26/277). The final diagnoses included suppurative cholangitis in 2 cases (1 was cured with conservative treatment, 1 underwent surgical therapy), acute severe pancreatitis in 2 cases, mild acute pancreatitis in 8 cases, bleeding during sphincterotomy in 4 cases, massive hemorrhage in gastrointestinal tract in 1 case, moderate bleeding after phincterotomy in 2 cases, transient abdominal pain and jaundice in 2 cases, cholangitis in 2 cases, transient cerebropathia in 2 cases, fever in 1 case. Mortality was not related to the endoscope. The complication rate in VNTPS technique group was 13.3% (4/30). The tip of the soft wire was broken in the pancreatic duct of 1 case. Pancreatitis was not found in PDGP. Mild to moderate bleeding occurred in 2 cases and cured after procedure (PDWN). One patient with bleeding during operation (PDWN) was also treated with norepinephrine rinse, submucosal injection of epinephrine and electric coagulation, no other severe complications were found. There were no differences between VNTPS technique group and conventional ERCP group(13.3% vs 9.3%, χ2 = 0.478, P > 0.05). These finding suggest that VNTPST was one of the approaches when standard techniques failed.

DISCUSSION The standard cannulation technique of ERCP can achieve satisfactory effects, especially application of ultra-slipping guide wire increases the successful rate for bile duct cannulation and declines the incidence of complications. In our study, 247 cases in TRERCP group were successfully treated with a successful rate of 89.1% (247/277). Complications occurred in 26 cases, the incidence of complications was 9.3% (26/277), which is consistent with the

Deng DH et al . Precut sphincterotomy for ERCP

reported data both in China and in foreign countries[13-15]. Although the successful rate for standard ERCP is high at present, the selective bile duct cannulation failure rate is 5%-10%. Due to anatomic and physiological factors such as shortage of common cholangiopancreatic duct, duodenal diverticulum, small ampullary orifice, or cervical of the ampulla, it is difficult to perform selective biliary cannulation. Pathologic conditions, such as Oddi’s sphincer stenosis, duodenal inflammation, ampulla and papillary neoplasms, impacted calculi, may result in cannulation failure. However, VNTPST plays a salvage role in solving such cannulation difficulties. In our study, the successful rate was 80% (24/30)and the incidence of complication was 13.3% in VNTPST group, which is in line with the reported PSP both in China and in foreign countries[13,15-17], indicating that as long as precuts are skillfully performed, VNTPST is safe and effective and plays an important role in increasing the ERCP successful rate. The precondition of PSP is that the tip of scalpel should be inserted into the papilla orifice. In general, it is often applied when pancreatic duct cannulation is performed repeatedly and TRERCP fails. The technique is suitable for type “Y” or type “Ⅴ” pancreaticobiliary ductal junction, especially for type “Y”. The advantage of PSP is that the direction and depth are easy to control, but complications such as severe pancreatitis may occur, because pancreatic duct edema may occur after PSP [17]. In our study, the pure current was applied to the MPSP precut, and the direction of incision was made at the 10-11 o’ clock direction, thus avoiding the occurrence of edema and trauma in pancreatic duct orifice and pancreatitis after MPSP. MPSP cannot be applied to the small papilla orifice since PLPTS cannot be inserted into the pancreatic duct. For type V in particular, sometimes it is not easy to achieve such a succees, PDWN or PUWN should be performed. For the impacted stones in duodenal ampulla or duodenal ampulla mass, it is not as convenient as needle knife. Needle-knife is the major tool for precut[17]. In this study, needle knife was used in 19 cases (19/30, 63.3%) with PDWN or PUWN or PDGP or comprehensive technology, the successful rate was 68.4% (13/19). During the procedure, the tip of the needle should be put in the middle of visual area. If cannulation of the CBD through the opening is difficult, cannulation can be achieved with needle knife technique in most cases. Due to carelessness of nurses, the soft wire was not replaced by a hard one. The tip of soft wire was broken off in the pancreatic duct (PDGP group) in 1 case, but there were no postoperative complications. Bleeding occurred in 1 case when small blood vessels were cut by needle-knife, but was stopped and no severe complication occurred. Our data indicate that NPK is as successful and safe as MPSP. In Katsinelos study, 68 cases underwent needle knife precut, bleeding occurred in 5 cases (7%) and AP in 3 cases (4%), which were treated with conservative therapy[18]. The complication rate in our study was 13.3%, which is in line with the reported data[12], there were no severe complications or death, indicating that the procedure is highly successful and quite safe. Compared with TRERCP,

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postsphincterotomy hemorrhage often occurred after needle knife precut, but it was minor and able to be treated with epinephrine rinse or nephrine injection. One patient had hemorrhage in our study and no adverse effect was found after treatment. Abdominal pain should be closely observed after precut to avoid perforation for which an abdominal image is necessary. Once perforation occurs, titanium clips, gastrointestinal decompression and fast should be taken. Compared with needle knife, although the depth of incision is easy to control and may not result in perforation, SNKP is not so convenient for duodenal ampulla calculus incarceration. When the direction is controlled by pancreatic duct guidewire, PDGP is good for protruded papilla but not for small aperture endoscope[19]. The papilla orifice often cannot be turned up for the duodenum adherence or malformation after abdominal part operation, UROT can move up the incision. When standard ERCP is difficult to turn up the papilla orifice, BERET can be performed. When the guide wire is inserted repeatedly into the pancreatic duct, PDGP can avoid repeated catheter insertion. When the bile cannulation is achieved, standard ERCP follows. To improve ERCP successful rate, when a single method cannot complete deep cannulation, CT can be used in various precuts to obtain cannulation. The reasons for the failure of cannulation in all kinds of precut are as follows: The bile duct not found in 1 case of OSS combined with cholelocholithiasis, the bile duct distorted by neoplasm in 1 case of pancreatic head carcinoma, duodenal papilla carcinoma diagnosed in 2 cases, unnecessary precut, 1 case of distal bile duct cancer and distal stricture of CBD. Based on our study, the operation indication should be strictly controlled[20]. Precutting should be avoided for diagnostic purposes because other available methods, such as MRCP and endosonography can provide diagnostic information. Precut should be avoided if the distal stricture of CBD is longer than 1.5 cm. When cannulnation of the common bile duct is not possible after precut, ERCP should be repeatedly performed 5-7 d later when edema caused by precut relieves and cannulation can be achieved. In conclusion, needle knife technique can be performed when orifice cannot be exposed for the inflamed papilla or ampullary edema for which PSP is not available. The exposed length of needle knife is determined according to the size and shape of papilla. The isolating sheath should be fixed by nurse, otherwise intraperitoneal perforation may occur. Capillary hemorrhage in the NPK can be stopped by electric coagulation combined with needle knife. Perforation may occur in subsequent EST after the precut in patients with cholelithiasis, therefore, the incision size should not be too big in subsequent EST. The precut should be slowly extended step by step, from ampullary mucosa to submucosa in layers, finally ended up with a seepage of bile detected or the bile duct sphincter tissue seen. Both PUWN and PDWN can be used in patients with duodenal ampulla calculus incarceration, whereas PDWN should be carefully performed in patients with small papilla. When distal stenosis of CBD is too long, needle knife should be cautiously used. For a right bile orifice, opening of the bile ducts may be found in the right side of precut. PUWN is suitable for the small papilla with a small orifice. www.wjgnet.com

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COMMENTS Background

McCune reported endoscopic retrograde cholangiopancreatography (ERCP) in 1968 for the first time, with the successful rate of 25%[1]. It was early applied in clinical practice in 1972[21], and the therapeutic ERCP (TERCP) was developed. Kawai and Nagai in Japan used endoscopic nasobiliary drainage (ENBD) for acute obstructed suppurate cholangitis (AOSC) patients and achieved success in 1978. In China, ERCP was introduced into Peking Union Hospital in 1974. Dr. Ming-Zhang Chen initially reported endoscopic sphincterotomy (EST), but it was not widely used for its complex technique and lower successful rate. With the advances in surgical techniques and equipments, the successful rate for ERCP was 84.0% in 1970s, and reached 93.5% in 1980s and 96.1% in 1990s in China[2], which is similar to the international reports [3,4]. TERCP was initially applied in early 1980s and has become an important method in treatment of biliary duct and pancreatic disorders. The therapeutic ERCP for patients with biliary duct and pancreatic disorders has achieved satisfactory curative effects on biliary duct and pancreatic diseases in China[8]. The successful rate for different diagnostic ERCP (DERCP) techniques depends on EST, which is associated with selective and deep cannulation of the bile duct. Due to anatomy, physiology factors and pathologic variation, the guide wire, catheter and scalpel cannot always be inserted into the bile duct successfully, leading to failure in TERCP. So it is the key step to various new type precut sphincterotomy techniques (VNTPST) in ERCP.

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Research frontiers

Precut was initially reported by Siegel[5], the successful rate for ERCP can go up with the success of precut. The so-call “precut” can be explained as following: when biliary cannulation is difficult to perform, papilla and bile duct sphincters need to be partially cut in advance for catheter insertion into the bile duct, then the procedures could be well done. So it is different from the general EST. It was reported that the successful rate for precut is 77%-91%[6,7]. The application of precut is limitted[8-11] because more complications occur. Since needle shape scalpel is very difficult to use and not safe during the precut papillotomy, few reports on its use are available.

9 10 11 12

Innovations and breakthroughs

We first designed and reported the mucosal bridge precut (Deng's precut) which is especially suitable for patients with inflamed papilla combined with up papillary diverticula. The technique can correct the cannulation angle, and make cannulation easy. It is safer than needle knife. We suggest that this procedure can be extensively used because it is of higher successful rate and safety.

13

14

Applications

The study suggests that when efforts using standard techniques have failed in ERCP and biliary access is required, Deng’s precut, a combined endoscopic “precut” technique is available. In experienced hands of ERCP, the ratio of ERCP can be increased with remarkable effects. Precuts appear to be as safe and effective as standard EST and can be widely performed in large and middle-sized hospitals.

Terminology

VNTPST: various new type precut sphincterotomy techniques; MPSP: modified pancreatic sphincter precutting; PDWN: precut down with needle; NDF: needle knife; PUWN: precut up with needle; PDGP: pancreatic duct guideline precut; Deng’s precut: mucosal bridge precut; UROT: up removal orifice technique; BERET: bending endoscope or rotating endoscope technique; PDGC: pancreatic duct guideline cannulation

Peer review

This is a well written paper. In the study, the authors retrospectively investigated the effect and safety of VNTPST in ERCP for difficult biliary duct cannulation (DBC). VNTPS and Deng’s precut are highly effective to get biliary access during ERCP for DBC. They can increase the successful rate for deep cannulation and decrease complications.

RFFERENCES 1. 2

Endoscopic cannulation of the ampulla of Vater: a preliminary report. By William S. McCune, Paul E. Shorb, and Herbert Moscovitz, 1968. Gastrointest Endosc 1988; 34: 278-280 Sun ZX, Xu GM, Li ZS, Xie SQ, Wang N, Tian Q, Wu RP,

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Yao YZ, Fang YQ. Practical value of endoscopic retrograde cholangiopancreatography in diagnosis and treatment of pancreaticobiliary disease. Dier Junyi Daxue Xuebao 1998; 19: 401-404 Cheng CL, Sherman S, Watkins JL, Barnett J, Freeman M, Geenen J, Ryan M, Parker H, Frakes JT, Fogel EL, Silverman WB, Dua KS, Aliperti G, Yakshe P, Uzer M, Jones W, Goff J, Lazzell-Pannell L, Rashdan A, Temkit M, Lehman GA. Risk factors for post-ERCP pancreatitis: a prospective multicenter study. Am J Gastroenterol 2006; 101: 139-147 Frank CD, Adler DG. Post-ERCP pancreatitis and its prevention. Nat Clin Pract Gastroenterol Hepatol 2006; 3: 680-688 Siegel JH. Precut papillotomy: a method to improve success of ERCP and papillotomy. Endoscopy 1980; 12: 130-133 Uchida N, Tsutsui K, Kamada H, Ogawa M, Fukuma H, Ezaki T, Aritomo Y, Kobara H, Ono M, Morishita A, Masaki T, Watanabe S, Nakatsu T, Kuriyama S. Pre-cutting using a noseless papillotome with independent lumens for contrast material and guidewire. J Gastroenterol Hepatol 2005; 20: 947-950 Schwacha H, Allgaier HP, Deibert P, Olschewski M, Allgaier U, Blum HE. A sphincterotome-based technique for selective transpapillary common bile duct cannulation. Gastrointest Endosc 2000; 52: 387-391 Hu B, Zhou DY, Gong B, Wang SZ, Zhang FM, Wang XL. Application of precut sphincterotomy in the diagnostic and therapeutic ERCP :a report of 73 cases. Shijie Huaren Xiaohua Zazhi 1999; 7: 1052-1054 Qin MF, Zhou FS, Wang ZY, Fan JD, Lu HZ. Analysis of 691 cases underwent endoscopic pre-cut papillotomy. Zhonghua Xiaohua Neijing Zazhi 2001; 18: 95-96 Jiang CB, Wang JB. Analysis of precut papillotomy in endoscopy. Xiandai Shiyong Yixue 2002; 14: 606 Zhang BY, Tian FZ, Wang Y, Huang DR, Gong L. Endoscopic sphincterotomy with needle-shaped knife: report of 476 cases. Hepatobiliary Pancreat Dis Int 2002; 1: 434-437 Akashi R, Kiyozumi T, Jinnouchi K, Yoshida M, Adachi Y, Sagara K. Pancreatic sphincter precutting to gain selective access to the common bile duct: a series of 172 patients. Endoscopy 2004; 36: 405-410 Ma SC, Yang XJ, Shen JW, Zhang XR, Zhang X Zhong B, Xie WF, Chen WZ, Shen YF, Li S. Analysis on 126 cases with bile duct diseases underwent sphincterotomy precut. Gan Dan Yi Waike Zazhi 1999; 11: 123-124 Vandervoort J, Soetikno RM, Tham TC, Wong RC, Ferrari AP Jr, Montes H, Roston AD, Slivka A, Lichtenstein DR, Ruymann FW, Van Dam J, Hughes M, Carr-Locke DL. Risk factors for complications after performance of ERCP. Gastrointest Endosc 2002; 56: 652-656 Pungpapong S, Kongkam P, Rerknimitr R, Kullavanijaya P. Experience on endoscopic retrograde cholangiopancreatography at tertiary referral center in Thailand: risks and complications. J Med Assoc Thai 2005; 88: 238-246 Tang SJ, Haber GB, Kortan P, Zanati S, Cirocco M, Ennis M, Elfant A, Scheider D, Ter H, Dorais J. Precut papillotomy versus persistence in difficult biliary cannulation: a prospective randomized trial. Endoscopy 2005; 37: 58-65 Kahaleh M, Tokar J, Mullick T, Bickston SJ, Yeaton P. Prospective evaluation of pancreatic sphincterotomy as a precut technique for biliary cannulation. Clin Gastroenterol Hepatol 2004; 2: 971-977 Katsinelos P, Mimidis K, Paroutoglou G, Christodoulou K, Pilpilidis I, Katsiba D, Kalomenopoulou M, Papagiannis A, Tsolkas P, Kapitsinis I, Xiarchos P, Beltsis A, Eugenidis N. Needle-knife papillotomy: a safe and effective technique in experienced hands. Hepatogastroenterology 2004; 51: 349-352 Draganov P, Devonshire DA, Cunningham JT. A new technique to assist in difficult bile duct cannulation at the time of endoscopic retrograde cholangiopancreatography. JSLS 2005; 9: 218-221 Larkin CJ, Huibregtse K. Precut sphincterotomy: indications, pitfalls, and complications. Curr Gastroenterol Rep 2001; 3: 147-153 Cotton PB. Cannulation of the papilla of Vater by endoscopy and retrograde cholangiopancreatography (ERCP). Gut 1972; 13: 1014-1025 S- Editor Zhu LH L- Editor Wang XL E- Editor Li LJ

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World J Gastroenterol 2007 August 28; 13(32): 4391-4393 World Journal of Gastroenterology ISSN 1007-9327 © 2007 WJG. All rights reserved.

