World Journal of Gastroenterology

Transcription

1 ISSN (print) ISSN (online) World Journal of Gastroenterology World J Gastroenterol 2012 December 14; 18(46):

2 Editorial Board The World Journal of Gastroenterology Editorial Board consists of 1352 members, representing a team of worldwide experts in gastroenterology and hepatology. They are from 64 countries, including Albania (1), Argentina (8), Australia (33), Austria (15), Belgium (14), Brazil (13), Brunei Darussalam (1), Bulgaria (2), Canada (21), Chile (3), China (82), Colombia (1), Croatia (2), Cuba (1), Czech (6), Denmark (9), Ecuador (1), Egypt (4), Estonia (2), Finland (8), France (29), Germany (87), Greece (22), Hungary (11), India (32), Indonesia (2), Iran (10), Ireland (6), Israel (13), Italy (124), Japan (140), Jordan (2), Kuwait (1), Lebanon (4), Lithuania (2), Malaysia (1), Mexico (11), Morocco (1), Moldova (1), Netherlands (32), New Zealand (2), Norway (13), Pakistan (2), Poland (11), Portugal (6), Romania (4), Russia (1), Saudi Arabia (3), Serbia (3), Singapore (11), Slovenia (1), South Africa (3), South Korea (46), Spain (43), Sri Lanka (1), Sweden (17), Switzerland (12), Thailand (1), Trinidad and Tobago (1), Turkey (30), United Arab Emirates (2), United Kingdom (95), United States (285), and Uruguay (1). HONORARY EDITORS-IN-CHIEF James L Boyer, New Haven Ke-Ji Chen, Beijing Martin H Floch, New Haven Bo-Rong Pan, Xi'an Eamonn M Quigley, Cork Rafiq A Sheikh, Sacramento Nicholas J Talley, Rochester EDITOR-IN-CHIEF Ferruccio Bonino, Pisa Myung-Hwan Kim, Seoul Kjell Öberg, Uppsala Matt Rutter, Stockton-on-Tees Andrzej S Tarnawski, Long Beach STRATEGY ASSOCIATE EDITORS-IN-CHIEF You-Yong Lu, Beijing Peter Draganov, Florida Hugh J Freeman, Vancouver Maria Concepción Gutiérrez-Ruiz, México Kazuhiro Hanazaki, Kochi Akio Inui, Kagoshima Kalpesh Jani, Baroda Javier San Martin, Punta del Este Natalia A Osna, Omaha Wei Tang, Tokyo Alan BR Thomson, Edmonton Harry Hua-Xiang Xia, Livingston John M Luk, Hong Kong Hiroshi Shimada, Yokohama GUEST EDITORIAL BOARD MEMBERS Jiunn-Jong Wu, Tainan Cheng-Shyong Wu, Chia-Yi Ta-Sen Yeh, Taoyuan Tsung-Hui Hu, Kaohsiung Chuah Seng-Kee, Kaohsiung I-Rue Lai, Taipei Jin-Town Wang, Taipei Ming-Shiang Wu, Taipei Teng-Yu Lee, Taichung Yang-Yuan Chen, Changhua Po-Shiuan Hsieh, Taipei Chao-Hung Hung, Kaohsiung Hon-Yi Shi, Kaohsiung Hui-kang Liu, Taipei Jen-Hwey Chiu, Taipei Chih-Chi Wang, Kaohsiung Wan-Long Chuang, Kaohsiung Wen-Hsin Huang, Taichung Hsu-Heng Yen, Changhua Ching Chung Lin, Taipei Chien-Jen Chen, Taipei Jaw-Ching Wu, Taipei Ming-Chih Hou, Taipei Kevin Cheng-Wen Hsiao, Taipei Chiun Hsu, Taipei Yu-Jen Chen, Taipei Chen Hsiu-Hsi Chen, Taipei Liang-Shun Wang, Taipei hun-fa Yang, Taichung Min-Hsiung Pan, Kaohsiung Chun- Hung Lin, Taipei Ming-Whei Yu, Taipei Chuen Hsueh, Taoyuan Hsiu-Po Wang, Taipei Lein-Ray Mo, Tainan Ming-Lung Yu, Kaohsiung MEMBERS OF THE EDITORIAL BOARD Albania Bashkim Resuli, Tirana Argentina Julio H Carri, Córdoba Bernabe Matias Quesada, Buenos Aires Bernardo Frider, Buenos Aires Maria Ines Vaccaro, Buenos Aires Eduardo de Santibañes, Buenos Aires Adriana M Torres, Rosario Carlos J Pirola, Buenos Aires Silvia Sookoian, Buenos Aires Australia Finlay A Macrae, Victoria David Ian Watson, Bedford Park Jacob George, Sydney Leon Anton Adams, Nedlands Minoti V Apte, Liverpool Andrew V Biankin, Sydney Filip Braet, Sydney Guy D Eslick, Sydney Michael A Fink, Melbourne Mark D Gorrell, Sydney Michael Horowitz, Adelaide John E Kellow, Sydney Daniel Markovich, Brisbane I January 7, 2012

3 Phillip S Oates, Perth Ross C Smith, Sydney Kevin J Spring, Brisbane Philip G Dinning, Koagarah Christopher Christophi, Melbourne Cuong D Tran, North Adelaide Shan Rajendra, Tasmania Rajvinder Singh, Adelaide William Kemp, Melbourne Phil Sutton, Melbourne Richard Anderson, Victoria Vance Matthews, Melbourne Alexander G Heriot, Melbourne Debbie Trinder, Fremantle Ian C Lawrance, Perth Adrian G Cummins, Adelaide John K Olynyk, Fremantle Alex Boussioutas, Melbourne Emilia Prakoso, Sydney Robert JL Fraser, Daw Park Austria Wolfgang Mikulits, Vienna Alfred Gangl, Vienna Dietmar Öfner, Salzburg Georg Roth, Vienna Herwig R Cerwenka, Graz Ashraf Dahaba, Graz Markus Raderer, Vienna Alexander M Hirschl, Wien Thomas Wild, Kapellerfeld Peter Ferenci, Vienna Valentin Fuhrmann, Vienna Kurt Lenz, Linz Markus Peck-Radosavljevic, Vienna Michael Trauner, Vienna Stefan Riss, Vienna Belgium Rudi Beyaert, Gent Inge I Depoortere, Leuven Olivier Detry, Liège Benedicte Y De Winter, Antwerp Etienne M Sokal, Brussels Marc Peeters, De Pintelaan Eddie Wisse, Keerbergen Jean-Yves L Reginster, Liège Mark De Ridder, Brussel Freddy Penninckx, Leuven Kristin Verbeke, Leuven Lukas Van Oudenhove, Leuven Leo van Grunsven, Brussels Philip Meuleman, Ghent Brazil Heitor Rosa, Goiania Roberto J Carvalho-Filho, Sao Paulo Damiao Carlos Moraes Santos, Rio de Janeiro Marcelo Lima Ribeiro, Braganca Paulista Eduardo Garcia Vilela, Belo Horizonte Jaime Natan Eisig, São Paulo Andre Castro Lyra, Salvador José Liberato Ferreira Caboclo, Brazil Yukie Sato-Kuwabara, São Paulo Raquel Rocha, Salvador Paolo R Salvalaggio, Sao Paulo Ana Cristina Simões e Silva, Belo Horizonte Joao Batista Teixeira Rocha, Santa Maria Brunei Darussalam Vui Heng Chong, Bandar Seri Begawan Bulgaria Zahariy Krastev, Sofia Mihaela Petrova, Sofia Canada Eldon Shaffer, Calgary Nathalie Perreault, Sherbrooke Philip H Gordon, Montreal Ram Prakash Galwa, Ottawa Baljinder Singh Salh, Vancouver Claudia Zwingmann, Montreal Alain Bitton, Montreal Pingchang Yang, Hamilton Michael F Byrne,Vancouver Andrew L Mason, Alberta John K Marshall, Hamilton Ontario Kostas Pantopoulos, Montreal Waliul Khan, Ontario Eric M Yoshida, Vancouver Geoffrey C Nguyen, Toronto Devendra K Amre, Montreal Tedros Bezabeh, Winnipeg Wangxue Chen, Ottawa Qiang Liu, Saskatoon Chile De Aretxabala Xabier, Santiago Marcelo A Beltran, La Serena Silvana Zanlungo, Santiago China Chi-Hin Cho, Hong Kong Chun-Qing Zhang, Jinan Ren Xiang Tan, Nanjing Fei Li, Beijing Hui-Jie Bian, Xi'an Xiao-Peng Zhang, Beijing Xing-Hua Lu, Beijing Fu-Sheng Wang, Beijing An-Gang Yang, Xi an Xiao-Ping Chen, Wuhan Zong-Jie Cui, Beijing Ming-Liang He, Hong Kong Yuk-Tong Lee, Hong Kong Qin Su, Beijing Jian-Zhong Zhang, Beijing Paul Kwong-Hang Tam, Hong Kong Wen-Rong Xu, Zhenjiang Chun-Yi Hao, Beijing San-Jun Cai, Shanghai Simon Law, Hong Kong Yuk Him Tam, Hong Kong De-Liang Fu, Shanghai Eric WC Tse, Hong Kong Justin CY Wu, Hong Kong Nathalie Wong, Hong Kong Jing Yuan Fang, Shanghai Yi-Min Mao, Shanghai Wei-Cheng You, Beijing Xiang-Dong Wang, Shanghai Xuan Zhang, Beijing Zhao-Shen Li, Shanghai Guang-Wen Cao, Shanghai En-min Li, Shantou Yu-Yuan Li, Guangzhou Fook Hong Ng, Hong Kong Hsiang-Fu Kung, Hong Kong Wai Lun Law, Hong Kong Eric CH Lai, Hong Kong Jun Yu, Hong Kong Ze-Guang Han, Shanghai Bian zhao-xiang, Hong Kong Wei-Dong Tong, Chongqing Colombia Germán Campuzano-Maya, Medellín Croatia Tamara Cacev, Zagreb Marko Duvnjak, Zagreb Cuba Damian C Rodriguez, Havana Czech Milan Jirsa, Praha Pavel Trunečka, Prague Jan Bures, Hradec Kralove Marcela Kopacova, Hradec Kralove Ondrej Slaby, Brno Radan Bruha, Prague Denmark Asbjørn M Drewes, Aalborg Leif Percival Andersen, Copenhagen Jan Mollenhauer, Odense C Morten Frisch, Copenhagen S Jorgen Rask-Madsen, Skodsborg Morten Hylander Møller, Holte Søren Rafaelsen, Vejle Vibeke Andersen, Aabenraa Ole Haagen Nielsen, Herlev Ecuador Fernando E Sempértegui, Quito Egypt Zeinab Nabil Ahmed Said, Cairo Hussein M Atta, El-Minia Asmaa Gaber Abdou, Shebein Elkom II January 7, 2012