CASE REPORT

Listeria monocytogenes following orthotopic liver transplantation: Central nervous system involvement and review of the literature Shugo Mizuno, Ivan R Zendejas, Alan I Reed, Robin D Kim, Richard J Howard, Alan W Hemming, Denise C Schain, Consuelo Soldevila-Pico, Roberto J Firpi, Shiro Fujita Shugo Mizuno, Ivan R Zendejas, Alan I Reed, Robin D Kim, Richard J Howard, Alan W Hemming, Shiro Fujita, Division of Transplantation and Hepatobiliary Surgery, Department of Surgery, University of Florida College of Medicine, Gainesville, FL 32610-0286, United States Denise C Schain, Division of Infectious Disease, Department of Medicine, University of Florida College of Medicine, Gainesville, FL 32610-0286, United States Consuelo Soldevila-Pico, Roberto J Firpi, Division of Gastroenterology, Department of Medicine, University of Florida College of Medicine, Gainesville, FL 32610-0286, United States Correspondence to: Shiro Fujita, MD, Department of Surgery, University of Florida College of Medicine, PO Box 100286, Gainesville, FL 32610-0286, United States. [email protected] Telephone: +1-352-2650606 Fax: +1-352-2650678  Accepted:2007-05-12 Received: 2007-04-26

Abstract Listeria monocytogene is a well-recognized cause of

bacteremia in immunocompromised individuals, including solid organ transplant recipients, but has been rarely reported following orthotopic liver transplantation. We describe a case of listeria meningitis that occurred within a week after liver transplantation. The patient developed a severe headache that mimicked tacrolimus encephalopathy, and was subsequently diagnosed with listeria meningitis by cerebrospinal fluid culture. The infection was successfully treated with three-week course of intravenous ampicillin. Recurrent hepatitis C followed and was successfully treated with interferon alfa and ribavirin. Fourteen cases of listeriosis after orthotopic liver transplantation have been reported in the English literature. Most reported cases were successfully treated with intravenous ampicillin. There were four cases of listeria meningitis, and the mortality of them was 50%. Early detection and treatment of listeria meningitis are the key to obtaining a better prognosis. © 2007 WJG . All rights reserved.

Key words: Listeria monocytogene ; Meningitis; Liver transplantation Mizuno S, Zendejas IR, Reed AI, Kim RD, Howard RJ, Hemming AW, Schain DC, Soldevila-Pico C, Firpi RJ, Fujita S. Listeria monocytogenes following orthotopic liver

transplantation: Central nervous system involvement and review of the literature. World J Gastroenterol 2007; 13(32): 4391-4393

http://www.wjgnet.com/1007-9327/13/4391.asp

INTRODUCTION Listeria monocytogene (L. monocytogene) is a well-known environmental organism that is most often transmitted to humans via contaminated foods such as milk and cheese, undercooked meat, or uncooked vegetables. Although L. monocytogene has been isolated from the stool of approximately 5% of healthy adults, disease caused by the microorganism occurs primarily in neonates, pregnant women, and immunocompromised individuals[1]. Immunosuppression following organ transplantation has been described as a risk factor for listeriosis and there are several reports in the literature of occurrence following renal and bone marrow transplantation[2,3]. Occurrence of listeriosis following liver transplantation (LT) has not been widely documented, especially listeria meningitis. We describe a case of listeria meningitis occurring in the early post-orthotopic liver transplant period and review the English literature on listeriosis following LT.

CASE REPORT A 53-year-old Caucasian male underwent orthotopic LT due to hepatitis C virus (HCV)-induced liver cirrhosis with associated hepatocellular carcinoma. He was doing well and afebrile before transplantation. White blood cell count (WBC), total bilirubin, creatinine, and prothrombin time international normalized ratio (PT INR) were 4500/mL, 0.8 mg/dL, 1.1 mg/dL, 1.1, respectively. The model for end-stage liver disease (MELD) score was 8. Cefazolin (6 g/d) was used for postoperative antimicrobial prophylaxis for two days. Posttransplant immunosuppression included oral tacrolimus and prednisone, and his early postoperative course was uncomplicated. On postoperative day five, he developed a severe, throbbing headache. His body temperature was normal, WBC was 6200/mL and a CT scan of the head revealed no abnormal findings. On the following day, his mental status gradually declined.

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(IU/mL)

(/mL)

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2000

WBC 12 000 8000 4000 0

August 28, 2007

ALT

1500 1000

AST

500 0

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 (POD)

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 (POD)

Figure 1 Laboratory profiles of this patient following liver transplantation. WBC: white blood cells; AST: aspartate aminotransferase; ALT: alanine aminotransferase; POD: postoperative day.

Table 1 Cases of listeria infection following liver transplantation Age (yr) Sex

Time postTx Clinical presentation

Clinical syndrome

Treatment

Outcome

Ref

66 39 63 66 46 55 57 56 41 47 8M UNK 67 13

32 mo 7d 62 d 50 d 3 yr 4 mo 20 mo 8 mo Unknown Unknown 10 d 14 d 21 d 4 mo

Bacteremia Bacteremia Bacteremia Bacteremia Bacteremia Bacteremia Bacteremia and hepatitis Bacteremia and hepatitis Endocarditis and septic pulmonary emboli Peritonitis and bacteremia Meningitis, peritonitis, epididymitis and orchitis Meningitis Meningitis Meningitis

Ampicillin Ampicillin Ampicillin Ampicillin Ampicillin Gentamicin TMP/SMZ Gentamicin Ampicillin Ampicillin Gentamicin Ampicillin Ampicillin Amikacin Ampicillin Unknown Unknown Ampicillin

Survived Survived Survived Survived Survived Survived Survived Survived Survived Survived Survived Survived Died Died

7 8 9 9 10 11 12 13 14 15 16 17 18 19

F F F M F F F M F F M UNK F F

Fever, right flank pain, anorexia Fever, abdominal pain Fever, abdominal pain Fever, abdominal pain Fever, diarrhea, recurrent Fever, confusion, diarrhea Fever with chill Fever, jaundice Fever, malaise Fever, malaise Fever, rash, irritability, dyspnea Unknown Unknown Fever, headache

Tacrolimus encephalopathy was suspected due to a high tacrolimus trough level (22.9 ng/mL), and administration of tacrolimus was temporarily discontinued. On the seventh postoperative day, the serum tacrolimus level decreased to 8.9 ng/mL, but his headache remained and he became unresponsive. A CT scan showed extensive acute hydrocephalus with no evidence of bleeding or herniation. His body temperature increased to 39.1℃. and his WBC elevated to 13 000/mL. The patient underwent lumbar puncture and analysis of cerebrospinal fluid (CSF) revealed a white blood cell count of 100/mL (69% segmented neutrophils and 18% lymphocytes), protein level of 39 mg/dL, and glucose level of 69 mg/dL. L. monocytogene was grown in culture from the CSF. Blood culture also yielded the growth of L. monocytogenes. The diagnosis of listeria meningitis was made and the patient was treated with a three-week course of ampicillin. The patient became afebrile and WBC decreased to 7600/mL on the following day. The patient’s neurological findings gradually improved but had persistent ventriculomegaly. Due to labile mental status and persistent hydrocephalus of a CT finding, a VP shunt was placed operatively on postoperative day (POD) 28. He had been stable since and his mental status became baseline. On POD 20, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) gradually increased (Figure 1), HCV RNA-b DNA level was markedly elevated to > 7 961 230 IU/mL, and the recurrence of HCV was diagnosed by liver biopsy. The patient was started on interferon alfa and ribavirin, and AST and ALT levels www.wjgnet.com

gradually decreased. He was discharged on the 48th d following operation.

DISCUSSION Immunosuppression following organ transplantation has been described as a risk factor for listeriosis and several cases have been described following bone marrow and renal transplantation [2,3]. Nolla-Salas et al[4] recently reported that liver cirrhosis should also be considered a predisposing factor for listeriosis. Fourteen cases of listeriosis following LT have been reported[5-17], in which, only four occurrences of meningitis were reported, three within three weeks and one within four months following transplantation (Table 1). The patient age ranged from eight months to 67 years (age not reported in one case). Clinical symptoms included fever, headache, and irritability. All patients with meningitis were treated with intravenous ampicillin with a 50% mortality, though all of the other listeriosis patients without meningitis survived after antibiotic treatment including either ampicillin or trimethoprim-sulfamethoxazole. According to the centers for disease control and prevention (CDC), L. monocytogene is the fifth most frequent cause of bacterial meningitis [18]. It was reported that listeriosis of the central nervous system is characterized by a high mortality rate (20%-50%). A recent prospective cohort study showed that the mortality rate even now is 17%[19]. Stamm et al[20] reported that, in patients with

Mizuno S et al. Listeria monocytogenes following orthotopic liver transplantation

listeriosis after kidney transplantation, meningitis is also associated with a significantly higher mortality (37%) than listeriosis without meningitis (11%). In our patient, the correct diagnosis was delayed because of the appropriate initial thought that the patient’s CNS symptoms were secondary to tacrolimus toxicity. It is well known that tacrolimus-associated encephalopathy demonstrates various symptoms, such as tremor, convulsions, drowsiness, headache, nausea, and cortical blindness. The risk of tacrolimus-associated encephalopathy is significantly correlated with the tacrolimus blood level. The mode of transmission of L. monocytogene is not clearly understood. It is well known that L. monocytogene is widespread in nature, being found commonly in soil, decaying vegetation, and as part of the fecal flora in many mammals. Elsner et al[10], however, studied nosocomial infections with L. monocytogenes in immunocompromised patients and suggested that nosocomial food-borne and person-to-person transmission could not be proven. While this patient stayed at our hospital, there were no additional cases of listeria infection among patients or hospital staff. Transmission from the donor organ was also excluded because the donor blood culture was negative for listeria infection, and according to the information of the united network for organ sharing (UNOS), the recipients who received other organs from the same donor did not develop listeriosis. We speculate that enteric previous colonization is the rule in patients with listeriosis. Vander et al[21] reported that inherent T cell dysfunction caused by HCV infection may increase the susceptibility to listeria infection. In our case, the patient underwent liver transplantation because of chronic HCV infection and hepatocellular carcinoma. He developed recurrent HCV infection as demonstrated by a very high HCV-RNA level in the early postoperative period (HCV RNA-b DNA > 7 961 230 IU/mL, HCV RNA-b DNA log > 6.89 log IU/ mL). This situation might have caused the patient to have increased susceptibility to listeria infection. Though our patient was successfully treated with ampicillin, it is important to diagnose and treat as early as possible because the prognosis of listeria meningitis is not favorable.

REFERENCES 1

2

3

MacGowan AP, Marshall RJ, MacKay IM, Reeves DS. Listeria faecal carriage by renal transplant recipients, haemodialysis patients and patients in general practice: its relation to season, drug therapy, foreign travel, animal exposure and diet. Epidemiol Infect 1991; 106: 157-166 Gellin BG, Broome CV, Bibb WF, Weaver RE, Gaventa S, Mascola L. The epidemiology of listeriosis in the United States--1986. Listeriosis Study Group. Am J Epidemiol 1991; 133: 392-401 Skogberg K, Syrjanen J, Jahkola M, Renkonen OV, Paavonen J, Ahonen J, Kontiainen S, Ruutu P, Valtonen V. Clinical presentation and outcome of listeriosis in patients with and without immunosuppressive therapy. Clin Infect Dis 1992; 14:

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8 9

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12

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14 15

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17 18

19 20

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815-821 Nolla-Salas J, Almela M, Gasser I, Latorre C, Salvado M, Coll P. Spontaneous Listeria monocytogenes peritonitis: a populationbased study of 13 cases collected in Spain. Am J Gastroenterol 2002; 97: 1507-1511 Rettally CA, Speeg KV. Infection with Listeria monocytogenes following orthotopic liver transplantation: case report and review of the literature. Transplant Proc 2003; 35: 1485-1487 Limaye AP, Perkins JD, Kowdley KV. Listeria infection after liver transplantation: report of a case and review of the literature. Am J Gastroenterol 1998; 93: 1942-1944 Elsner HA, Tenschert W, Fischer L, Kaulfers PM. Nosocomial infections by Listeria monocytogenes: analysis of a cluster of septicemias in immunocompromised patients. Infection 1997; 25: 135-139 Peetermans WE, Endtz HP, Janssens AR, van den Broek PJ. Recurrent Listeria monocytogenes bacteraemia in a liver transplant patient. Infection 1990; 18: 107-108 Spitzer PG, Hammer SM, Karchmer AW. Treatment of Listeria monocytogenes infection with trimethoprimsulfamethoxazole: case report and review of the literature. Rev Infect Dis 1986; 8: 427-430 Vargas V, Aleman C, de Torres I, Castells L, Gavalda J, Margarit C, Esteban R, Guardia J. Listeria monocytogenesassociated acute hepatitis in a liver transplant recipient. Liver 1998; 18: 213-215 Bourgeois N, Jacobs F, Tavares ML, Rickaert F, Deprez C, Liesnard C, Moonens F, Van de Stadt J, Gelin M, Adler M. Listeria monocytogenes hepatitis in a liver transplant recipient: a case report and review of the literature. J Hepatol 1993; 18: 284-289 Avery RK, Barnes DS, Teran JC, Wiedemann HP, Hall G, Wacker T, Guth KJ, Frost JB, Mayes JT. Listeria monocytogenes tricuspid valve endocarditis with septic pulmonary emboli in a liver transplant recipient. Transpl Infect Dis 1999; 1: 284-287 Chapoutot C, Perney P, Pageaux GP, Lefebvre A, Souche B, Blanc F. Spontaneous Listeria monocytogenes peritoneal infection complicating hepatic transplantation. Gastroenterol Clin Biol 1996; 20: 700-702 von Schnakenburg C, Hinrichs B, Fuchs J, Kardorff R. Posttransplant epididymitis and orchitis following Listeria monocytogenes septicaemia. Pediatr Transplant 2000; 4: 156-158 Paya CV, Hermans PE, Washington JA 2nd, Smith TF, Anhalt JP, Wiesner RH, Krom RA. Incidence, distribution, and outcome of episodes of infection in 100 orthotopic liver transplantations. Mayo Clin Proc 1989; 64: 555-564 Pouyet M, Ducerf C, Gaussorgues P, Salord F, Sirodot M, Caillon P, Dubois JM, Rivoire M, Baulieux J, Bouletreau P. Fulminant and subfulminant hepatitis treated by orthotopic transplantation of the liver. Apropos of 10 cases. Chirurgie 1989; 115: 533-539 Louria DB, Blevins A, Armstrong D. Listeria infections. Ann N Y Acad Sci 1970; 174: 545-551 Wenger JD, Hightower AW, Facklam RR, Gaventa S, Broome CV. Bacterial meningitis in the United States, 1986: report of a multistate surveillance study. The Bacterial Meningitis Study Group. J Infect Dis 1990; 162: 1316-1323 Brouwer MC, van de Beek D, Heckenberg SG, Spanjaard L, de Gans J. Community-acquired Listeria monocytogenes meningitis in adults. Clin Infect Dis 2006; 43: 1233-1238 Stamm AM, Dismukes WE, Simmons BP, Cobbs CG, Elliott A, Budrich P, Harmon J. Listeriosis in renal transplant recipients: report of an outbreak and review of 102 cases. Rev Infect Dis 1982; 4: 665-682 Vander T, Medvedovsky M, Hallevy C, Golzman G, Herishanu Y. Listeria monocytogenes meningitis in a patient with chronic hepatitis C infection, treated by interferon alfa and ribavirin. J Infect 2003; 46: 70-71 S- Editor Zhu LH L- Editor Wang XL

E- Editor Ma WH

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World J Gastroenterol 2007 August 28; 13(32): 4394-4397 World Journal of Gastroenterology ISSN 1007-9327 © 2007 WJG. All rights reserved.

CASE REPORT

Fulminant hepatic failure in a case of autoimmune hepatitis in hepatitis C during peg-interferon-alpha 2b plus ribavirin treatment Takayuki Kogure, Yoshiyuki Ueno, Koji Fukushima, Futoshi Nagasaki, Jun Inoue, Eiji Kakazu, Yasunori Matsuda, Osamu Kido, Yu Nakagome, Osamu Kimura, Noriyuki Obara, Yuta Wakui, Takao Iwasaki, Tooru Shimosegawa Takayuki Kogure, Yoshiyuki Ueno, Koji Fukushima, Futoshi Nagasaki, Jun Inoue, Eiji Kakazu, Yasunori Matsuda, Osamu Kido, Yu Nakagome, Osamu Kimura, Noriyuki Obara, Yuta Wakui, Takao Iwasaki, Tooru Shimosegawa, Division of Gastroenterology, Tohoku University Graduate School of Medicine, 1-1 Seiryo, Aobaku, Sendai City 980-8574, Japan Correspondence to: Yoshiyuki Ueno, MD, PhD, Division of Gastroenterology, Tohoku University Graduate School of Medicine, 1-1 Seiryo, Aoba, Sendai City 980-8574, Japan. [email protected] Telephone: +81-22-7177171 Fax: +81-22-7177177  Accepted: 2007-05-12 Received: 2007-05-08

infection is pegylated interferon (peg-IFN) in combination with ribavirin (RBV) [1]. This treatment exerts a variety of immuno-modulatory effects and may unmask the underlying autoimmune diseases such as autoimmune thyroiditis and rheumatoid arthritis[2,3]. In a similar fashion, interferon (IFN) was reported to induce or exacerbate autoimmune hepatitis (AIH) [4-7]. We describe a patient who developed fulminant hepatic failure with AIH during the treatment with peg-IFN and RBV. The patient had no prior history of autoimmune diseases and the severe hepatitis occurred after the HCV RNA level decreased to below the detection limit.

Abstract

CASE REPORT

A 27-year-old Caucasian female with hepatitis C virus (HCV) infection treated with interferon (IFN) who developed severe autoimmune hepatitis (AIH) is described. The infecting viral strain was of genotype Ib and the pre-treatment HCV viral load was at a high level. The patient was treated with pegylated IFN-alpha 2b and ribavirin, and her HCV-RNA became negative at wk 12, but after that she developed fulminant hepatic failure. The patient recovered after steroid pulse therapy consisting of methylprednisolone 1000 mg/d for three days which was administered twice. A needle liver biopsy revealed the typical pathological findings of AIH.

Patient The patient was a 27-year-old Caucasian woman from South Caucasian Country. She had never consumed alcohol. She had not received blood transfusions or undergone surgery. Her sister and mother were receiving treatment for Basedow's disease, but she had no manifestation of Basedow's disease or other autoimmune diseases.

© 2007 WJG . All rights reserved.

Key words: Autoimmune hepatitis; Interferon; Ribavirin; Hepatitis C virus; Anti-viral therapyA; Acute liver failure Kogure T, Ueno Y, Fukushima K, Nagasaki F, Inoue J, Kakazu E, Matsuda Y, Kido O, Nakagome Y, Kimura O, Obara N, Wakui Y, Iwasaki T, Shimosegawa T. Fulminant hepatic failure in a case of autoimmune hepatitis in hepatitis C during peg-interferon-alpha 2b plus ribavirin treatment. World J Gastroenterol 2007; 13(32): 4394-4397

http://www.wjgnet.com/1007-9327/13/4394.asp

INTRODUCTION The most effective treatment for hepatitis C virus (HCV) www.wjgnet.com

Present illness She was found to be infected with hepatitis C virus during her pregnancy and was referred to our department in October 2005. She hoped to receive IFN therapy to reduce the risk of transmission to her second baby. The HCV genotype was Ib and the HCV RNA viral load was 4600 kIU/mL (high range) (Table 1). Alanine aminotransferase (ALT) was 47 IU/L (normal < 35 IU/L). The markers of hepatic functional reserve were within normal ranges and ultrasonography of the liver indicated no abnormal findings. After delivery, interferon therapy was commenced with peg-IFN-alpha 2b (Peg-Intron®, Schering-Plough Corp., NJ, USA) and RBV (Rebetol®, Schering-Plough) on January 30, 2006. The clinical course of this anti-viral therapy is illustrated in Figure 1. The initial doses consisted of 100 µg peg-IFN per week and 600 mg RBV per day. This therapy was well tolerated with minimal adverse effects and HCV RNA became negative at wk 12. Around the same time, a rise of transaminase was noted: ALT, 83 IU/L (normal < 35 IU/L); and asparate aminotransferase (AST), 56 IU/L (normal < 30 IU/L). The dosage of peg-

Kogure T et al . Fulminant AIH during peg IFN + ribavirin

4395

Table 1 Laboratory findings before interferon treatment WBC Hb Plt PT

5300 /μL 11.0 g/dL 452 × 103 /μL 101.8%

AST ALT LDH ALP γ-GTP ChE

Total bilirubin Direct bilirubin Total protein Albumin BUN Creatinine Uric acid Na K Cl Total cholesterol Glucose

39 IU/L 47 IU/L 176 IU/L 278 IU/L 11 IU/L 386 IU/L

0.6 mg/dL 0.1 mg/dL 7.7 g/dL 4.2 g/dL 16 mg/dL 0.8 mg/dL 3.4 mg/dL 140 mEq/L 4.5 mEq/L 104 mEq/L 133 mg/dL 77 mg/dL

HCV-Ab HCV-RNA HCV genotype HBs-Ag HBs-Ab ANA ASMA AMA IgG IgA IgM HLA-DR allele

Positive 4600 kIU/L Ib Negative Negative × 80 Negative Negative 1635 mg/dL 108 mg/dL 283 mg/dL DR17/DR13 DRB1*0301/*1302

WBC: white blood cell; Hb: hemoglobin; Plt: platelet; PT: prothrombin time; AST: asparate aminotransferase; ALT: alanine aminotransferase; LDH: lactate dehidrogenase; ALP: alkaline phosphatase; γ-GTP: gamma glutamil transpeptidase; ChE: choline esterase; BUN: blood urea nitrogen; HCV: hepatitis C virus; HBs-Ag: hepatitis B virus surface antigen; HBs-Ab: hepatitis B virus surface antibody; ANA: antinuclear antibody; ASMA: anti-smooth muscle antibody; AMA: anti-mitochondrial antibody; Ig: immunoglobulin; HLA: human leukocyte antigen.

Table 2 Laboratory findings at onset of acute liver failure WBC Hb Plt PT APTT

5800/μL 12.2 g/dL 290 × 103 /μL 41.4% 44.4 sec

AST ALT LDH ALP γ-GTP ChE Total bilirubin Direct bilirubin Total protein Albumin

311 IU/L 280 IU/L 245 IU/L 571 IU/L 108 IU/L 124 IU/L 5.5 mg/dL 3.3 mg/dL 7.4 g/dL 3.1 g/dL

BUN Creatinine Uric acid Na K Cl Total cholesterol Triglyceride Glucose CRP

5.0 mg/dL 0.7 mg/dL 2.8 mg/dL 138 mEq/L 3.9 mEq/L 105 mEq/L 175 mg/dL 67 mg/dL 127 mg/dL 0.2 mg/dL

Tyrosin BCAA

262 μmol/L 313 μmol/L

TSH T3 T4

0.499 μIU/mL 1.66 pg/mL 0.55 ng/dL

ANA ASMA LKM-1 AMA IgG IgA IgM

× 160 Negative Positive Negative 2436mg/dL 175 mg/dL 280 mg/dL

HCV-RNA HBs-Ag HBs-Ab HBc-Ab IgM HAV EBV HSV CMV

13 kIU/l Negative Negative Negative Negative Negative Negative Negative

APTT: activated partial thromboplastin time; CRP: C-reactive protein; BCAA: branched-chain amino acid; TSH: thyroid-stimulating hormone; LKM-1: antibody to microsomes type 1; HBc-Ab: hepatitis B virus core antibody; HAV: hepatitis A virus; EBV: Epstein-Barr virus; HSV: herpes simplex virus; CMV: cytomegalovirus.

AST ALT (IU/L) 1000

T-Bil (mg/dL) 10

Peg-IFN α2b + ribavirin

5

500 0 2006

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Figure 1 Clinical course of peg-interferon (peg-IFN) plus ribavirin therapy for hepatitis C virus infection. ALT: Alanine aminotransferase; AST: asparate aminotransferase; T-Bil: total bilirubin.

IFN was reduced to 80 µg at wk 17, and 60 µg at wk 18 because of the persistent increase of ALT, and finally, the treatment was discontinued at wk 19. Glycyrrhizinate

( S t r o n g e r n e o - m i n o p h a g e n C ®, M i n o p h a g e n Pharmaceutical Co., Ltd., Tokyo, Japan) was administrated intravenously, and ALT indicated a decreasing tendency (peak ALT was 815 IU/L). After that, she showed jaundice and a prolongation of the prothrombin time (PT) was noted. Her human leukocyte antigen (HLA)-DR serotypes were DR17 and DR13. She was admitted to our hospital on June 30, 2006. Physical findings on admission Physical examination revealed: height 167 cm, weight 56 kg, blood pressure 118/64 mmHg, body temperature 37.3℃, and clear consciousness. The bulber conjunctiva was slightly icteric. No peripheral edema, vascular spiders, and flapping tremor were observed. Clinical course On admission, elevations of transaminases (ALT 280 IU/L, AST 311 IU/L) (Table 2) and total bilirubin (5.5 www.wjgnet.com

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that, the hepatic encephalopathy improved gradually. The ALT level rose again on July 14, and steroid pulse therapy was administered again. The ALT showed a decreasing tendency again, after which she started oral intake. PT, BUN, and UA had improved gradually. She was discharged from our hospital on August 10. A needle liver biopsy was performed on October 16 and it showed the typical pathological features of autoimmune hepatitis. Interface hepatitis with infiltration of inflammatory cells including plasma cells and rosette formation, were found but no biliary change was noted (Figure 4). The serum ALT levels were normalized over the following six months by 10 mg of prednisolone per day.

DISCUSSION Figure 3 A, B: Abdominal CT at the onset of hepatic encephalopathy; C, D: Enhanced abdominal MRI.

mg/mL) and decrease of blood urea nitrogen (BUN), uric acid (UA), and PT were detected. Hepatitis A, hepatitis B, cytomegalovirus, herpes simplex, and Epstein-Barr virusesl were negative. Anti-nuclear antibody (ANA) was × 160 (normal < × 80) and immunoglobulin G (IgG) 2436 mg/mL (normal < 1695 mg/mL), which were × 80 and 1635 mg/mL before the IFN treatment. Antibody to liver/kidney microsomes type 1 (LKM-1) was positive. Steroid pulse therapy (methylpredonisolone 1000 mg/d for three days) was performed (Figure 2). Nevertheless, hepatic encephalopathy appeared on July 3. Abdominal computed tomography indicated massive necrosis of hepatocytes (Figure 3). Brain magnetic resonance imaging showed no abnormal findings. The findings of her electric encephalogram were typical of metabolic encephalopathy. Total parenteral nutrition was started. The protein load was restricted and lactulose enemas were performed. After www.wjgnet.com

IFN therapy for patients with HCV infection has been reported to induce or exacerbate AIH [4-7] . In the present case, severe hepatitis occurred after HCV RNA had decreased to below the detection limit after treatment with peg-IFN with RBV for 12 wk. The patient presented the typical clinical features of serum aminotransferase elevation, positive ANA and LKM-1, and hypergammaglobulinemia, and responsiveness to glucocorticoid therapy. The pathological findings showed the typical features of AIH, which included interface hepatitis with infiltration of plasma cells, and rossete formation. One of the explanations for the occur rence of autoimmunity in HCV patients is the loss of self-tolerance due to molecular mimicry between viral proteins and self-antigen[8]. HCV infection is known to be related to autoimmune disease, and chronic hepatitis C patients show autoantibodies such as ANA and LKM-1. HCV core protein has been reported to show homology with cytochrome P450IID6, which could be recognized by LKM-1 antibodies as seen in type 2 AIH[9,10]. Recently, AIH has been found to occur in patients during IFN therapy after liver transplantation for HCV liver cirrhosis[11,12].