4 Maha Maher Shehata, Mansoura Estonia Riina Salupere, Tartu Tamara Vorobjova, Tartu Finland Saila Kauhanen, Turku Pauli Antero Puolakkainen, Turku Minna Nyström, Helsinki Juhani Sand, Tampere Jukka-Pekka Mecklin, Jyvaskyla Lea Veijola, Helsinki Kaija-Leena Kolho, Helsinki Thomas Kietzmann, Oulu France Boris Guiu, Dijon Baumert F Thomas, Strasbourg Alain L Servin, Châtenay-Malabry Patrick Marcellin, Paris Jean-Jacques Tuech, Rouen Francoise L Fabiani, Angers Jean-Luc Faucheron, Grenoble Philippe Lehours, Bordeaux Stephane Supiot, Nantes Lionel Bueno, Toulouse Flavio Maina, Marseille Paul Hofman, Nice Abdel-Majid Khatib, Paris Annie Schmid-Alliana, Nice cedex 3 Frank Zerbib, Bordeaux Cedex Rene Gerolami Santandera, Marseille Sabine Colnot, Paris Catherine Daniel, Lille Cedex Thabut Dominique, Paris Laurent Huwart, Paris Alain Braillon, Amiens Bruno Bonaz, Grenoble Evelyne Schvoerer, Strasbourg M Coeffier, Rouen Mathias Chamaillard, Lille Hang Nguyen, Clermont-Ferrand Veronique Vitton, Marseille Alexis Desmoulière, Limoges Juan Iovanna, Marseille Germany Hans L Tillmann, Leipzig Stefan Kubicka, Hannover Elke Cario, Essen Hans Scherubl, Berlin Harald F Teutsch, Ulm Peter Konturek, Erlangen Thilo Hackert, Heidelberg Jurgen M Stein, Frankfurt Andrej Khandoga, Munich Karsten Schulmann, Bochum Jutta Elisabeth Lüttges, Riegelsberg Wolfgang Hagmann, Heidelberg Hubert Blum, Freiburg Thomas Bock, Berlin Christa Buechler, Regensburg Christoph F Dietrich, Bad Mergentheim Ulrich R Fölsch, Kiel Nikolaus Gassler, Aachen Markus Gerhard, Munich Dieter Glebe, Giessen Klaus R Herrlinger, Stuttgart Eberhard Hildt, Berlin Joerg C Hoffmann, Ludwigshafen Joachim Labenz, Siegen Peter Malfertheiner, Magdeburg Sabine Mihm, Göttingen Markus Reiser, Bochum Steffen Rickes, Magdeburg Andreas G Schreyer, Regensburg Henning Schulze-Bergkamen, Heidelberg Ulrike S Stein, Berlin Wolfgang R Stremmel, Heidelberg Fritz von Weizsäcker, Berlin Stefan Wirth, Wuppertal Dean Bogoevski, Hamburg Bruno Christ, Halle/Saale Peter N Meier, Hannover Stephan Johannes Ott, Kiel Arndt Vogel, Hannover Dirk Haller, Freising Jens Standop, Bonn Jonas Mudter, Erlangen Jürgen Büning, Lübeck Matthias Ocker, Erlangen Joerg Trojan, Frankfurt Christian Trautwein, Aachen Jorg Kleeff, Munich Christian Rust, Munich Claus Hellerbrand, Regensburg Elke Roeb, Giessen Erwin Biecker, Siegburg Ingmar Königsrainer, Tübingen Jürgen Borlak, Hannover Axel M Gressner, Aachen Oliver Mann, Hamburg Marty Zdichavsky, Tübingen Christoph Reichel, Bad Brückenau Nils Habbe, Marburg Thomas Wex, Magdeburg Frank Ulrich Weiss, Greifswald Manfred V Singer, Mannheim Martin K Schilling, Homburg Philip D Hard, Giessen Michael Linnebacher, Rostock Ralph Graeser, Freiburg Rene Schmidt, Freiburg Robert Obermaier, Freiburg Sebastian Mueller, Heidelberg Andrea Hille, Goettingen Klaus Mönkemüller, Bottrop Elfriede Bollschweiler, Köln Siegfried Wagner, Deggendorf Dieter Schilling, Mannheim Joerg F Schlaak, Essen Michael Keese, Frankfurt Robert Grützmann, Dresden Ali Canbay, Essen Dirk Domagk, Muenster Jens Hoeppner, Freiburg Frank Tacke, Aachen Patrick Michl, Marburg Alfred A Königsrainer, Tübingen Kilian Weigand, Heidelberg Mohamed Hassan, Duesseldorf Gustav Paumgartner, Munich Philipe N Khalil, Munich Martin Storr, Munich Greece Andreas Larentzakis, Athens Tsianos Epameinondas, Ioannina Elias A Kouroumalis, Heraklion Helen Christopoulou-Aletra, Thessaloniki George Papatheodoridis, Athens Ioannis Kanellos, Thessaloniki Michael Koutsilieris, Athens T Choli-Papadopoulou, Thessaloniki Emanuel K Manesis, Athens Evangelos Tsiambas, Ag Paraskevi Attiki Konstantinos Mimidis, Alexandroupolis Spilios Manolakopoulos, Athens Spiros Sgouros, Athens Ioannis E Koutroubakis, Heraklion Stefanos Karagiannis, Athens Spiros Ladas, Athens Elena Vezali, Athens Dina G Tiniakos, Athens Ekaterini Chatzaki, Alexandroupolis Dimitrios Roukos, Ioannina George Sgourakis, Athens Maroulio Talieri, Athens Hungary Peter L Lakatos, Budapest Yvette Mándi, Szeged Ferenc Sipos, Budapest György M Buzás, Budapest László Czakó, Szeged Peter Hegyi, Szeged Zoltan Rakonczay, Szeged Gyula Farkas, Szeged Zsuzsa Szondy, Debrecen Gabor Veres, Budapest Zsuzsa Schaff, Budapest India Philip Abraham, Mumbai Sri P Misra, Allahabad Ramesh Roop Rai, Jaipur Nageshwar D Reddy, Hyderabad Rakesh Kumar Tandon, New Delhi Jai Dev Wig, Chandigarh Uday C Ghoshal, Lucknow Pramod Kumar Garg, New Delhi Barjesh Chander Sharma, New Delhi Gopal Nath, Varanasi Bhupendra Kumar Jain, Delhi Devinder Kumar Dhawan, Chandigarh Ashok Kumar, Lucknow Benjamin Perakath, Tamil Nadu Debidas Ghosh, Midnpore Pankaj Garg, Panchkula Samiran Nundy, New Delhi Virendra Singh, Chandigarh Bikash Medhi, Chandigarh Radha K Dhiman, Chandigarh Vandana Panda, Mumbai Vineet Ahuja, New Delhi SV Rana, Chandigarh III January 7, 2012