Kogure T et al . Fulminant AIH during peg IFN + ribavirin

In the present case, AIH occurred during IFN therapy and the patient developed fulminant hepatic failure when HCV RNA had decreased to below the detection limit. To our knowledge, there is no report of an HCV patient who developed fulminant hepatic failure after peg-IFN with RBV therapy. Peg-IFN with RBV is an established therapy for chronic hepatitis C patients but AIH should be considered as a potential complication of therapy leading to severe hepatitis. Especially, longer treatment duration (48 wk) and prolonged elevation of serum IFN levels in the pegylated-IFN could contribute to the development of autoimmune phenomenon. Liver biopsy in the early phase of acute liver injury to determine whether immunnosuppression therapy may be required for such patients. In Asians, the development of AIH during antiviral therapy is believed to be rare, although the potential risk should be taken into consideration if female young Caucasian case is treated like this report. However, the treatment of recurrence of HCV after cessation of antiviral therapy due to the emergence of autoimmune hepatitis like our case is a very difficult clinical decision to make. Probably, the interferon based antiviral therapy, the most standard anti-viral agent, could remain as the mainstream regimen for next decade. The balance between antiviral effects and possible autoimmune phenomena could be key factors as described previously[13,14]. Small molecules such as HCV protease inhibitor, either as monotherapy or combined with other small molecules, could be the first choice for the treatment of the current case in future.

REFERENCES 1 2 3

Strader DB, Wright T, Thomas DL, Seeff LB. Diagnosis, management, and treatment of hepatitis C. Hepatology 2004; 39: 1147-1171 Conrad B. Potential mechanisms of interferon-alpha induced autoimmunity. Autoimmunity 2003; 36: 519-523 Steegmann JL, Requena MJ, Martin-Regueira P, De La Camara R, Casado F, Salvanes FR, Fernandez Ranada JM. High incidence of autoimmune alterations in chronic myeloid

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leukemia patients treated with interferon-alpha. Am J Hematol 2003; 72: 170-176 Shindo M, Di Bisceglie AM, Hoofnagle JH. Acute exacerbation of liver disease during interferon alfa therapy for chronic hepatitis C. Gastroenterology 1992; 102: 1406-1408 Papo T, Marcellin P, Bernuau J, Durand F, Poynard T, Benhamou JP. Autoimmune chronic hepatitis exacerbated by alpha-interferon. Ann Intern Med 1992; 116: 51-53 Todros L, Saracco G, Durazzo M, Abate ML, Touscoz G, Scaglione L, Verme G, Rizzetto M. Efficacy and safety of interferon alfa therapy in chronic hepatitis C with autoantibodies to liver-kidney microsomes. Hepatology 1995; 22: 1374-1378 Garcia-Buey L, Garcia-Monzon C, Rodriguez S, Borque MJ, Garcia-Sanchez A, Iglesias R, DeCastro M, Mateos FG, Vicario JL, Balas A. Latent autoimmune hepatitis triggered during interferon therapy in patients with chronic hepatitis C. Gastroenterology 1995; 108: 1770-1777 Kita H, Mackay IR, Van De Water J, Gershwin ME. The lymphoid liver: considerations on pathways to autoimmune injury. Gastroenterology 2001; 120: 1485-1501 Yamamoto AM, Cresteil D, Boniface O, Clerc FF, Alvarez F. Identification and analysis of cytochrome P450IID6 antigenic sites recognized by anti-liver-kidney microsome type-1 antibodies (LKM1). Eur J Immunol 1993; 23: 1105-1111 Kammer AR, van der Burg SH, Grabscheid B, Hunziker IP, Kwappenberg KM, Reichen J, Melief CJ, Cerny A. Molecular mimicry of human cytochrome P450 by hepatitis C virus at the level of cytotoxic T cell recognition. J Exp Med 1999; 190: 169-176 Cholongitas E, Samonakis D, Patch D, Senzolo M, Burroughs AK, Quaglia A, Dhillon A. Induction of autoimmune hepatitis by pegylated interferon in a liver transplant patient with recurrent hepatitis C virus. Transplantation 2006; 81: 488-490 Kontorinis N, Agarwal K, Elhajj N, Fiel MI, Schiano TD. Pegylated interferon-induced immune-mediated hepatitis post-liver transplantation. Liver Transpl 2006; 12: 827-830 Festi D, Sandri L, Mazzella G, Roda E, Sacco T, Staniscia T, Capodicasa S, Vestito A, Colecchia A. Safety of interferon beta treatment for chronic HCV hepatitis. World J Gastroenterol 2004; 10: 12-16 Lodato F, Tame MR, Colecchia A, Racchini C, Azzaroli F, D'Errico A, Casanova S, Pinna A, Roda E, Mazzella G. Systemic lupus erythematosus following virological response to peginterferon alfa-2b in a transplanted patient with chronic hepatitis C recurrence. World J Gastroenterol 2006; 12: 4253-4255 S- Editor Liu Y L- Editor Ma JY E- Editor Yin DH

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World J Gastroenterol 2007 August 28; 13(32): 4398-4400 World Journal of Gastroenterology ISSN 1007-9327 © 2007 WJG. All rights reserved.

CASE REPORT

Successful treatment of hypovascular advanced hepatocellular carcinoma with lipiodol-targetting intervention radiology Kazutaka Kurokohchi, Akihiro Deguchi, Tsutomu Masaki, Takashi Himoto, Hirohito Yoneyama, Mitsuyoshi Kobayashi, Tsuyoshi Maeta, Takaaki Kiuchi, Fumikazu Kohi, Hisaaki Miyoshi, Tomohiko Taminato, Shigeki Kuriyama Kazutaka Kurokohchi, Akihiro Deguchi, Tsutomu Masaki, Takashi Himoto, Hirohito Yoneyama, Mitsuyoshi Kobayashi, H i s a a k i M i y o s h i , S h i g e k i Ku r i ya m a , D e p a r t m e n t o f Gastroenterology and Neurology, Kagawa University School of Medicine, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan Kazutaka Kurokohchi, Tomohiko Taminato, Department of Laboratory Medicine, Kagawa University School of Medicine, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan Tsuyoshi Maeta, Fumikazu Kohi, Department of Gastroenterology, KKR Takamatsu Hospital, 4-18 Tenjinmae, Takamatsu, Kagawa 760-0018, Japan Takaaki Kiuchi, Department of Radiology, KKR Takamatsu Hospital, 4-18 Tenjinmae, Takamatsu, Kagawa 760-0018, Japan Correspondence to: Kazutaka Kurokohchi, Department of Gastroenterology and Neurology, Kagawa University School of Medicine, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan. [email protected] Telephone: +81-87-8912156 Fax: +81-87-8912158 Received: 2007-04-19 Accepted: 2007-05-21

Abstract We report a case of hypovascular advanced hepatocellular carcinoma (HCC) successfully treated with a novel combination therapy of percutaneous ethanollipiodol injection (PELI) and intervention radiology (IVR), lipiodol-targetting IVR (Lipi-IVR). The present case had a hypovascular HCC (3 cm in diameter) located in the S6 region of the liver. Although the tumor was not detectable at all by both of early and late phase of helical dynamic computed tomography (CT), it could be detected by ultrasonography (US) as a low echoic space occupying lesion (SOL) beside the gallbladder and right kidney. Serum levels of alpha fetoprotein (AFP) and AFP-L3 were extremely high. Combination therapy of PELI, firstly reported in our department, and IVR (PELI and IVR, lipiodol-targetting IVR) was performed twice for the treatment. PELI could effectively visualize the location of the tumor for IVR treatment and show the presence of a thin blood vessel branching from the right hepatic artery flowing into the lipiodol deposit. After treatment, the serum levels of AFP and AFP-L3 were rapidly decreased to normal and maintained for more than eight months. Thus, this case expressing the tremendous effect might give us insight into the effectiveness of the novel combination therapy of PELI and IVR for the treatment of hypovascular HCC. © 2007 WJG . All rights reserved. www.wjgnet.com

Key words: Hypovascular hepatocellular carcinoma; Percutaneous ethanol-lipiodol injection therapy; Intervention radiology Kurokohchi K, Deguchi A, Masaki T, Himoto T, Yoneyama H, Kobayashi M, Maeta T, Kiuchi T, Kohi F, Miyoshi H, Taminato T, Kuriyama S. Successful treatment of hypovascular advanced hepatocellular carcinoma with lipiodol-targetting intervention radiology. World J Gastroenterol 2007; 13(32): 4398-4400

http://www.wjgnet.com/1007-9327/13/4398.asp

INTRODUCTION Hepatocellular carcinoma (HCC) is still one of the most progressive malignancies resistant to treatment [1]. Although several treatment modalities are currently used for the treatment of HCC, radiofrequency ablation (RFA) now plays a central role in local control of hepatic malignancies[2-4]. To improve the therapeutic effects of RFA, we innovated the novel combination therapy of percutaneous ethanol injection and RFA[5-8] and showed that this combination therapy could induce wider coagulated necrosis with smaller energy requirement [9]. Moreover, injection of the mixture of ethanol and lipiodol (PELI) is effective for the treatment of HCC difficult to treat with RFA[10,11]. Although HCC is usually a hypervascular tumor, some HCCs known as hypovascular HCC have no blood vessel. Hypovascular HCC is difficult to treat with intervention radiology (IVR) due to the lack of the feeding artery into the tumor. Here, we report a case of hypovascular HCC totally eliminated with combination therapy of PELI and IVR and normal levels of AFP and AFP-L3 were maintained for a long period of time.

CASE REPORT In April, 2006, a 56-year old woman positive for hepatitis B virus was admitted because of a recurrence of HCC. Four years ago, the patient underwent radiofrequency ablation (RFA) therapy for HCC located in the S6 region of the liver. The tumor was eliminated and serum levels of AFP and AFP-L3 were maintained within the normal range over the following four years. However, the levels of AFP and AFP-L3 started to gradually increase at the beginning of 2006. To detect recurrent tumor, helical dynamic computed tomography (CT) and ultrasonography

Kurokohchi K et al. Successful treatment of advanced HCC with lipiodol-targetting intervention radiology

A

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Figure 1 Helical dynamic CT in the late phase before tumor targetting by lipiodol (A), US of the right hepatic lobe before tumor targetting by lipiodol (B), plain CT after tumor targetting by lipiodol (C), and angiogram of the celiac artery after tumor targeting by lipiodol (D). RHA: right hepatic artery; GB: gall bladder; KID: kidney.

(US) were performed. Although US could detect a low echoic space occupying lesion (SOL) of 3 cm in diameter beside the gallbladder and right kidney (Figure 1A), the tumor was not detectable at all by helical dynamic CT (Figure 1B), indicating that it was not a hypervascular, but a hypovascular HCC. The hypovascularity suggested that the tumor could not be detected by angiography. Consequently, we injected a mixture of ethanol and lipiodol, a technique pioneered in our department in 2004[10,11], by a US-guided approach to mark the main tumor and make it visible in angiography. Plain CT showed a massive lipiodol deposit in the tumor area (Figure 1C). The serum level of of AFP was decreased to 101 ng/mL while the serum level AFP-L3 was unchanged after injection of the mixture of ethanol and lipiodol. An angiogram of the celiac artery, following lipiodol treatment, clearly showed the location of the tumor (Figure 1D, arrow heads) along with a thin vessel branching from the right hepatic artery and flowing into the lipiodol deposit (Figure 1D, arrow). Next, 5-fluorouracil (5-FU) was injected into the thin blood vessel after a super-selective canulation. After 5-FU was injected twice, the serum levels of AFP and AFP-L3 were finally decreased to 2.8 ng/mL and 0%, respectively, in August, 2006. These markers were still within the normal range in April 2007.

DISCUSSION Despite intensive efforts to develop treatment modalities for HCC, the prognosis of HCC remains relatively poor[12]. RFA plays a central role in the local control of HCC and a number of attempts have been made to enhance the therapeutic effect of RFA by combining RFA with transcatheter arterial chemoembolization[13,14]. We have recently reported successfully treated HCC cases, of



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which 1 had diffusely distributed HCC accompanied with tumor thrombus and was treated with combination therapy of 5-FU and pegylated IFN [15]. Another case had a large-size encapsulated lump of HCC and was treated with transarterial chemoembolization followed by the combination of PELI and RFA [16]. The present case had a hypovascular HCC which was different from those previously reported. Although HCC is usually a hyper vascular tumor, hypovascular HCC resistant to IVR can still be observed. Logically, administration of anticancer chemical reagents by IVR should not be effective against hypovascular HCC due to a low flow of the arterial blood into the tumor. We modified percutaneous ethanol injection therapy to produce percutaneous ethanol-lipiodol injection therapy (PELIT) for HCC that cannot be imaged by US or CT. By utilizing PELI, the tumor in this patient was made visible by angiography and injection of 5-fluorouracil was sufficient to dramatically decrease both AFP and AFP-L3 levels to the normal range, demonstrating that PELI can reveal the presence of small blood vessels flowing into HCC even though a hypovascular tumor diagnosed by CT and importantly can mark the precise location of the tumor at angiography. Dramatic therapeutic effects seen in this patient may result from the direct tumor targeting by lipiodol or the enhancement of anti-tumor activity of 5-FU by lipiodol. Effect of 5-FU may be enhanced by filling the tumor with lipiodol prior to the injection of 5-FU. Thus, injection of 5-FU after lipiodol injection may open up new avenues for the treatment of advanced HCC. This new technology may be effective for the treatment of both hypovascular and hypervascular HCC. Further study is needed to elucidate it.

REFERENCES 1 2 3

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Okuda K. Hepatocellular carcinoma. J Hepatol 2000; 32: 225-237 Curley SA, Izzo F, Ellis LM, Nicolas Vauthey J, Vallone P. Radiofrequency ablation of hepatocellular cancer in 110 patients with cirrhosis. Ann Surg 2000; 232: 381-391 Livraghi T, Goldberg SN, Lazzaroni S, Meloni F, Ierace T, Solbiati L, Gazelle GS. Hepatocellular carcinoma: radiofrequency ablation of medium and large lesions. Radiology 2000; 214: 761-768 Goldberg SN, Gazelle GS, Compton CC, Mueller PR, Tanabe KK. Treatment of intrahepatic malignancy with radiofrequency ablation: radiologic-pathologic correlation. Cancer 2000; 88: 2452-2463 Kurokohchi K, Watanabe S, Masaki T, Hosomi N, Funaki T, Arima K, Yoshida S, Miyauchi Y, Kuriyama S. Combined use of percutaneous ethanol injection and radiofrequency ablation for the effective treatment of hepatocelluar carcinoma. Int J Oncol 2002; 21: 841-846 Kurokohchi K, Watanabe S, Masaki T, Hosomi N, Funaki T, Arima K, Yoshida S, Nakai S, Murota M, Miyauchi Y, Kuriyama S. Combination therapy of percutaneous ethanol injection and radiofrequency ablation against hepatocellular carcinomas difficult to treat. Int J Oncol 2002; 21: 611-615 Kurokohchi K, Masaki T, Watanabe S, Nakai S, Deguchi A, Morishita A, Yoneyama H, Ohgi T, Ono M, Yoshitake A, Kako T, Ohmachi N, Kiuchi T, Maeta T, Yoshida M, Mori Y, Kohi F, Kuriyama S. Time-lag performance of radiofrequency ablation after percutaneous ethanol injection for the treatment of hepatocellular carcinoma. Int J Oncol 2006; 28: 971-976

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Kurokohchi K, Hirai S, Ohgi T, Ono M, Yoshitake A, Ebara K, Kitamura Y, Kasai Y, Maeta T, Kiuchi T, Masaki T, Yoneyama H, Kohi F, Kuriyama S. Thoracoscopic ethanol injection and radiofrequency ablation for the treatment of hepatocellular carcinoma located immediately under the diaphragm. Int J Oncol 2006; 29: 375-380 Kurokohchi K, Watanabe S, Masaki T, Hosomi N, Miyauchi 9 Y, Himoto T, Kimura Y, Nakai S, Deguchi A, Yoneyama H, Yoshida S, Kuriyama S. Comparison between combination therapy of percutaneous ethanol injection and radiofrequency ablation and radiofrequency ablation alone for patients with hepatocellular carcinoma. World J Gastroenterol 2005; 11: 1426-1432 10 Kurokohchi K, Masaki T, Miyauchi Y, Hosomi N, Yoneyama H, Yoshida S, Himoto T, Deguchi A, Nakai S, Inoue H, Watanabe S, Kuriyama S. Efficacy of combination therapies of percutaneous or laparoscopic ethanol-lipiodol injection and radiofrequency ablation. Int J Oncol 2004; 25: 1737-1743 11 Kurokohchi K, Masaki T, Miyauchi Y, Funaki T, Yoneyama H, Miyoshi H, Yoshida S, Himoto T, Morishita A, Uchida N, Watanabe S, Kuriyama S. Percutaneous ethanol and lipiodol injection therapy for hepatocellular carcinoma. Int J Oncol 2004; 24: 381-387

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Bruix J, Hessheimer AJ, Forner A, Boix L, Vilana R, Llovet JM. New aspects of diagnosis and therapy of hepatocellular carcinoma. Oncogene 2006; 25: 3848-3856 13 Rossi S, Garbagnati F, Lencioni R, Allgaier HP, Marchiano A, Fornari F, Quaretti P, Tolla GD, Ambrosi C, Mazzaferro V, Blum HE, Bartolozzi C. Percutaneous radio-frequency thermal ablation of nonresectable hepatocellular carcinoma after occlusion of tumor blood supply. Radiology 2000; 217: 119-126 14 Buscarini L, Buscarini E, Di Stasi M, Quaretti P, Zangrandi A. Percutaneous radiofrequency thermal ablation combined with transcatheter arterial embolization in the treatment of large hepatocellular carcinoma. Ultraschall Med 1999; 20: 47-53 15 Kurokohchi K, Takaguchi K, Kita K, Masaki T, Kuriyama S. Successful treatment of advanced hepatocellular carcinoma by combined administration of 5-fluorouracil and pegylated interferon-alpha. World J Gastroenterol 2005; 11: 5401-5403 16 Kurokohchi K, Hosomi N, Yoshitake A, Ohgi T, Ono M, Maeta T, Kiuchi T, Matsumoto I, Masaki T, Yoneyama H, Kohi F, Kuriyama S. Successful treatment of large-size advanced hepatocellular carcinoma by transarterial chemoembolization followed by the combination therapy of percutaneous ethanollipiodol injection and radiofrequency ablation. Oncol Rep 2006; 16: 1067-1070 S- Editor Zhu LH L- Editor Wang XL

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World J Gastroenterol 2007 August 28; 13(32): 4401-4404 World Journal of Gastroenterology ISSN 1007-9327 © 2007 WJG. All rights reserved.

CASE REPORT

Hepatocellular carcinoma with chronic B-type hepatitis complicated by autoimmune hemolytic anemia: A case report Toshie Okada, Keiichi Kubota, Junji Kita, Masato Kato, Tokihiko Sawada Toshie Okada, Keiichi Kubota, Junji Kita, Masato Kato, Tokihiko Sawada, Department of Gastro-enterological Surgery, Dokkyo University School of Medicine, Tochigi, Japan Correspondence to: Keiichi Kubota, MD, PhD, Department of Gastroenterological Surgery, Dokkyo University School of Medicine, Kitakobayashi 880, Mibu, Shimotsuga, Tochigi 321-0293, Japan. [email protected] Telephone: +81-282-861111 Fax: +81-282-866317 Received: 2007-04-09 Accepted: 2007-04-26

Okada T, Kubota K, Kita J, Kato M, Sawada T. Hepatocellular carcinoma with chronic B-type hepatitis complicated by autoimmune hemolytic anemia: A case report. World J Gastroenterol 2007; 13(32): 4401-4404

http://www.wjgnet.com/1007-9327/13/4401.asp

INTRODUCTION Abstract A 57-year-old man consulted a local hospital because of a persistent slight fever. At the age of 37 years he was diagnosed having B-type hepatitis, but left the liver dysfunction untreated. Twenty years later, he was diagnosed having chronic hepatitis B, hepatocellular carcinoma (HCC) and macrocytic anemia, and referred to our hospital for further investigation. A HCC with a maximum diameter of 5.2 cm was detected in segment 8. Results of blood tests included 1.8 mg/dL serum total bilirubin, 0.9 mg/dL bilirubin, less than 10 mg/dL haptoglobin, 7.9 g/dL hemoglobin, 130 fL MCV, and 14.5% reticulocytes. A bone marrow sample showed erythroid hyperplasia. The direct Coombs test gave a positive result. We diagnosed the anemia as autoimmmune hemolytic anemia (AIHA), for which prednisolone could not be administered due to positivity for HBsAg and HBeAg. After preparation of washed blood cells for later transfusion, the patient underwent systematic resection of segment 8. The cut surface of the resected specimen demonstrated an encapsulated yellow-brownish tumor measuring 52 mm × 40 mm which was diagnosed pathologicaly as moderately th differentiated HCC. On the 9 postoperative day, the patient’s temperature rose to 38℃, and exacerbated hemolysis was observed. The maximum total bilirubin value was 5.8 mg/dL and minimum hemoglobin level was 4.6 g/dL. He tolerated this period without blood transfusion. Currently he is being followed up as an outpatient, and shows no signs of HCC recurrence or symptoms of anemia. AIHA associated with HBV infection has been described in only three previous cases, and the present case is the first in which surgery was performed for accompanying HCC. © 2007 WJG . All rights reserved.

Key words: Hepatocellular carcinoma; B-type hepatitis; Auto-immune hemolytic anemia

Autoimmune hemolytic anemia (AIHA) is a disease characterized by hemolysis caused by auto-antibodies targeting erythrocytes, and is exacerbated by stress [1]. In this report, we describe a patient with AIHA who underwent resection of a hepatocellular carcinoma (HCC), but subsequently showed improvement of anemia.

CASE REPORT A 57-year-old man consulted a local hospital because of a slight fever for 2 wk. At the age of 37 years, he was diagnosed having liver dysfunction due to B-type hepatitis, but had left it untreated for 20 years. His blood chemistry data showed liver dysfunction and macrocytic anemia, and a liver tumor was found by abdominal ultrasonography. He was, therefore, referred to our hospital in November, 2003. On physical examination, his conjunctivae were anemic. The liver was palpable one finger-breadth below the right costal margin, but the spleen was not palpable. His family history showed that his elder brother died of liver disease at the age of 53, but the details were unknown. The patient underwent abdominal surgery twice (appendectomy for appendicitis at the age of 19 and cholecystectomy for cholelithiasis at 35) and received no blood transfusion prior to admission to our hospital. Blood tests revealed impaired liver function, hemolysis and macrocytic anemia (Table 1). The AFP and PIVKAⅡ levels were also high. The indocyaningreen (ICG) retention rate at 15 min was 13%. HBs antigen, HBs antibody, Hbe antigen, HBe antibody and HBc antibody were positive. HBV-DNA PCR showed viral proliferation. Serological tests for syphilis, HCV antibody, antinuclear and antiDNA antibodies, HAM test and sugar water test all gave negative results. However, direct and indirect Coombs tests gave positive results, and the cold agglutinin titer increased 256-fold. A bone marrow sample showed erythroid hyperplasia. Abdominal ultrasonography and computed tomography www.wjgnet.com

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Table 1 Blood test data AST 67 U/L ALT 64 U/L LDH 318 U/L (LDH1: 36.1%, LDH2: 34.2%) T. Bil 1.8 mg/dL (D.Bil: 0.9 mg/dL, I.Bil: 0.9 mg/dL) TP 6.2 g/dL Alb 3.8 g/dL ICGR15 13% AFP 869 ng/mL PIVKA-II 301 AU/mL Direct Coombs test Positive Anti-nuclear antibody Negative HAM test Negative Sugar water test Negative Cold agglutinin titer 256 times

WBC RBC Reticulocyte HGB HCT MCV PLT PT% HBsAg HBsAb HBeAg HBeAB HBcAb HBV-DNA PCR TPHA HCVAb

2700 × 106/L 199 × 1010/L 14.5% 7.9 g/dL 23.6% 130 fL 6 × 109/L 68% 69 COI 172 COI 108 COI 40 Inh% 100 Inh% 5.9 LGE/mL Negative Negative

(CT) revealed a liver tumor with a maximum diameter of 5.2 cm in segment 8, which was enhanced by contrast material (Figure 1A). Abdominal angiography showed a hypervascular liver tumor (Figure 1B), but portography demonstrated no abnormality. Under a diagnosis of HCC, chronic B-type hepatitis and AIHA, the patient underwent systematic resection of segment 8. The operation time was 5 h 42 min and bleeding volume was 642 mL. Blood transfusion was not required. The cut surface of the resected liver specimen (S8) demonstrated an encapsulated yellow-brownish tumor measuring 52 mm × 40 mm (Figure 2). Histology showed the characteristics of HCC with moderately differentiated neoplastic cells in a trabecular pattern. The tumor was solitary, but as vascular invasion was present, the classification was T2N0M0 according to the UICC Manual of Clinical Oncology (English Edition)[2]. T he patient recovered steadily, but on the 9 th postoperative day, his temperature rose to 38℃, and antibiotics were administered. At the same time, exacerbation of hemolysis was observed, the maximum total bilir ubin value was 5.8 mg/dL and minimum hemoglobin level was 4.6 g/dL. He tolerated this period without blood transfusion, and the symptoms gradually improved over the course of about one week. On the 26th postoperative day, he received two units of washed blood cells due to increased activity and was discharged three days later (Figure 3). For treatment of B-type hepatitis, lamivudine (100 mg/d) was administered from the 29th postoperative day, and the blood tests for HB virus subsequently became negative about four months later. Because of improvement of the anemia with a hemoglobin level of 9.3 g/dL 9.7 g/dL, prednisolone was not administered for treatment of AIHA after surgery. The patient is currently being followed up as an outpatient, and shows no signs of HCC recurrence.

DISCUSSION AIHA is a disease in which erythrocytes are injured by an auto-antibody that reacts with an antigen on the www.wjgnet.com

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erythrocyte membrane. This results in both intra- and extravascular hemolysis, and anemia[1]. AIHA is classified into “warm” or “cold” AIHA according to the temperature at which the antibody acts. IgG or complement on erythrocytes can be detected by direct Coombs test. Several reasons for the appearance of auto-antibody have been suggested. (1) Erythrocytes are recognized as “non-self ” as a result of a change of their surface antigen; (2) An antibody originally directed to an invasive microorganism cross-reacts with an erythrocyte antigen; (3) Immunity tolerance fails due to an abnormality of the immunity response system; (4) The clone responsible for producing an auto-antibody increases in a monoclonal or polyclonal manner. However, the fundamental causes or mechanisms are unclear. AIHA has been repor ted to be associated with several liver diseases, including autoimmune hepatitis in 15 cases[3-7], C-type hepatitis in 11[8-18], B-type hepatitis in 2[19,20], cytomegalovirus hepatitis in one[21], and A-type hepatitis in 1[22]. Autoimmune responses may play a major role in inducing the combination of the two diseases. Further more, there are a number of reports of HBV infections associated with polyarteritis nodosa, membranous glomerulonephritis, and Gianotti disease[23,24]. An autoimmune reaction participates in all these diseases. Anemia deteriorated in 6 of 11 patients with C-type hepatitis complicated by AIHA, because of interferon therapy for chronic active C-type hepatitis. These findings suggest that activation of the immunological response can lead to production of auto-antibody directed against erythrocytes, thus worsening AIHA. Before presentation, our patient had suffered from chronic HBV infection for at least 20 years. Therefore, it can be speculated that the patient’s immune response was stimulated by HBV, thus contributing to production of the anti-erythrocyte antibody and induction of AIHA. The primary treatment for AIHA is steroid medication. If this is ineffective, splenectomy or an immunosuppressant can be considered instead. Although treatment of AIHA before surgery may be advisable, our patient could not administer prednisolone due to the presence of HBV. Therefore, we selected surgical treatment as the first option. This is the first reported case of AIHA in which surgery was performed for an accompanying HCC. For patients with AIHA, transfusion of washed blood cells is desirable. However, irrespective of the type of transfusion performed, hemolysis occurs. Therefore, in this case, blood transfusion was given only once. Although splenectomy has been performed for many patients with AIHA, surgery for tumors complicating AIHA has been reported for only two patients with renal cell carcinoma [25], and 19 patients with benign ovarian cysts[26,27]. In the case of renal cell carcinoma complicated by AIHA, prompt resolution of AIHA occurred after surgical excision. In 17 of the 19 cases of benign ovarian cyst, the AIHA also improved after surgical excision. For AIHA complicated by dermoid cyst, steroid treatment may sometimes be slightly effective. There is one case report of a Japanese patient who underwent transcatheter

Okada T et al. HCC with autoimmune hemolytic anemia

A1



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A2

Enhanced early phase

Plain

A3

B

Figure 1 Helical dynamic abdominal CT revealing a liver tumor with a maximum diameter of 5.2 cm in segment 8 (A) and abdominal angiography showing hypervascularity in it (B).