5 Deepak N Amarapurkar, Mumbai Abhijit Chowdhury, Kolkata Jasbir Singh, Kurukshetra B Mittal, Lucknow Sundeep Singh Saluja, New Delhi Pradyumna Kumar Mishra, Mumbai Runu Chakravarty, Kolkata Nagarajan Perumal, New Delhi Indonesia David handojo Muljono, Jakarta Andi Utama, Tangerang Iran Seyed-Moayed Alavian, Tehran Reza Malekzadeh, Tehran Peyman Adibi, Isfahan Alireza Mani, Tehran Seyed Mohsen Dehghani, Shiraz Mohammad Abdollahi, Tehran Majid Assadi, Bushehr Arezoo Aghakhani, Tehran Marjan Mohammadi, Tehran Fariborz Mansour-Ghanaei, Rasht Ireland Ross McManus, Dublin Billy Bourke, Dublin Catherine Greene, Dublin Ted Dinan, Cork Marion Rowland, Dublin Israel Abraham R Eliakim, Haifa Simon Bar-Meir, Tel Hashomer Ami D Sperber, Beer-Sheva Boris Kirshtein, Beer Sheva Mark Pines, Bet Dagan Menachem Moshkowitz, Tel-Aviv Ron Shaoul, Haifa Shmuel Odes, Beer Sheva Sigal Fishman, Tel Aviv Alexander Becker, Afula Assy Nimer, Safed Eli Magen, Ashdod Amir Shlomai, Tel-Aviv Italy Mauro Bortolotti, Bologna Gianlorenzo Dionigi, Varese Fiorucci Stefano, Perugia Roberto Berni Canani, Naples Ballarin Roberto, Modena Bruno Annibale, Roma Vincenzo Stanghellini, Bologna Giovanni B Gaeta, Napoli Claudio Bassi, Verona Mauro Bernardi, Bologna Giuseppe Chiarioni, Valeggio Michele Cicala, Rome Dario Conte, Milano Francesco Costa, Pisa Giovanni D De Palma, Naples Giammarco Fava, Ancona Francesco Feo, Sassari Edoardo G Giannini, Genoa Fabio Grizzi, Milan Salvatore Gruttadauria, Palermo Pietro Invernizzi, Milan Ezio Laconi, Cagliari Giuseppe Montalto, Palermo Giovanni Musso, Torino Gerardo Nardone, Napoli Valerio Nobili, Rome Raffaele Pezzilli, Bologna Alberto Piperno, Monza Anna C Piscaglia, Roma Piero Portincasa, Bari Giovanni Tarantino, Naples Cesare Tosetti, Porretta Terme Alessandra Ferlini, Ferrara Alessandro Ferrero, Torino Donato F Altomare, Bari Giovanni Milito, Rome Giuseppe Sica, Rome Guglielmo Borgia, Naples Giovanni Latella, L'Aquila Salvatore Auricchio, Naples Alberto Biondi, Rome Alberto Tommasini, Trieste Antonio Basoli, Roma Giuliana Decorti, Trieste Marco Silano, Roma Michele Reni, Milan Pierpaolo Sileri, Rome Achille Iolascon, Naples Alessandro Granito, Bologna Angelo A Izzo, Naples Giuseppe Currò, Messina Pier Mannuccio Mannucci, Milano Marco Vivarelli, Bologna Massimo Levrero, Rome Massimo Rugge, Padova Paolo Angeli, Padova Silvio Danese, Milano Antonello Trecca, Rome Antonio Gasbarrini, Rome Cesare Ruffolo, Treviso Massimo Falconi, Verona Fausto Catena, Bologna Francesco Manguso, Napoli Giancarlo Mansueto, Verona Luca Morelli, Trento Marco Scarpa, Padova Mario M D'Elios, Florence Francesco Luzza, Catanzaro Franco Roviello, Siena Guido Torzilli, Rozzano Milano Luca Frulloni, Verona Lucia Malaguarnera, Catania Lucia Ricci Vitiani, Rome Mara Massimi, L'Aquila Mario Pescatori, Rome Mario Rizzetto, Torino Mirko D Onofrio, Verona Nadia Peparini, Rome Paola De Nardi, Milan Paolo Aurello, Rome Piero Amodio, Padova Riccardo Nascimbeni, Brescia Vincenzo Villanacci, Brescia Vittorio Ricci, Pavia Silvia Fargion, Milan Luigi Bonavina, Milano Oliviero Riggio, Rome Fabio Pace, Milano Gabrio Bassotti, Perugia Giulio Marchesini, Bologna Roberto de Franchis, Milano Giovanni Monteleone, Rome C armelo Scarpignato, Parma Luca VC Valenti, Milan Urgesi Riccardo, Rome Marcello Persico, Naples Antonio Moschetta, Bari Luigi Muratori, Bologna Angelo Zullo, Roma Vito Annese, Florence Simone Lanini, Rome Alessandro Grasso, Savona Giovanni Targher, Verona Domenico Girelli, Verona Alessandro Cucchetti, Bologna Fabio Marra, Florence Michele Milella, Rome Francesco Franceschi, Rome Giuseppina De Petro, Brescia Salvatore Leonardi, Catania Cristiano Simone, Santa Maria Imbaro Bernardino Rampone, Salerno Francesco Crea, Pisa Walter Fries, Messina Antonio Craxì, Palermo Gerardo Rosati, Potenza Mario Guslandi, Milano Gianluigi Giannelli, Bari Paola Loria, Modena Paolo Sorrentino, Avellino Armando Santoro, Rozzano Gabriele Grassi, Trieste Antonio Orlacchio, Rome Japan Tsuneo Kitamura, Chiba Katsutoshi Yoshizato, Higashihiroshima Masahiro Arai, Tokyo Shinji Tanaka, Hiroshima Keiji Hirata, Kitakyushu Yoshio Shirai, Niigata Susumu Ohmada, Maebashi Kenichi Ikejima, Tokyo Masatoshi Kudo, Osaka Yoshiaki Murakami, Hiroshima Masahiro Tajika, Nagoya Kentaro Yoshika, Toyoake Kyoichi Adachi, Izumo Yasushi Adachi, Sapporo Takafumi Ando, Nagoya Akira Andoh, Otsu Hitoshi Asakura, Tokyo Mitsuhiro Fujishiro, Tokyo Toru Hiyama, Higashihiroshima Yutaka Inagaki, Kanagawa Hiromi Ishibashi, Nagasaki Shunji Ishihara, Izumo Toru Ishikawa, Niigata Yoshiaki Iwasaki, Okayama Terumi Kamisawa, Tokyo IV January 7, 2012

6 Norihiro Kokudo, Tokyo Shin Maeda, Tokyo Yasushi Matsuzaki, Ibaraki Kenji Miki, Tokyo Hiroto Miwa, Hyogo Yoshiharu Motoo, Kanazawa Kunihiko Murase, Tusima Atsushi Nakajima, Yokohama Yuji Naito, Hisato Nakajima, Tokyo Hiroki Nakamura, Yamaguchi Shotaro Nakamura, Fukuoka Mikio Nishioka, Niihama Hirohide Ohnishi, Akita Kazuichi Okazaki, Osaka Morikazu Onji, Ehime Satoshi Osawa, Hamamatsu Hidetsugu Saito, Tokyo Yutaka Saito, Tokyo Yasushi Sano, Kobe Tomohiko Shimatani, Kure Yukihiro Shimizu, Toyama Shinji Shimoda, Fukuoka Masayuki Sho, Nara Hidekazu Suzuki, Tokyo Shinji Togo, Yokohama Satoshi Yamagiwa, Niigata Takayuki Yamamoto, Yokkaichi Hiroshi Yoshida, Tokyo Norimasa Yoshida, Kyoto Akihito Nagahara, Tokyo Hiroaki Takeuchi, Kochi Keiji Ogura, Tokyo Kotaro Miyake, Tokushima Mitsunori Yamakawa, Yamagata Naoaki Sakata, Sendai Naoya Kato, Tokyo Satoshi Mamori, Hyogo Shogo Kikuchi, Aichi Shoichiro Sumi, Kyoto Susumu Ikehara, Osaka Taketo Yamaguchi, Chiba Tokihiko Sawada, Tochigi Tomoharu Yoshizumi, Fukuoka Toshiyuki Ishiwata, Tokyo Yasuhiro Fujino, Akashi Yasuhiro Koga, Isehara city Yoshihisa Takahashi, Tokyo Yoshitaka Takuma, Okayama Yutaka Yata, Maebashi-city Itaru Endo, Yokohama Kazuo Chijiiwa, Miyazaki Kouhei Fukushima, Sendai Masahiro Iizuka, Akita Mitsuyoshi Urashima, Tokyo Munechika Enjoji, Fukuoka Takashi Kojima, Sapporo Takumi Kawaguchi, Kurume Yoshiyuki Ueno, Sendai Yuichiro Eguchi, Saga Akihiro Tamori, Osaka Atsushi Masamune, Sendai Atsushi Tanaka, Tokyo Hitoshi Tsuda, Tokyo Takashi Kobayashi, Tokyo Akimasa Nakao, Nagogya Hiroyuki Uehara, Osaka Masahito Uemura, Kashihara Satoshi Tanno, Sapporo Toshinari Takamura, Kanazawa Yohei Kida, Kainan Masanori Hatakeyama, Tokyo Satoru Kakizaki, Gunma Shuhei Nishiguchi, Hyogo Yuichi Yoshida, Osaka Manabu Morimoto, Japan Mototsugu Kato, Sapporo Naoki Ishii, Tokyo Noriko Nakajima, Tokyo Nobuhiro Ohkohchi, Tsukuba Takanori Kanai, Tokyo Kenichi Goda, Tokyo Mitsugi Shimoda, Mibu Zenichi Morise, Nagoya Hitoshi Yoshiji, Kashihara Takahiro Nakazawa, Nagoya Utaroh Motosugi, Yamanashi Nobuyuki Matsuhashi, Tokyo Yasuhiro Kodera, Nagoya Takayoshi Ito, Tokyo Yasuhito Tanaka, Nagoya Haruhiko Sugimura, Hamamatsu Hiroki Yamaue, Wakayama Masao Ichinose, Wakayama Takaaki Arigami, Kagoshima Nobuhiro Zaima, Nara Naoki Tanaka, Matsumoto Satoru Motoyama, Akita Tomoyuki Shibata, Toyoake Tatsuya Ide, Kurume Tsutomu Fujii, Nagoya Osamu Kanauchi, Toky Atsushi Irisawa, Aizuwakamatsu Hikaru Nagahara, Tokyo Keiji Hanada, Onomichi Keiichi Mitsuyama, Fukuoka Shin Maeda, Yokohama Takuya Watanabe, Niigata Toshihiro Mitaka, Sapporo Yoshiki Murakami, Kyoto Tadashi Shimoyama, Hirosaki Jordan Ismail Matalka, Irbid Khaled Jadallah, Irbid Islam Khan, Safat Kuwait Lebanon Bassam N Abboud, Beirut Rami Moucari, Beirut Ala I Sharara, Beirut Rita Slim, Beirut Lithuania Giedrius Barauskas, Kaunas Limas Kupcinskas, Kaunas Malaysia Andrew Seng Boon Chua, Ipoh Mexico Saúl Villa-Trevio, México Omar Vergara-Fernandez, Mexico Diego Garcia-Compean, Monterrey Arturo Panduro, Jalisco Miguel Angel Mercado, Distrito Federal Richard A Awad, Mexico Aldo Torre Delgadillo, México Paulino Martínez Hernández Magro, Celaya Carlos A Aguilar-Salinas, Mexico Jesus K Yamamoto-Furusho, Mexico Morocco Samir Ahboucha, Khouribga Moldova Igor Mishin, Kishinev Netherlands Ulrich Beuers, Amsterdam Albert Frederik Pull ter Gunne, Tilburg Jantine van Baal, Heidelberglaan Wendy Wilhelmina Johanna de Leng, Utrecht Gerrit A Meijer, Amsterdam Lee Bouwman, Leiden J Bart A Crusius, Amsterdam Frank Hoentjen, Haarlem Servaas Morré, Amsterdam Chris JJ Mulder, Amsterdam Paul E Sijens, Groningen Karel van Erpecum, Utrecht BW Marcel Spanier, Arnhem Misha Luyer, Sittard Pieter JF de Jonge, Rotterdam Robert Christiaan Verdonk, Groningen John Plukker, Groningen Maarten Tushuizen, Amsterdam Wouter de Herder, Rotterdam Erwin G Zoetendal, Wageningen Robert J de Knegt, Rotterdam Albert J Bredenoord, Nieuwegein Annemarie de Vries, Rotterdam Astrid van der Velde, Ede Lodewijk AA Brosens, Utrecht James CH Hardwick, Leiden Loes van Keimpema, Nijmegen WJ de Jonge, Amsterdam Zuzana Zelinkova, Rotterdam LN van Steenbergen, Eindhoven Frank G Schaap, Amsterdam Jeroen Maljaars, Leiden New Zealand Andrew S Day, Christchurch Max S Petrov, Auckland Norway Espen Melum, Oslo V January 7, 2012