Angiography

Enhanced late phase

Table 2 Cases of AIHA complications Complicated disease

Number of complications by AIHA

A-type hepatitis B-type hepatitis C-type hepatitis Autoimmune hepatitis CMV hepatitis Renal cell carcinoma Benign ovarian tumor HCC

Figure 2 Gross appearance of resected specimen. The cut surface of the resected liver specimen (S8) showed an encapsulated yellow-brownish tumor measuring 52 mm × 40 mm and 250 g.

30

HBG: g/dL HCT: % I-Bil: mg/dL

HBG HCT I-Bil

20

10

  0

↑0

↑9

↑19

↑26↑29

Figure 3 Postoperative course. ↑0: Operation, ↑9:Fever up, ↑19: Sleep out, ↑26: Blood transfusion of washed blood cells, ↑29: Discharged and started intake of lamivudine.

1 2 11 14 1 1a 19b 2c

In patients with renal cell carcinoma (a) and ovarian tumor (b), AIHA improved after resection of them. In two cases of HCC complicated by AIHA (c), one is our case and the other is a case that revealed the symptom of AIHA after TAE for HCC.

the partial liver resection, and developed the symptoms of AIHA after this treatment (Table 2) [8] . It is thus unprecedented for liver resection to be performed for a patient with AIHA. In our patient also, anemia improved after surgical excision without treatment of AIHA. In the cases of ovarian tumor mentioned above, AIHA might be regarded as a concomitant symptom of dermoid cyst, i.e. a para-neoplastic syndrome. In our patient, however, we were unable to clearly demonstrate whether AIHA was a para-neoplastic syndrome due to HCC. Our patient is now being followed up on an outpatient basis, and shows no signs of hepatitis vir us, HCC recurrence or symptoms of anemia. This is the first report of safe and successful resection of a complicating liver tumor in a patient with AIHA.

REFERENCES hepatic arterial embolizaion for recurrence of HCC after

1

Neff AT. Autoimmune hemolytic anemias. In: Greer JP, www.wjgnet.com

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Rodgers GM, Foerster J, Paraskevas F, Lukens JN, Glader B. Wintrobe’s Clinical Hematology. 11th ed. Philadelphia: Lippincott Williams & Wilkins, 2004: 1157-1182 Zhao YT, Steven AC. Liver cancer. In: Pollock RE, Doroshow 2 JH, Khayat D, Nakao A, O’Sullivan B. UICC Manual of Clinical Oncology. 8th ed. New Jersey: John Wiley & Sons, Inc., 2004: 405-427 Kondo H, Kajii E, Oyamada T, Kasahara Y. Direct antiglobulin 3 test negative autoimmune hemolytic anemia associated with autoimmune hepatitis. Int J Hematol 1998; 68: 439-443 Yokoyama T, Matsunaga K, Kawai T, Tsuji T, Ueda A, Ohno J, 4 Ishigatsubo Y, Tani K, Yokota S, Miyachi K, Ohkubo T. A case of autoimmune hepatitis type 2 complicated with autoimmune hemolytic anemia. Jpn J Clin Immun 1994; 17: 199-204 Ogawa M, Ueda S, Yokosuka O. Autoimmune hemolytic 5 anemia as a sequela of autoimmune hepatitis. Rheumatology 1996; 15: 74-78 Miyatake H , H i r a s a k i S , T a k a g u c h i K . A u t o i m m u n e 6 Hepatitis Associated with Autoimmune Hemolytic Anemia. Kagawatyubyouishi 2001; 20: 16-21 Urganci N, Akyildiz B, Yildirmak Y, Ozbay G. A case of 7 autoimmune hepatitis and autoimmune hemolytic anemia following hepatitis A infection. Turk J Gastroenterol 2003; 14: 204-207 Fukutomi Y, Kojima Y, Nakamura H, Yamazaki T, Tanaka Y, 8 Yasuda N, Watanabe Y, Kawase K, Ohnishi H, Yamada M, Shimizu M. A case of liver cirrhosis type C and hepatocellular carcinoma accompanied by autoimmune hemolytic anemia. Kanzo 2002; 43: 207-211 Giannoccaro F, Pace L, Antonaci S, Schiraldi O. Autoimmune 9 hemolytic anemia in chronic hepatitis C virus infection treated with alpha-interferon: a therapy-related event or extra-hepatic immunologic manifestation? Recenti Prog Med 1999; 90: 592-594 10 Etienne A, Gayet S, Vidal F, Poullin P, Brunet C, Harle JR, Kaplanski G. Severe hemolytic anemia due to cold agglutinin complicating untreated chronic hepatitis C: efficacy and safety of anti-CD20 (rituximab) treatment. Am J Hematol 2004; 75: 243-245 11 Katayama T, Naitou Y, Kusaka M, Ochiai S, Yoshida M, Kaito K, Masuoka H, Shimada T, Nishiwaki K, Kobayashi M. Autoimmune hemolytic anemia reactivated by alphainterferon therapy in a case of chronic active C-type hepatitis. Rinsho Ketsueki 1995; 36: 339-346 12 Kazuta Y, Watanabe N, Sagawa K, Kobayashi H, Kojima T, Funabashi H, Moritoh T, Kasukawa R. A case of autoimmune hemolytic anemia induced by IFN-beta therapy for type-C chronic hepatitis. Fukushima J Med Sci 1995; 41: 43-49 13 Yamaura T, Yoshizawa K, Rokuhara A, Nishizawa Y, Matsumoto A, Tanaka E, Kiyosawa K. A case of chronic

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hepatitis C complicated by Coombs-negative autoimmune hemolytic anemia during interferon treatment. Kanzo 2001; 42: 465-470 14 Ohsawa I, Uehara Y, Hashimoto S, Endo M, Fujita T, Ohi H. Autoimmune hemolytic anemia occurred prior to evident nephropathy in a patient with chronic hepatitis C virus infection: case report. BMC Nephrol 2003; 4: 7 15 Rizzi R, Lucarella FP, Ruggieri GB, L'Abbate M. Autoimmune hemolytic anemia presenting during treatment of chronic hepatitis C with interferon alpha. Ann Ital Med Int 2003; 18: 107-110 16 Moccia F, Tognoni E, Boccaccio P. Autoimmune hemolytic anemia in chronic hepatitis C virus infection: an unusual extrahepatic autoimmune manifestation. Ann Ital Med Int 2001; 16: 256-259 17 Chao TC, Chen CY, Yang YH, Chen PM, Chang FY, Lee SD. Chronic hepatitis C virus infection associated with primary warm-type autoimmune hemolytic anemia. J Clin Gastroenterol 2001; 33: 232-233 18 Srinivasan R. Autoimmune hemolytic anemia in treatmentnaive chronic hepatitis C infection. J Clin Gastroenterol 2001; 32: 245-247 19 Yoshioka K, Miyata H. Autoimmune haemolytic anaemia in an asymptomatic carrier of hepatitis B virus. Arch Dis Child 1980; 55: 233-234 20 Fagiolo E, Vigevani F, Pozzetto U. High cytokine serum levels in patients with autoimmune hemolytic anemia (AIHA). Immunol Invest 1994; 23: 449-456 21 Cataldo F, Marasa R, Maltese I, Gueci G, Violante M, Albeggiani A. Autoimmune hemolytic anemia in hepatitis due to cytomegalovirus in a 5-month-old nursing infant. Minerva Pediatr 1987; 39: 803-806 22 Ibe M, Rude B, Gerken G, Meyer zum Buschenfelde KH, Lohse AW. Coombs-negative severe hemolysis associated with hepatitis A. Z Gastroenterol 1997; 35: 567-569 23 Kidd-Ljunggren K, Miyakawa Y, Kidd AH. Genetic variability in hepatitis B viruses. J Gen Virol 2002; 83: 1267-1280 24 Sherlock S, Dooley J. Disease of the Liver and Biliary System. 10th ed. Oxford: Blackwell Sci Pub, 1997: 265-302 25 Lands R, Foust J. Renal cell carcinoma and autoimmune hemolytic anemia. South Med J 1996; 89: 444-445 26 Payne D, Muss HB, Homesley HD, Jobson VW, Baird FG. Autoimmune hemolytic anemia and ovarian dermoid cysts: case report and review of the literature. Cancer 1981; 48: 721-724 27 Agarwal V, Sachdev A, Singh R, Lehl S, Basu S. Autoimmune hemolytic anemia associated with benign ovarian cyst: a case report and review of literature. Indian J Med Sci 2003; 57: 504-506 S- Editor Liu Y L- Editor Wang XL

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CASE REPORT

Spontaneous rupture of the liver in a patient with chronic hepatitis B and D Ching-Jung Liu, Rong-Nan Chien, Cho-Li Yen, Jia-Jang Chang Ching-Jung Liu, Rong-Nan Chien, Cho-Li Yen, Jia-Jang Chang, Liver Research Unit, Chang Gung Memorial Hospital and Institute of Technology, Keelung, Taiwan, China Correspondence to: Rong-Nan Chien, Professor, Liver Research Unit, Chang Gung Memorial Hospital and Institute of Technology, Keelung 222, Mai-Chin Road Keelung, Taiwan, China. [email protected] Telephone: +886-2-24313131-3171 Fax: +886-2-24335342 Received: 2007-04-25 Accepted: 2007-06-09

Abstract Spontaneous rupture of the liver is a rare condition with serious consequences, if not recognized and treated in time. It has been reported as a complication of several disorders, including benign or malignant liver tumors, connective tissue disease, infiltrating liver disease, preeclampsia, and post anticoagulant therapy. We report a case of spontaneous rupture of liver in a noncirrhotic, chronic hepatitis B and D patient presenting with acute hemoperitoneum and shock. The subcapsular hematoma and rupture of liver were documented by image studies. The patients' condition gradually stabilized after fluid resuscitation. The reported case and literature review suggest that spontaneous rupture of liver must be considered in a differential diagnosis of acute hemoperitoneum. A high index of suspicion and early diagnosis with imaging are critically important. © 2007 WJG . All rights reserved.

Key words: Spontaneous liver rupture; Hemoperitoneum; Hepatitis B Liu CJ, Chien RN, Yen CL, Chang JJ. Spontaneous rupture of the liver in a patient with chronic hepatitis B and D. World J Gastroenterol 2007; 13(32): 4405-4407

http://www.wjgnet.com/1007-9327/13/4405.asp

INTRODUCTION Atraumatic spontaneous r upture of the liver is an uncommon but important clinical entity associated with a high morbidity and mortality. Possible causes include infections such as hydatid disease, infiltrating liver diseases such as amyloidosis, inflammatory disorders such as vasculitis, malignant and benign tumors, and pregnancy

related conditions[1]. We describe a case of spontaneous liver rupture, which mimics the rupture of hepatocellular carcinoma (HCC), in a patient with dual viral infection of hepatitis B and D viruses. To the best of our knowledge, this is the first case of spontaneous liver rupture occurred in a patient with dual viral infection at non-cirrhotic stage.

CASE REPORT This 43-year-old male taxi driver was diagnosed to have aortic dissection and underwent ascending aortic graft surgery at a medical center 8 years ago, with sequellae of paraplegia and urine incontinence. He had diabetes mellitus and hypertension with regular medication control at our hospital and was found to be seropositive for hepatitis B surface antigen (HBsAg) during the past 10 years, but he paid no attention to it. Unfortunately, he was admitted because of nausea, vomiting and intermittent right upper abdominal dull pain for one week. The pain could radiate to right upper back and neck, 10-20 min in duration, not related to exercise and relieved spontaneously. He denied any trauma history prior to admission. The pain became more severe with sharp and tearing like features in the midnight prior to admission. On arrival, he was sick, pale and mild short of breath. His blood pressure was 106/66 mmHg, heart rate 102 beats/min, temperature 35.7℃, and respiratory rate 24/min. Tenderness on epigastric and right upper quadrant of abdomen without rebound pain or shift dullness was demonstrated. The liver span was 12 cm in right middle clavicle line. The laboratory tests at emergency room showed 12.9 g/dL hemoglobin, 38.9% hematocrit, 23.8 × 109/L leukocytes, 80 × 109/L platelets, 38 U/L (normal < 34 U/L) aspartate transaminase, 59 U/L (normal < 36 U/L) alanine transaminase, 1.2 mg/dL (normal < 1.3 mg/ dL) total bilirubin, 85 U/L (normal 28-94 U/L) alkaline phosphatase, 147 U/L (normal < 26 U/L) γ -glutamyl transpeptidase, 13.3 s (control: 10.8 s) prothrombin time, 2.5 g/dL albumin, 83 U/L (normal < 137 U/L) amylase, 230 U/L (normal < 190 U/L) lipase, 17.9 mg/dL blood urea nitrogen, 2.4 mg/dL creatinine, and 5 ng/mL α-fetoprotein by radioimmunoassay. Unfortunately, fever, chills and hypotension occurred 12 h after admission. His blood pressure dropped to 64/43 mmHg, heart rate was 120 beats/min, respiratory rate was 25/min and central venous pressure was 4 cm H2O. Follow-up hemoglobin fell to 5.8 g/dL, and hematocrit to 18%. HBsAg and antibody to hepatitis delta virus were positive by radioimmunoassay (Ausria Ⅱ, and Anti-Delta, Abbott Laboratories, Chicago, www.wjgnet.com

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Figure 1 Abdominal CT on admission showing a subcapular hematoma and a ruptured cleft at the right posterior-superior segment of liver.