7 Trine Olsen, Tromsø Eyvind J Paulssen, Tromsø Rasmus Goll, Tromsø Asle W Medhus, Oslo Jon Arne Søreide, Stavanger Kjetil Soreide, Stavanger Reidar Fossmark, Trondheim Trond Peder Flaten, Trondheim Olav Dalgard, Oslo Ole Høie, Arendal Magdy El-Salhy, Bergen Jørgen Valeur, Oslo Pakistan Shahab Abid, Karachi Syed MW Jafri, Karachi Poland Beata Jolanta Jablońska, Katowice Halina Cichoż-Lach, Lublin Tomasz Brzozowski, Cracow Hanna Gregorek, Warsaw Marek Hartleb, Katowice Stanislaw J Konturek, Krakow Andrzej Dabrowski, Bialystok Jan Kulig, Kraków Julian Swierczynski, Gdansk Marek Bebenek, Wroclaw Dariusz M Lebensztejn, Bialystok Portugal Ricardo Marcos, Porto Guida Portela-Gomes, Estoril Ana Isabel Lopes, Lisboa Codex Raquel Almeida, Porto Rui Tato Marinho, Lisbon Ceu Figueiredo, Porto Romania Dan L Dumitrascu, Cluj Adrian Saftoiu, Craiova Andrada Seicean, Cluj-Napoca Anca Trifan, Iasi Russia Vasiliy I Reshetnyak, Moscow Saudi Arabia Ibrahim A Al Mofleh, Riyadh Abdul-Wahed Meshikhes, Qatif Faisal Sanai, Riyadh Serbia Tamara M Alempijevic, Belgrade Dusan M Jovanovic, Sremska Kamenica Zoran Krivokapic, Belgrade Singapore Brian Kim Poh Goh, Singapore Khek-Yu Ho, Singapore Fock Kwong Ming, Singapore Francis Seow-Choen, Singapore Kok Sun Ho, Singapore Kong Weng Eu, Singapore Madhav Bhatia, Singapore London Lucien Ooi, Singapore Wei Ning Chen, Singapore Richie Soong, Singapore Kok Ann Gwee, Singapore Slovenia Matjaz Homan, Ljubljana South Africa Rosemary Joyce Burnett, Pretoria Michael Kew, Cape Town Roland Ndip, Alice South Korea Byung Chul Yoo, Seoul Jae J Kim, Seoul Jin-Hong Kim, Suwon Marie Yeo, Suwon Jeong Min Lee, Seoul Eun-Yi Moon, Seoul Joong-Won Park, Goyang Hoon Jai Chun, Seoul Myung-Gyu Choi, Seoul Sang Kil Lee, Seoul Sang Yeoup Lee, Gyeongsangnam-do Won Ho Kim, Seoul Dae-Yeul Yu, Daejeon Donghee Kim, Seoul Sang Geon Kim, Seoul Sun Pyo Hong, Geonggi-do Sung-Gil Chi, Seoul Yeun-Jun Chung, Seoul Ki-Baik Hahm, Incheon Ji Kon Ryu, Seoul Kyu Taek Lee, Seoul Yong Chan Lee, Seoul Seong Gyu Hwang, Seongnam Seung Woon Paik, Seoul Sung Kim, Seoul Hong Joo Kim, Seoul Hyoung-Chul Oh, Seoul Nayoung Kim, Seongnam-si Sang Hoon Ahn, Seoul Seon Hahn Kim, Seoul Si Young Song, Seoul Young-Hwa Chung, Seoul Hyo-Cheol Kim, Seoul Kwang Jae Lee, Swon Sang Min Park, Seoul Young Chul Kim, Seoul Do Hyun Park, Seoul Dae Won Jun, Seoul Dong Wan Seo, Seoul Soon-Sun Hong, Incheon Hoguen Kim, Seoul Ho-Young Song, Seoul Joo-Ho Lee, Seoul Jung Eun Lee, Seoul Jong H Moon, Bucheon Spain Eva Vaquero, Barcelona Andres Cardenas, Barcelona Laureano Fernández-Cruz, Barcelona Antoni Farré, Spain Maria-Angeles Aller, Madrid Raul J Andrade, Málaga Fernando Azpiroz, Barcelona Josep M Bordas, Barcelona Antoni Castells, Barcelona Vicente Felipo, Valencia Isabel Fabregat, Barcelona Angel Lanas, Zaragoza Juan-Ramón Larrubia, Guadalajara María IT López, Jaén Jesús M Prieto, Pamplona Mireia Miquel, Sabadell Ramon Bataller, Barcelona Fernando J Corrales, Pamplona Julio Mayol, Madrid Matias A Avila, Pamplona Juan Macías, Seville Juan Carlos Laguna Egea, Barcelona Juli Busquets, Barcelona Belén Beltrán, Valencia José Manuel Martin-Villa, Madrid Lisardo Boscá, Madrid Luis Grande, Barcelona Pedro Lorenzo Majano Rodriguez, Madrid Adolfo Benages, Valencia Domínguez-Muñoz JE, Santiago de Compostela Gloria González Aseguinolaza, Navarra Javier Martin, Granada Luis Bujanda, San Sebastián Matilde Bustos, Pamplona Luis Aparisi, Valencia José Julián calvo Andrés, Salamanca Benito Velayos, Valladolid Javier Gonzalez-Gallego, León Ruben Ciria, Córdoba Francisco Rodriguez-Frias, Barcelona Manuel Romero-Gómez, Sevilla Albert Parés, Barcelona Joan Roselló-Catafau, Barcelona Sri Lanka Arjuna De Silva, Kelaniya Sweden Stefan G Pierzynowski, Lund Hanns-Ulrich Marschall, Stockholm Lars A Pahlman, Uppsala Helena Nordenstedt, Stockholm Bobby Tingstedt, Lund Evangelos Kalaitzakis, Gothenburg Lars Erik Agréus, Huddinge Annika Lindblom, Stockholm VI January 7, 2012

8 Roland Andersson, Lund Zongli Zheng, Stockholm Mauro D'Amato, Huddinge Greger Lindberg, Stockholm Pär Erik Myrelid, Linköping Sara Lindén, Göteborg Sara Regnér, Malmö Åke Nilsson, Lund Switzerland Jean L Frossard, Geneva Andreas Geier, Zürich Bruno Stieger, Zürich Pascal Gervaz, Geneva Paul M Schneider, Zurich Felix Stickel, Berne Fabrizio Montecucco, Geneva Inti Zlobec, Basel Michelangelo Foti, Geneva Pascal Bucher, Geneva Andrea De Gottardi, Berne Christian Toso, Geneva Thailand Weekitt Kittisupamongkol, Bangkok Trinidad and Tobago Shivananda Nayak, Mount Hope Turkey Tarkan Karakan, Ankara Yusuf Bayraktar, Ankara Ahmet Tekin, Mersin Aydin Karabacakoglu, Konya Osman C Ozdogan, Istanbul Özlem Yilmaz, Izmir Bülent Salman, Ankara Can GONEN, Kutahya Cuneyt Kayaalp, Malatya Ekmel Tezel, Ankara Eren Ersoy, Ankara Hayrullah Derici, Balıkesir Mehmet Refik Mas, Etlik-Ankara Sinan Akay, Tekirdag A Mithat Bozdayi, Ankara Metin Basaranoglu, Istanbul Mesut Tez, Ankara Orhan Sezgin, Mersin Mukaddes Esrefoglu, Malatya Ilker Tasci, Ankara Kemal Kismet, Ankara Selin Kapan, Istanbul Seyfettin Köklü, Ankara Murat Sayan, Kocaeli Sabahattin Kaymakoglu, Istanbul Yucel Ustundag, Zonguldak Can Gonen, Istanbul Yusuf Yilmaz, Istanbul Müge Tecder-Ünal, Ankara İlhami Yüksel, Ankara United Arab Emirates Fikri M Abu-Zidan, Al-Ain Sherif M Karam, Al-Ain United Kingdom Anastasios Koulaouzidis, Edinburgh Sylvia LF Pender, Southampton Hong-Xiang Liu, Cambridge William Dickey, Londonderry Simon D Taylor-Robinson, London James Neuberger, Birmingham Frank I Tovey, London Kevin Robertson, Glasgow Chew Thean Soon, Manchester Geoffrey Burnstock, London Vamsi R Velchuru, United Kingdom Simon Afford, Birmingham Navneet K Ahluwalia, Stockport Lesley A Anderson, Belfast Anthony TR Axon, Leeds Jim D Bell, London Alastair D Burt, Newcastle Tatjana Crnogorac-Jurcevic, London Daniel R Gaya, Edinburgh William Greenhalf, Liverpool Indra N Guha, Southampton Stefan G Hübscher, Birmingham Robin Hughes, London Pali Hungin, Stockton Janusz AZ Jankowski, Oxford Peter Karayiannis, London Patricia F Lalor, Birmingham Giorgina Mieli-Vergani, London D Mark Pritchard, Liverpool Marco Senzolo, Padova Roger Williams, London M H Ahmed, Southampton Christos Paraskeva, Bristol Emad M El-Omar, Aberdeen A M El-Tawil, Birmingham Anne McCune, Bristol Charles B Ferguson, Belfast Chin Wee Ang, Liverpool Clement W Imrie, Glasgow Dileep N Lobo, Nottingham Graham MacKay, Glasgow Guy Fairbairn Nash, Poole Ian Lindsey, Oxford Jason CB Goh, Birmingham Jeremy FL Cobbold, London Julian RF Walters, London Jamie Murphy, London John Beynon, Swansea John B Schofield, Kent Anil George, London Aravind Suppiah, East Yorkshire Basil Ammori, Salford Catherine Walter, Cheltenham Chris Briggs, Sheffield Jeff Butterworth, Shrewsbury Nawfal Hussein, Nottingham Patrick O'Dwyer, Glasgow Rob Glynne-Jones, Northwood Sharad Karandikar, Venkatesh Shanmugam, Derby Yeng S Ang, Wigan Alberto Quaglia, London Andrew Fowell, Southampton Gianpiero Gravante, Leicester Piers Gatenby, London Kondragunta Rajendra Prasad, Leeds Sunil Dolwani, Cardiff Andrew McCulloch Veitch, Wolverhampton Brian Green, Belfast Noriko Suzuki, Middlesex Richard Parker, North Staffordshire Shahid A Khan, London Akhilesh B Reddy, Cambridge Jean E Crabtree, Leeds John S Leeds, Sheffield Paul Sharp, London Sumita Verma, Brighton Thamara Perera, Birmingham Donald Campbell McMillan, Glasgow Kathleen B Bamford, London Helen Coleman, Belfast Eyad Elkord, Manchester Mohammad Ilyas, Nottingham Simon R Carding, Norwich Ian Chau, Sutton Claudio Nicoletti, Norwich Hendrik-Tobias Arkenau, London Muhammad Imran Aslam, Leicester Giuseppe Orlando, Oxford John S Leeds, Aberdeen S Madhusudan, Nottingham Amin Ibrahim Amin, Dunfermline David C Hay,Edinburgh Alan Burns, London United States Tauseef Ali, Oklahoma City George Y Wu, Farmington Josef E Fischer, Boston Thomas Clancy, Boston John Morton, Stanford Luca Stocchi, Cleveland Kevin Michael Reavis, Orange Shiu-Ming Kuo, Buffalo Gary R Lichtenstein, Philadelphia Natalie J Torok, Sacramento Scott A Waldman, Philadelphia Georgios Papachristou, Pittsburgh Carla W Brady, Durham Robert CG Martin, Louisville Eugene P Ceppa, Durham Shashi Bala, Worcester Imran Hassan, Springfield Klaus Thaler, Columbia Andreas M Kaiser, Los Angeles Shawn D Safford, Norfolk Massimo Raimondo, Jacksonville Kazuaki Takabe, Richmond VA Stephen M Kavic, Baltimore T Clark Gamblin, Pittsburgh BS Anand, Houston Ananthanarayanan M, New York Anthony J Bauer, Pittsburgh Edmund J Bini, New York Xian-Ming Chen, Omaha Ramsey Chi-man Cheung, Palo Alto Parimal Chowdhury, Arkansas Mark J Czaja, New York VII January 7, 2012