Figure 2 Follow-up CT 5 mo later showing the decreased size of subcapsular hematoma (3.5 cm).

IL, USA). Antibody to hepatitis C virus by the third generation enzyme immunoassay kit (AxSYM ® HCV. Version 3.0 Abbott Lab) was negative. Abdominal ultrasonography (US) revealed intra-abdominal fluid collection and a 15 cm right subcapsular hematoma over a non-cirrhotic liver. Abdominal computed tomography (CT) and magnetic resonance imaging (MRI) revealed a right subcapsular hematoma and a ruptured at the right posterior-superior segment of liver (Figure 1) besides a type B aortic dissecting aneurysm. No hepatic aneurysmal lesions were detected in abdominal CT or MRI studies. Coronary arteriography revealed coronary arterial disease. Aortogram revealed chronic complex aortic dissection without rupture. Selective catheterization of the bleeding branch of the hepatic artery was unsuccessful due to tortuous vessels. US guided paracentesis from the right subcapsular hematoma revealed fresh blood with clotting immediately. The patients’ general conditions, including hemogram, body temperature, heart rate, respiratory rate and blood pressure gradually improved after blood transfusion and intravenous fluid resuscitation. He was discharged 13 d after symptoms subsided, and followed up at our outpatient clinic regularly. Follow-up CT 5 mo later showed a 3.5 cm low density liver mass over the right posterior-superior segment (Figure 2). Biopsy of the mass showed only hematoma component without malignancy. The pathology of liver rather than the mass showed chronic persistent hepatitis with the histological activity index (necro-inflammatory score 3 and fibrosis score 0). Liver biochemical tests and alpha-fetoprotein value were normal. The liver hematoma became smaller (1 cm) one year later by CT study. Now, he enjoys his life 4 years after initial diagnosis and no hepatic lesion was detected during the subsequent follow-up.

Otherwise, it is spontaneous[3]. However, in the literature regardless of underlying disease, atraumatic liver rupture is synonymous with spontaneous liver rupture. The clinical manifestations of atramatic acute hemoperitoneum, right subcapsular hematoma of liver, and evidence of chronic hepatitis without HCC in the present case strongly suggest that he was a victim of spontaneous liver rupture. Certainly, it is very difficult to distinguish between atraumatic spontaneous rupture and rupture following trivial trauma. HCC rupture is the most frequently reported cause of spontaneous liver rupture [4,5]. From a review of 70 Chinese patients with spontaneous liver rupture reported by Chen et al[5], the major cause is HCC (85.7%) followed by adenoma (5.7%), cirrhosis (4.3%), hemangioma (2.9%), and metastatic liver tumor (2.9%)[5]. Review of the literature disclosed that other conditions associated with spontaneous liver rupture include other benign and malignant liver tumors, pregnancy related disorders (acute fatty liver, preeclampsia and eclampsia of pregnancy[6,7], amyloidosis [2], peliosis hepatitis, autoimmune diseases (systemic lupus erythematous, rheumatoid arthritis, EhlersDanlos syndrome, polyarteritis nodosa)[8,9], anticoagulant therapy, liver transplantation[1], aortic aneurysm surgery[10], peritoneal hemodialysis[11], and perforated gastric ulcer[12]. Atraumatic liver r upture is usually preceded by subcapsular hematoma [6] . Epigastric or right upper quadrant abdominal pain is the most common symptom of subcapsular hepatic hemorrhage. Patients may suffer from pain for several days or weeks before rupture of the capsule. Physical examination usually reveals hepatomegaly, right upper quadrant tenderness. Abdominal distention, peritoneal signs and shock are usually present when rupture Occurs[2,4]. The diagnosis of spontaneous liver rupture is often confirmed by imaging studies in a stable patient because initial clinical suspicion is low. In a hemodynamically unstable patient, an abdominal US in an emergency room may demonstrate ascites in the peritoneal cavity and a mass lesion in liver. Hepatic subcapsular hematoma can be diagnosed by abdominal US, MRI or nuclear scan, but abdominal CT is the most sensitive and specific imaging examination[13]. Abdominal US can usually distinguish liver rupture from biliary tract disease, and can diagnose a subcapsular hematoma. However, it is difficult

DISCUSSION Hepatic rupture is mostly caused by trauma. Clinical conditions inducing he patomeg aly, for example, amyloidosis, malaria, venous stasis and enlarged liver tumors, predispose to traumatic rupture [2]. Atraumatic liver rupture is a rare condition with serious consequences, if not recognized and treated in time. Strictly speaking, it is pathologic if the patient has an underlying disease. www.wjgnet.com

Liu CJ et al . Spontaneous rupture of liver

to distinguish hepatic hematoma from liver abscess by US only and these two entities should be differentiated by other methods such as abdominal CT or paracentesis. Paracentesis can also document blood in the peritoneal cavity when rupture occurs [6]. Control of bleeding is the crucial management in patients with spontaneous liver rupture. Treatment options include conservative treatment, transcatheter hepatic arterial embolization, or surgery. In patients with spontaneous HCC rupture, surgery can achieve hemostasis in more than 95% of cases[5] and selective surgical intervention is better than an aggressive surgical approach[14]. Transcatheter hepatic arterial embolization gives usually only temporary effect with surgical intervention in ruptured amyloid livers [2]. However, the mortality is the highest with surgery and the lowest with transcatheter hepatic arterial embolization of a bleeding hepatic artery when rupture occurs in pregnant women [7]. These reports suggest that the best choice of treatment depends on the underlying etiology and individualized patient’s condition. The exact mechanism of spontaneous liver rupture is controversial. Spontaneous liver rupture in amyloidosis has been thought to be due to liver enlargement, rigidity of hepatic parenchyma, and vascular fragility from amyloid involvement [15]. Spontaneous liver rupture in pregnancy may be due to hepatic infarction resulting from gross ischemia and obstruction to the sinusoidal blood flow by deposited fibrin and relative hypovolemia[6]. In HCC or other malignant liver tumors, the pathogenesis of spontaneous liver rupture may be attributed to the overlying normal liver parenchyma splitting from the expanding tumor growth, a tear in tumor surface, rupture of parasitic feeding artery, or hemorrhage caused by tumor necrosis locating near the liver surface[16]. Venous congestion secondary to obstruction of hepatic venous outflow of the tumor by thrombi, together with rich and fragile arterial supply may contribute to the rupture of HCC[17]. We have previously reported a spontaneous spleen rupture patient with hepatitis B virus related cirrhosis[18], and two cases of spontaneous liver rupture in cirrhosis were also mentioned by Chen et al[5]. Portal hypertension may play an important role in these 3 cases. To the best of our knowledge, the present case is the first case of spontaneous liver rupture in a non-cirrhotic liver disease. Liver biopsy of this patient showed fibrosis score 0 by histological activity index scoring system[19], excluding the possibility of liver cirrhosis, even though a possible sample bias was present. Li et al[9] speculated that spontaneous liver rupture in polyarteritis nodosa is due to massive bleeding from an aneurysmal intrahepatic artery. The present case suffered from chronic aortic dissecting aneurysm and perhaps concomitant vascular lesions, such as a small intrahepatic aneurysm along with hepatitis. However, abdominal CT and MRI revealed no hepatic aneurysmal lesions. Detailed history and careful physical examination excluded traumatic injury. The definite cause of liver rupture in our case remains unclear. In summary, spontaneous liver rupture is a medical emergency. This case report suggests that the possibility of



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spontaneous liver rupture should be considered if a patient presents with acute hemoperitoneum. It is a diagnostic and therapeutic challenge for the clinician because of its rarity and potential lethal entity. The optimal choice of treatment depends on the underlying etiology. Understanding the common causes of spontaneous liver rupture helps the clinician to make a correct judgment and management in time.

REFERENCES 1

2

3 4 5 6 7

8 9 10 11 12 13 14

15 16

17 18 19

Mistry BM, Solomon H, Garvin PJ, Durham RM, Turnage S, Bacon BR, Galvin N, Varma CR. Spontaneous rupture of the liver upon revascularization during transplantation. Transplantation 2000; 69: 2214-2218 Bujanda L, Beguiristain A, Alberdi F, Cosme A, Ruiz de la Hermosa J, Gutierrez-Stampa, Arenas JI. Spontaneous rupture of the liver in amyloidosis. Am J Gastroenterol 1997; 92: 1385-1386 Hyun BH, Varga CF, Rubin RJ. Spontaneous and pathologic rupture of the spleen. Arch Surg 1972; 104: 652-657 Miyamoto M, Sudo T, Kuyama T. Spontaneous rupture of hepatocellular carcinoma: a review of 172 Japanese cases. Am J Gastroenterol 1991; 86: 67-71 Chen ZY, Qi QH, Dong ZL. Etiology and management of hemmorrhage in spontaneous liver rupture: a report of 70 cases. World J Gastroenterol 2002; 8: 1063-1066 Ralston SJ, Schwaitzberg SD. Liver hematoma and rupture in pregnancy. Semin Perinatol 1998; 22: 141-148 Rinehart BK, Terrone DA, Magann EF, Martin RW, May WL, Martin JN Jr. Preeclampsia-associated hepatic hemorrhage and rupture: mode of management related to maternal and perinatal outcome. Obstet Gynecol Surv 1999; 54: 196-202 Pettersson T, Lepantalo M, Friman C, Ahonen J. Spontaneous rupture of the liver in rheumatoid arthritis. Scand J Rheumatol 1986; 15: 348-349 Li AK, Rhodes JM, Valentine AR. Spontaneous liver rupture in polyarteritis nodosa. Br J Surg 1979; 66: 251-252 de Cossart L. Spontaneous rupture of the liver; an unexpected cause of death after aortic aneurysm surgery. J Cardiovasc Surg (Torino) 1988; 29: 483-485 Colucci P, Fortis D, Nicolini A, Banfi G, Scalamogna A, De Vecchi AF. Severe hemoperitoneum caused by spontaneous liver rupture: a case report. Perit Dial Int 2002; 22: 417-419 Tsokos M, Schulz F. Non-traumatic liver rupture due to a perforated gastric ulcer. Int J Legal Med 1999; 112: 321-323 Casillas VJ, Amendola MA, Gascue A, Pinnar N, Levi JU, Perez JM. Imaging of nontraumatic hemorrhagic hepatic lesions. Radiographics 2000; 20: 367-378 Leung KL, Lau WY, Lai PB, Yiu RY, Meng WC, Leow CK. Spontaneous rupture of hepatocellular carcinoma: conservative management and selective intervention. Arch Surg 1999; 134: 1103-1107 Greipp PR, Kyle RA, Bowie EJ. Factor-X deficiency in amyloidosis: a critical review. Am J Hematol 1981; 11: 443-450 Chearanai O, Plengvanit U, Asavanich C, Damrongsak D, Sindhvananda K, Boonyapisit S. Spontaneous rupture of primary hepatoma: report of 63 cases with particular reference to the pathogenesis and rationale treatment by hepatic artery ligation. Cancer 1983; 51: 1532-1536 Ong GB, Taw JL. Spontaneous rupture of hepatocellular carcinoma. Br Med J 1972; 4: 146-149 Chien RN, Liaw YF. Pathological rupture of spleen in hepatitis B virus-related cirrhosis. Am J Gastroenterol 1993; 88: 1793-1795 Knodell RG, Ishak KG, Black WC, Chen TS, Craig R, Kaplowitz N, Kiernan TW, Wollman J. Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology 1981; 1: 431-435 S- Editor Liu Y L- Editor Wang XL

E- Editor Lu W

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World J Gastroenterol 2007 August 28; 13(32): 4408 World Journal of Gastroenterology ISSN 1007-9327 © 2007 WJG. All rights reserved.

ACKNOWLEDGMENTS

Acknowledgments to Reviewers of World Journal of Gastroenterology Many reviewers have contributed their expertise and time to the peer review, a critical process to ensure the quality of World Journal of Gastroenterology. The editors and authors of the articles submitted to the journal are grateful to the following reviewers for evaluating the articles (including those were published and those were rejected in this issue) during the last editing period of time.

Frank A Anania, Professor Emory University School of Medicine, Division of Division Digestive Diseases, 615 Michael Street, Room 255 Whitehead Biomedical Research Building, Atlanta, GA 30322, United States Masahiro Arai, MD, PhD Department of Gastroenterology, Toshiba General Hospital, 6-3-22 Higashi-ooi, Shinagawa-ku, Tokyo 140-8522, Japan Hitoshi Asakura, Director, Emeritus Professor International Medical Information Center, Shinanomachi Renga Bldg.35, Shinanomachi, Shinjukuku, Tokyo 160-0016, Japan Tomasz Brzozowski, Professor Department of Physiology, Jagiellonian University Medical College, 16 Grzegorzecka Str, Cracow 31-531, Poland Andres Cardenas Institut de Malalties Digestives i Metaboliques Hospital Clinic, Villaroel 170, Esc 7-4, Barcelona 08036, Spain John Y Chiang, MD, PhD, Professor Department of Biochemistry and Molecular Pathology, Northeastern Ohio Univ. College of Medicine, 4209 State Route 44, PO Box 95, Rootstown, OH 44272, United States Parimal Chowdhury, Professor Department of Physiology and Biophysics, College of Medicine University of Arkansas for Medical Sciences, 4301 W Markham Street Little Rock, Arkansas 72205, United States Dario Conte, Professor GI Unit-IRCCS Osp. Maggiore, Chair of Gastroenterology, Via F. Sforza, 35, Milano 20122, Italy Francesco Costa, Dr Dipartimento di Medicina Interna-UO di Gastroenterologia Università di Pisa-Via Roma, 67-56122-Pisa, Italy John Cuppoletti, Professor Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine 231 Albert Sabin Way, Cincinnati OH 45267-0576, United States Curt Einarsson, Professor Department of Medicine, Karolinska institute, Karolinska University Hospital Huddinge, Dept of Gastroenterology and Hepatology, K 63, Huddinge SE-141 86, Sweden

William Greenhalf, PhD Division of Surgery and Oncology, University of Liverpool, UCD Building, 5th Floor, Royal Liverpool University Hospital, Daulby Street, Liverpool, L69 3GA, United Kingdom Naohiko Harada, PhD Department of Gastroenterology, Fukuoka Higashi Medical Center, Chidori 1-1-1, Koga, Fukuoka 811-3195, Japan Leonard R Johnson, Professor Department of Physiology, University Tennessee College of Medicine, 894 Union Ave, Memphis, TN 38163, United States Ali Keshavarzian, MD Josephine M. Dyrenforth Professor of  Medicine Professor of  Pharmacology  and Molecular Biophysics & Physiolog y Director, Dig estive Diseases  and Nutrition Vice Chairman of  Medicine for Academic and Research Affairs Rush University Medical Center 1725 W. Harrison, Suite 206, Chicago, IL  60612, United States Ton Lisman, PhD Thrombosis and Haemostasis Laboratory, Department of Haematology G.03.550, University Medical Centre, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands Kevin McGrath, MD Division of Gastroenterology, Hepatology and Nutrition, University of Pittsburgh Medical Center, M2, C wing, PUH, 200 Lothrop St, Pittsburgh, PA 15213, United States Yoshiharu Motoo, MD, PhD, FACP, FACG, Professor and Chairman Department of Medical Oncology, Kanazawa Medical University,1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan Nageshwar Duvvuru Reddy, Professor Asian Institute of Gastroenterology, 6-3-652, Somajiguda, Hyderabad-500 082, India Gerhard Rogler, Dr, Professor Department of Internal Medicine, University of Zürich, Zürich 8091, Switzerland Mitsuo Shimada, Professor Department of Digestive and Pediatric Surgery, Tokushima University, Kuramoto 3-18-15, Tokushima 770-8503, Japan Rudolf E Stauber, Professor Department of Internal Medicine, Medical University Graz, Division of Gastroenterology and Hepatology, Auenbruggerplatz 15, A-8036 Graz, Austria Liping Su, MD, PhD Department of Pathology, The University of Chicago, 5841 S. Maryland, MC 1089, Chicago, IL 60637, United States Seng-Lai Tan, Principal Scientist Eli Lilly and Company, Indianapolis 46285, United States Roberto Testa, Professor Department of Internal Medicine, University of Genoa, Viale Benedetto XV 6, Genoa 16132, Italy Stefan Wirth, Professor, Dr Children’s Hospital, Heusnerstt. 40, Wuppertal 42349, Germany

Fabio Farinati, MD Surgical And Gastroenterological Sciences, University of Padua, Via Giustiniani 2, Padua 35128, Italy

Harry HX Xia, PhD, MD Novartis Pharmaceuticals Corporation, One Health Plaza, East Hanover, NJ 07936-1080, United States

Kazuma Fujimoto, Professor Department of Internal Medicine, Saga Medical School, Nabeshima, Saga, Saga 849-8501, Japan

Jian-Zhong Zhang, Professor Department of Pathology and Laboratory Medicine, Beijing 306 Hospital, 9 North Anxiang Road, PO Box 9720, Beijing 100101, China

World J Gastroenterol 2007 August 28; 13(32): 4409 World Journal of Gastroenterology ISSN 1007-9327 © 2007 WJG. All rights reserved.

Online Submissions: wjg.wjgnet.com www.wjgnet.com [email protected]

Meetings MAJOR MEETINGS COMING UP Meeting Falk Research Workshop: Morphogenesis and Cancerogenesis of the Liver 25-26 January 2007 Goettingen [email protected] Meeting Canadian Digestive Diseases Week (CDDW) 16-20 February 2007 Banff-AB [email protected] www.cag-acg.org/cddw/cddw2007. htm Meeting Falk Symposium 158: Intestinal Inflammation and Colorectal Cancer 23-24 March 2007 Sevilla [email protected] Meeting BSG Annual Meeting 26-29 March 2007 Glasgow www.bsg.org.uk/

NEXT 6 MONTHS Meeting 42nd Annual Meeting of the European Association for the Study of the Liver 11-15 April 2007 Barcelona [email protected] www.easl.ch/liver-meeting/ Meeting Falk Symposium 159: IBD 2007 - Achievements in Research and Clinical Practice 4-5 May 2007 Istanbul [email protected] Meeting European Society for Paediatric Gastroenterology, Hepatology and Nutrition Congress 2007 9-12 May 2007 Barcelona [email protected] Digestive Disease Week 19-24 May 2007 Washington Convention Center, Washington DC Meeting Gastrointestinal Endoscopy Best Practices: Today and Tomorrow, ASGE Annual Postgraduate Course at DDW 23-24 May 2007 Washington-DC [email protected] Meeting ESGAR 2007 18th Annual Meeting and Postgraduate Course 12-15 June 2007 Lisbon [email protected] Meeting Falk Symposium 160: Pathogenesis and Clinical Practice in

Gastroenterology 15-16 June 2007 Portoroz [email protected] Meeting ILTS 13th Annual International Congress 20-23 June 2007 Rio De Janeiro www.ilts.org Meeting 9th World Congress on Gastrointestinal Cancer 27-30 June 2007 Barcelona [email protected]

EVENTS AND MEETINGS IN 2007 Meeting Falk Research Workshop: Morphogenesis and Cancerogenesis of the Liver 25-26 January 2007 Goettingen [email protected] Meeting Canadian Digestive Diseases Week (CDDW) 16-20 February 2007 Banff-AB [email protected] www.cag-acg.org/cddw/cddw2007. htm Meeting Falk Symposium 158: Intestinal Inflammation and Colorectal Cancer 23-24 March 2007 Sevilla [email protected] Meeting BSG Annual Meeting 26-29 March 2007 Glasgow www.bsg.org.uk/ Meeting 42nd Annual Meeting of the European Association for the Study of the Liver 11-15 April 2007 Barcelona [email protected] www.easl.ch/liver-meeting/ Meeting Falk Symposium 159: IBD 2007 - Achievements in Research and Clinical Practice 4-5 May 2007 Istanbul [email protected] Meeting European Society for Paediatric Gastroenterology, Hepatology and Nutrition Congress 2007 9-12 May 2007 Barcelona [email protected] Meeting Gastrointestinal Endoscopy Best Practices: Today and Tomorrow, ASGE Annual Postgraduate Course at DDW 23-24 May 2007 Washington-DC [email protected] Meeting ESGAR 2007 18th Annual Meeting and Postgraduate Course 12-15 June 2007 Lisbon [email protected]

Meeting Falk Symposium 160: Pathogenesis and Clinical Practice in Gastroenterology 15-16 June 2007 Portoroz [email protected] Meeting ILTS 13th Annual International Congress 20-23 June 2007 Rio De Janeiro www.ilts.org Meeting 9th World Congress on Gastrointestinal Cancer 27-30 June 2007 Barcelona [email protected] Meeting 15th International Congress of the European Association for Endoscopic Surgery 4-7 July 2007 Athens [email protected] congresses.eaes-eur.org/ Meeting 39th Meeting of the European Pancreatic Club 4-7 July 2007 Newcastle www.e-p-c2007.com Meeting XXth International Workshop on Heliobacter and related bacteria in cronic degistive inflammation 20-22 September 2007 Istanbul www.heliobacter.org Meeting Falk Workshop: Mechanisms of Intestinal Inflammation 10 October 2007 Dresden [email protected] Meeting Falk Symposium 161: Future Perspectives in Gastroenterology 11-12 October 2007 Dresden [email protected] Meeting Falk Symposium 162: Liver Cirrhosis - From Pathophysiology to Disease Management 13-14 October 2007 Dresden [email protected] American College of Gastroenterology Annual Scientific Meeting 12-17 October 2007 Pennsylvania Convention Center Philadelphia, PA Meeting APDW 2007 - Asian Pacific Digestive Disease Week 2007 15-18 October 2007 Kobe [email protected] www.apdw2007.org 15th United European Gastroenterology Week, UEGW 27-31 October 2007 Le Palais des Congrès de Paris, Paris, France Meeting The Liver Meeting ® 2007 57th Annual Meeting of the American Association for the Study of Liver Diseases

2-6 November 2007 Boston-MA www.aasld.org Gastro 2009, World Congress of Gastroenterology and Endoscopy London, United Kingdom 2009

World J Gastroenterol 2007 August 28; 13(32): 4410-4412 World Journal of Gastroenterology ISSN 1007-9327 © 2007 WJG. All rights reserved.

Online Submissions: wjg.wjgnet.com www.wjgnet.com [email protected]

Instructions to authors GENERAL INFORMATION World Journal of Gastroenterology (WJG, World J Gastroenterol ISSN 1007-9327 CN 14-1219/R) is a weekly journal of more than 48 000 circulation, published on the 7th, 14th, 21st and 28th of every month. Original Research, Clinical Trials, Reviews, Comments, and Case Reports in esophageal cancer, gastric cancer, colon cancer, liver cancer, viral liver diseases, etc., from all over the world are welcome on the condition that they have not been published previously and have not been submitted simultaneously elsewhere.

Indexed and abstracted in

Current Contents®/Clinical Medicine, Science Citation Index Expanded (also known as SciSearch®) and Journal Citation Reports/Science Edition, Index Medicus, MEDLINE and PubMed, Chemical Abstracts, EMBASE/ Excerpta Medica, Abstracts Journals, Nature Clinical Practice Gastroenterology and Hepatology, CAB Abstracts and Global Health. ISI JCR 2003-2000 IF: 3.318, 2.532, 1.445 and 0.993.

Published by WJG

SUBMISSION OF MANUSCRIPTS Manuscripts should be typed double-spaced on A4 (297 mm × 210 mm) white paper with outer margins of 2.5 cm. Number all pages consecutively, and start each of the following sections on a new page: Title Page , Abstract, Introduction, Materials and Methods, Results, Discussion, acknowledgements, References, Tables, Figures and Figure Legends. Neither the editors nor the Publisher is responsible for the opinions expressed by contributors. Manuscripts formally accepted for publication become the permanent property of WJG, and may not be reproduced by any means, in whole or in part without the written permission of both the authors and the Publisher. We reserve the right to put onto our website and copy-edit accepted manuscripts. Authors should also follow the guidelines for the care and use of laboratory animals of their institution or national animal welfare committee. Authors should retain one copy of the text, tables, photographs and illustrations, as rejected manuscripts will not be returned to the author(s) and the editors will not be responsible for the loss or damage to photographs and illustrations in mailing process.

Online submission

Online submission is strongly advised. Manuscripts should be submitted through the Online Submission System at: http://www.wjgnet.com/index.jsp. Authors are highly recommended to consult the ONLINE INSTRUCTIONS TO AUTHORS (http://www.wjgnet.com/wjg/help/instructions.jsp) before attempting to submit online. Authors encountering problems with the Online Submission System may send an email you describing the problem to wjg@ wjgnet.com for assistance. If you submit your manuscript online, do not make a postal contribution. A repeated online submission for the same manuscript is strictly prohibited.

Postal submission

Send 3 duplicate hard copies of the full-text manuscript typed double-spaced on A4 (297 mm × 210 mm) white paper together with any original photographs or illustrations and a 3.5 inch computer diskette or CD-ROM containing an electronic copy of the manuscript including all the figures, graphs and tables in native Microsoft Word format or *.rtf format to:

line spacing and in word size 12 with ample margins. The letter font is Tahoma. For authors from China, one copy of the Chinese translation of the manuscript is also required (excluding references). Style should conform to our house format. Required information for each of the manuscript sections is as follows:

Title page

Full manuscript title, running title, all author(s) name(s), affiliations, institution(s) and/or department(s) where the work was accomplished, disclosure of any financial support for the research, and the name, full address, telephone and fax numbers and email address of the corresponding author should be included. Titles should be concise and informative (removing all unnecessary words), emphasize what is new, and avoid abbreviations. A short running title of less than 40 letters should be provided. List the author(s)’ name(s) as follows: initial and/or first name, middle name or initial(s) and full family name.

Abstract

An informative, structured abstract of no more than 350 words should accompany each manuscript. Abstracts for original contributions should be structured into the following sections: AIM: Only the purpose should be included. METHODS: The materials, techniques, instruments and equipments, and the experimental procedures should be included. RESULTS: The observatory and experimental results, including data, effects, outcome, etc. should be included. Authors should present P value where necessary, and the significant data should accompany. CONCLUSION: Accurate view and the value of the results should be included. The format of structured abstracts is at: http://www.wjgnet.com/wjg/ help/11.doc

Key words

Please list 5-10 key words that could reflect content of the study mainly from Index Medicus.

Text

For most article types, the main text should be structured into the following sections: INTRODUCTION, MATERIALS AND METHODS, RESULTS and DISCUSSION, and should include in appropriate Figures and Tables. Data should be presented in the body text or in Figures and Tables, but not in both.

Illustrations

Figures should be numbered as 1, 2, 3 and so on, and mentioned clearly in the main text. Provide a brief title for each figure on a separate page. No detailed legend should be involved under the figures. This part should be added into the text where the figures are applicable. Digital images: black and white photographs should be scanned and saved in TIFF format at a resolution of 300 dpi; color images should be saved as CMYK (print files) but not as RGB (screen-viewing files). Place each photograph in a separate file. Print images: supply images of size no smaller than 126 mm × 85 mm printed on smooth surface paper; label the image by writing the Figure number and orientation using an arrow. Photomicrographs: indicate the original magnification and stain in the legend. Digital Drawings: supply files in EPS if created by freehand and illustrator, or TIFF from photoshops. EPS files must be accompanied by a version in native file format for editing purposes. Existing line drawings should be scanned at a resolution of 1200 dpi and as close as possible to the size where they will appear when printed. Please use uniform legends for the same subjects. For example: Figure 1 Pathological changes of atrophic gastritis after treatment. A: ...; B: ...; C: ...; D: ...; E: ...; F: ...; G: ...

Tables

Editorial Office

Three-line tables should be numbered as 1, 2, 3 and so on, and mentioned clearly in the main text. Provide a brief title for each table. No detailed legend should be included under the tables. This part should be added into the text where the tables are applicable. The information should complement but not duplicate that contained in the text. Use one horizontal line under the title, a second under the column heads, and a third below the Table, above any footnotes. Vertical and italic lines should be omitted.

World Journal of Gastroenterology

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Notes in tables and illustrations

MANUSCRIPT PREPARATION All contributions should be written in English. All articles must be submitted using a word-processing software. All submissions must be typed in 1.5

Data that are not statistically significant should not be noted. aP