9 Conor P Delaney, Cleveland Sharon DeMorrow, Temple Bijan Eghtesad, Cleveland Alessandro Fichera, Chicago Glenn T Furuta, Aurora Jean-Francois Geschwind, Baltimore Shannon S Glaser, Temple Ajay Goel, Dallas James H Grendell, New York Anna S Gukovskaya, Los Angeles Jamal A Ibdah, Columbia Atif Iqbal, Omaha Hajime Isomoto, Rochester Hartmut Jaeschke, Kansas Leonard R Johnson, Memphis Rashmi Kaul, Tulsa Ali Keshavarzian, Chicago Miran Kim, Providence Burton I Korelitz, New York Richard A Kozarek, Seattle Alyssa M Krasinskas, Pittsburgh Ming Li, New Orleans Zhiping Li, Baltimore Chen Liu, Gainesville Michael R Lucey, Madison James D Luketich, Pittsburgh Patrick M Lynch, Houston Willis C Maddrey, Dallas Mercedes Susan Mandell, Aurora Wendy M Mars, Pittsburgh Laura E Matarese, Pittsburgh Lynne V McFarland, Washington Stephan Menne, New York Didier Merlin, Atlanta George Michalopoulos, Pittsburgh James M Millis, Chicago Pramod K Mistry, New Haven Emiko Mizoguchi, Boston Peter L Moses, Burlington Masaki Nagaya, Boston Robert D Odze, Boston Stephen JD O Keefe, Pittsburgh Zhiheng Pei, New York Raymund R Razonable, Minnesota Basil Rigas, New York Richard A Rippe, Chapel Hill Philip Rosenthal, San Francisco Stuart Sherman, Indianapolis Christina Surawicz, Seattle Wing-Kin Syn, Durham Yvette Taché, Los Angeles K-M Tchou-Wong, New York George Triadafilopoulos, Stanford Chung-Jyi Tsai, Lexington Andrew Ukleja, Florida Arnold Wald, Wisconsin Irving Waxman, Chicago Steven D Wexner, Weston Jackie Wood, Ohio Jian Wu, Sacramento Zobair M Younossi, Virginia Liqing Yu, Winston-Salem Ruben Zamora, Pittsburgh Michael E Zenilman, New York Michael A Zimmerman, Colorado Beat Schnüriger, California Clifford S Cho, Madison R Mark Ghobrial, Texas Anthony T Yeung, Philadelphia Chang Kim, West Lafayette Balamurugan N Appakalai, Minneapolis Aejaz Nasir, Tampa Ashkan Farhadi, Irvine Kevin E Behrns, Gainesville Joseph J Cullen, Iowa City David J McGee, Shreveport Anthony J Demetris, Pittsburgh Dimitrios V Avgerinos, New York Dong-Hui Li, Houston Eric S Hungness, Chicago Giuseppe Orlando, Winston Salem Hai-Yong Han, Phoenix Huanbiao Mo, Denton Jong Park, Tampa Justin MM Cates, Nashville Charles P Heise, Madison Craig D Logsdon, Houston Ece A Mutlu, Chicago Jessica A Davila, Houston Rabih M Salloum, Rochester Amir Maqbul Khan, Marshall Bruce E Sands, Boston Chakshu Gupta, Saint Joseph Ricardo Alberto Cruciani, New York Mariana D Dabeva, Bronx Edward L Bradley III, Sarasota Martín E Fernández-Zapico, Rochester Henry J Binder, New Haven John R Grider, Richmond Ronnie Fass, Tucson Dinesh Vyas, Washington Wael El-Rifai, Nashville Craig J McClain, Louisville Christopher Mantyh, Durham Daniel S Straus, Riverside David A Brenner, San Diego Eileen F Grady, San Francisco Ekihiro Seki, La Jolla Fang Yan, Nashville Fritz Francois, New York Giamila Fantuzzi, Chicago Guang-Yin Xu, Galveston Jianyuan Chai, Long Beach JingXuan Kang, Charlestown Le Shen, Chicago Lin Zhang, Pittsburgh Mitchell L Shiffman, Richmond Douglas K Rex, Indianapolis Bo Shen, Cleveland Edward J Ciaccio, New York Jean S Wang, Saint Louis Bao-Ting Zhu, Kansas Tamir Miloh, Phoenix Eric R Kallwitz, Chicago Yujin Hoshida, Cambridge C Chris Yun, Atlanta Alan C Moss, Boston Oliver Grundmann, Gainesville Linda A Feagins, Dallas Chanjuan Shi, Nashville Xiaonan Han, Cincinnati William R Brugge, Boston Richard W McCallum, El Paso Lisa Ganley-Leal, Boston Lin-Feng Chen, Urbana Elaine Y Lin, New York Julian Abrams, New York Arun Swaminath, New York Huiping Zhou, Richmond Korkut Uygun, Boston Anupam Bishayee, Signal Hill C Bart Rountree, Hershey Avinash Kambadakone, Boston Courtney W Houchen, Oklahoma Joshua R Friedman, Philadelphia Justin H Nguyen, Jackonville Sophoclis Alexopoulos, Los Angeles Suryakanth R Gurudu, Scottsdale Wei Jia, Kannapolis Yoon-Young Jang, Baltimore Ourania M Andrisani, West Lafayette Roderick M Quiros, Bethlehem Timothy R Koch, Washington Adam S Cheifetz, Boston Lifang Hou, Chicago Thiru vengadam Muniraj, Pittsburgh Dhiraj Yadav, Pittsburgh Ying Gao, Rockville John F Gibbs, Buffalo Aaron Vinik, Norfolk Charles Thomas, Oregon Robert Jensen, Bethesda John W Wiley, Ann Arbor Jonathan Strosberg, Tampa Randeep Singh Kashyap, New York Kaye M Reid Lombardo, Rochester Lygia Stewart, San Francisco Martin D Zielinski, Rochester Matthew James Schuchert, Pittsburgh Michelle Lai, Boston Million Mulugeta, Los Angeles Patricia Sylla, Boston Pete Muscarella, Columbus Raul J Rosenthal, Weston Robert V Rege, Dallas Roberto Bergamaschi, New York Ronald S Chamberlain, Livingston Alexander S Rosemurgy, Tampa Run Yu, Los Angeles Samuel B Ho, San Diego Sami R Achem, Florida Sandeep Mukherjee, Omaha Santhi Swaroop Vege, Rochester Scott Steele, Fort Lewis Steven Hochwald, Gainesville Udayakumar Navaneethan, Cincinnati Radha Krishna Yellapu, New York Rupjyoti Talukdar, Rochester Shi-Ying Cai, New Haven Thérèse Tuohy, Salt Lake City Tor C Savidge, Galveston William R Parker, Durham Xiaofa Qin, Newark Zhang-Xu Liu, Los Angeles Adeel A Butt, Pittsburgh Dean Y Kim, Detroit Denesh Chitkara, East Brunswick Mohamad A Eloubeidi, Alabama JiPing Wang, Boston Oscar Joe Hines, Los Angeles Jon C Gould, Madison Kirk Ludwig, Wisconsin Mansour A Parsi, Cleveland VIII January 7, 2012

10 Perry Shen, Winston-Salem Piero Marco Fisichella, Maywood Marco Giuseppe Patti, Chicago Michael Leitman, New York Parviz M Pour, Omaha Florencia Georgina Que, Rochester Richard Hu, Los Angeles Robert E Schoen, Pittsburgh Valentina Medici, Sacramento Wojciech Blonski, Philadelphia Yuan-Ping Han, Los Angeles Grigoriy E Gurvits, New York Robert C Moesinger, Ogden Mark Bloomston, Columbus Bronislaw L Slomiany, Newark Laurie DeLeve, Los Angeles Michel M Murr, Tampa John Marshall, Columbia Wilfred M Weinstein, Los Angeles Jonathan D Kaunitz, Los Angeles Josh Korzenik, Boston Kareem M Abu-Elmagd, Pittsburgh Michael L Schilsky, New Haven John David Christein, Birmingham Mark A Zern, Sacramento Ana J Coito, Los Angeles Golo Ahlenstiel, Bethesda Smruti R Mohanty, Chicago Victor E Reyes, Galveston CS Pitchumoni, New Brunswick Yoshio Yamaoka, Houston Sukru H Emre, New Haven Branko Stefanovic, Tallahassee Jack R Wands, Providence Wen Xie, Pittsburgh Robert Todd Striker, Madison Shivendra Shukla, Columbia Laura E Nagy, Cleveland Fei Chen, Morgantown Kusum K Kharbanda, Omaha Pal Pacher, Rockville Pietro Valdastri, Nashville IX January 7, 2012

11 S Contents Weekly Volume 18 Number 46 December 14, 2012 EDITORIAL 6693 Prevention and treatment of hepatic encephalopathy: Focusing on gut microbiota Garcovich M, Zocco MA, Roccarina D, Ponziani FR, Gasbarrini A FIELD OF VISION 6701 Glycogenotic hepatocellular carcinoma with glycogen-ground-glass hepatocytes: A heuristically highly relevant phenotype Bannasch P 6709 Targeting late SV40 factor: Is the achilles heel of hepatocarcinogenesis revealed? Shlomai A TOPIC HIGHLIGHT 6712 Thinking in three's: Changing surgical patient safety practices in the complex modern operating room Gibbs VC 6720 Modern treatment of gastric gastrointestinal stromal tumors Roggin KK, Posner MC 6729 Adult to adult living related liver transplantation: Where do we currently stand? Carlisle EM, Testa G 6737 Multidisciplinary approach for patients with esophageal cancer Villaflor VM, Allaix ME, Minsky B, Herbella FA, Patti MG 6747 Laparoscopic rectal cancer surgery: Where do we stand? Krane MK, Fichera A 6756 Minimally invasive approaches for the treatment of inflammatory bowel disease Zoccali M, Fichera A 6764 Impact of minimally invasive surgery on the treatment of benign esophageal disorders Bello B, Herbella FA, Allaix ME, Patti MG December 14, 2012 Volume 18 Issue 46

12 Contents World Journal of Gastroenterology Volume 18 Number 46 December 14, 2012 GUIDELINES FOR BASIC SCIENCE 6771 Hepatic expression and cellular distribution of the glucose transporter family Karim S, Adams DH, Lalor PF GUIDELINES FOR CLINICAL PRACTICE 6782 Utility of faecal calprotectin analysis in adult inflammatory bowel disease Smith LA, Gaya DR REVIEW 6790 Have guidelines addressing physical activity been established in nonalcoholic fatty liver disease? Finelli C, Tarantino G ORIGINAL ARTICLE 6801 Ileocecal valve dysfunction in small intestinal bacterial overgrowth: A pilot study Miller LS, Vegesna AK, Sampath AM, Prabhu S, Kotapati SK, Makipour K 6809 Schizandra arisanensis extract attenuates cytokine-mediated cytotoxicity in insulin-secreting cells Hsu YS, Kuo YH, Cheng HL, Flatt PR, Liu HK 6819 Moxibustion inhibits interleukin-12 and tumor necrosis factor alpha and modulates intestinal flora in rat with ulcerative colitis Wang XM, Lu Y, Wu LY, Yu SG, Zhao BX, Hu HY, Wu HG, Bao CH, Liu HR, Wang JH, Yao Y, Hua XG, Guo HY, Shen LR BRIEF ARTICLE 6829 Minimally invasive treatment of pancreatic necrosis Bello B, Matthews JB 6836 Dynamic magnetic resonance defecography in 10 asymptomatic volunteers Schreyer AG, Paetzel C, Fürst A, Dendl LM, Hutzel E, Müller-Wille R, Wiggermann P, Schleder S, Stroszczynski C, Hoffstetter P 6843 Improved techniques for double-balloon-enteroscopy-assisted endoscopic retrograde cholangiopancreatography Osoegawa T, Motomura Y, Akahoshi K, Higuchi N, Tanaka Y, Hisano T, Itaba S, Gibo J, Yamada M, Kubokawa M, Sumida Y, Akiho H, Ihara E, Nakamura K 6850 Is laparoscopy equal to laparotomy in detecting and treating small bowel injuries in a porcine model? Shan CX, Ni C, Qiu M, Jiang DZ II December 14, 2012 Volume 18 Issue 46

13 Contents World Journal of Gastroenterology Volume 18 Number 46 December 14, Analysis of TLR4 and TLR2 polymorphisms in inflammatory bowel disease in a Guangxi Zhuang population Chen L, Lin MJ, Zhan LL, Lv XP CASE REPORT 6861 Puncture injection of para-toluenesulfonamide combined with chemoembolization for advanced hepatocellular carcinoma He Q, Kuang AR, Guan YS, Liu YQ ACKNOWLEDGMENTS I Acknowledgments to reviewers of World Journal of Gastroenterology I Meetings APPENDIX I-VI Instructions to authors ABOUT COVER Editorial Board Member of World Journal of Gastroenterology, Hartmut Jaeschke, Professor, Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Blvd, MS 1018, Kansas City, KS 66160, United States FLYLEAF I-IX Editorial Board EDITORS FOR THIS ISSUE Responsible Assistant Editor: Shuai Ma Responsible Electronic Editor: Li Xiong Proofing Editor-in-Chief: Lian-Sheng Ma Responsible Science Editor: Xin-Zhen Huang Proofing Editorial Office Director: Jin-Lei Wang NAME OF JOURNAL World Journal of Gastroenterology ISSN AND EISSN ISSN (print) ISSN (online) LAUNCH DATE October 1, 1995 FREQUENCY Weekly RESPONSIBLE INSTITUTION Department of Science and Technology of Shanxi Province SPONSOR Taiyuan Research and Treatment Center for Digestive Diseases, 77 Shuangta Xijie, Taiyuan , Shanxi Province, China EDITING Editorial Board of World Journal of Gastroenterology Room 903, Building D, Ocean International Center, No. 62 Dongsihuan Zhonglu, Chaoyang District, Beijing , China Telephone: Fax: [email protected] EDITOR-IN-CHIEF Ferruccio Bonino, MD, PhD, Professor of Gastroenterology, Director of Liver and Digestive Disease Division, Department of Internal Medicine, University of Pisa, Director of General Medicine 2 Unit University Hospital of Pisa, Via Roma 67, Pisa, Italy Myung-Hwan Kim, MD, PhD, Professor, Head, Department of Gastroenterology, Director, Center for Biliary Diseases, University of Ulsan College of Medicine, Asan Medical Center, Pungnap-2dong, Songpa-gu, Seoul , South Korea Kjell Öberg, MD, PhD, Professor, Department of Endocrine Oncology, Uppsala University Hospital, SE Uppsala, Sweden Matt D Rutter, MBBS, MD, FRCP, Consultant Gastroenterologist, Senior Lecturer, Director, Tees Bowel Cancer Screening Centre, University Hospital of North Tees, Durham University, Stockton-on-Tees, Cleveland TS19 8PE, United Kingdom Andrzej S Tarnawski, MD, PhD, DSc (Med), Professor of Medicine, Chief Gastroenterology, VA Long Beach Health Care System, University of California, Irvine, CA, 5901 E. Seventh Str., Long Beach, CA 90822, United States EDITORIAL OFFICE Jian-Xia Cheng, Director Jin-Lei Wang, Vice Director World Journal of Gastroenterology Room 903, Building D, Ocean International Center, No. 62 Dongsihuan Zhonglu, Chaoyang District, Beijing , China Telephone: Fax: [email protected] PUBLISHER Baishideng Publishing Group Co., Limited Room 1701, 17/F, Henan Building, No.90 Jaffe Road, Wanchai, Hong Kong, China Fax: Telephone: [email protected] PRINT SUBSCRIPTION RMB 300 Yuan for each issue, RMB Yuan for one year. PUBLICATION DATE December 14, 2012 COPYRIGHT 2012 Baishideng. Articles published by this Open- Access journal are distributed under the terms of the Creative Commons Attribution Non-commercial License, which permits use, distribution, and reproduction in any medium, provided the original work is properly cited, the use is non commercial and is otherwise in compliance with the license. 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 wjgnet.com/ /g_info_ htm ONLINE SUBMISSION III December 14, 2012 Volume 18 Issue 46

14 Online Submissions: doi: /wjg.v18.i World J Gastroenterol 2012 December 14; 18(46): ISSN (print) ISSN (online) 2012 Baishideng. All rights reserved. EDITORIAL Prevention and treatment of hepatic encephalopathy: Focusing on gut microbiota Matteo Garcovich, Maria Assunta Zocco, Davide Roccarina, Francesca Romana Ponziani, Antonio Gasbarrini Matteo Garcovich, Maria Assunta Zocco, Davide Roccarina, Francesca Romana Ponziani, Antonio Gasbarrini, Department of Internal Medicine, Catholic University of Sacred Heart, Rome, Italy Author contributions: Garcovich M, Zocco MA and Gasbarrini A contributed to the article design, drafting and revision; Roccarina D and Ponziani FR contributed to the literature research, writing and revision, all authors approved the version to be published. Correspondence to: Matteo Garcovich, MD, Department of Internal Medicine, Catholic University of Sacred Heart, Rome, Largo A. Gemelli 8, Rome, Italy. [email protected] Telephone: Fax: Received: April 3, 2012 Revised: July 30, 2012 Accepted: August 4, 2012 Published online: December 14, 2012 Key words: Hepatic encephalopathy; Gut microbiota; Cirrhosis; Non-absorbable disaccharides; Rifaximin Peer reviewers: Peter Ferenci, Professor, Department of Internal Medicine IV, Division of Gastroenterology and Hepatology, Waehringer Guertel 18-20, Vienna A-1090, Austria; Philip Rosenthal, MD, Professor of Pediatrics and Surgery, UCSF, 500 Parnassus Avenue, Box 0136, MU 4-East, San Francisco, CA , United States Garcovich M, Zocco MA, Roccarina D, Ponziani FR, Gasbarrini A. Prevention and treatment of hepatic encephalopathy: Focusing on gut microbiota. World J Gastroenterol 2012; 18(46): Available from: URL: com/ /full/v18/i46/6693.htm DOI: org/ /wjg.v18.i Abstract The gut flora plays an important role in the pathogenesis of the complications of cirrhosis. Hepatic encephalopathy (HE) represents a broad continuum of neuropsychological dysfunction in patients with acute or chronic liver disease and/or porto-systemic shunting of blood flow and it manifests with progressive deterioration of the superior neurological functions. The pathophysiology of this disease is complex, as it involves overproduction and reduced metabolism of various neurotoxins, particularly ammonia. Management of HE is diversified and requires several steps: elimination of precipitating factors, removal of toxins, proper nutritional support, modulation of resident fecal flora and downregulation of systemic and gut-derived inflammation. This review will provide an overview of gut barrier function and the influence of gut-derived factors on HE, focusing on the role of gut microbiota in the pathogenesis of HE and the recent literature findings on its therapeutic manipulation Baishideng. All rights reserved. INTRODUCTION Humans have been proposed to be a meta-organisms consisting of a huge number of bacterial cells that are metabolically and immunologically integrated with somatic cells [1]. This interaction is especially important in the gastrointestinal tract, where the commensal bacteria, known as intestinal microbiota, are an integral component of human gut physiology and, together with the intestinal mucosa, form an important barrier against pathogens. Gut homeostasis and physiology are closely linked to the liver since it receives the intestinal blood content through the portal system and influences intestinal functions through bile secretion into the lumen [2]. Thus, alterations of gut barrier seem to play an important role in the pathogenesis and progression of liver damage [3]. Understanding of both partners in this normal gut-liver interaction is critical to the development of new therapeutic modalities to treat or prevent liver disease and its complications. This review will provide an overview of gut barrier function and the influence of gut-derived factors on hepatic encephalopathy (HE), fo December 14, 2012 Volume 18 Issue 46

15 Garcovich M et al. Hepatic encephalopathy and gut microbiota cusing on the role of gut microbiota in the pathogenesis of HE and the recent literature findings on its therapeutic manipulation. HOST-MICROBIOTA CROSS-TALK The human intestine provides residence to more than bacteria, a number which is 10 times the number of somatic cells in the human body [4,5]. Microorganisms start colonizing the gut immediately after birth and are characterized by a succession of different population until a stable, adult microbiota has been established. In this bacterial community anaerobes are more abundant than aerobes and the majority of the species are from the genera Bacteroidetes and Firmicutes [6]. Bacterial density and types differ substantially from lower small intestine to distal colon, and are regulated by physiological conditions. The specific populations also vary among individuals and in the same individual during periods of illness or dietary changes [7]. Microarray analysis of intestinal transcriptional responses and molecular taxonomic methodologies have greatly increased our understanding of the gut microbiota composition, activities and functions [1,8,9]. In a healthy individual the host/microbiota relationship is characterized by a homeostatic symbiosis, in which the host provides nutrients and a stable environment and, in turn, the microbiota ensures optimal epithelial functioning. GUT-LIVER AXIS Receiving most of its blood supply from the intestine through the portal circulation, the liver is exposed to gut-derived toxins, including bacteria and bacterial products, and must be prepared to react against these potential systemic pathogens. For this purpose it contains a large number of resident immune cells including macrophages, dendritic cells, lymphocytes, natural killer cells. These cells act together with other non-parenchymal cells like endothelial and stellate cells to produce an organized response to these potentially highly inflammatory factors [10,11]. The role of immune cells during inflammatory response or chronic liver injury and the potential impact of gut-derived toxins on these processes have been extensively studied [2]. In particular bacterial overgrowth and altered intestinal permeability result in high plasmatic levels of bacterial endotoxins, such as lipopolysaccharide (LPS), peptidoglycan, and various lipopeptides also termed pathogen-associated molecular patterns. Endotoxemia could be responsible for initiation of the liver damage, through its interaction with specific recognition receptors, the toll like receptors on the surface of immune cells. These receptors contribute to adaptive immune response and regulation of inflammation and represent a link between intestinal flora changes, endotoxemia, and liver damage [12]. Liver cirrhosis is characterized by several abnormalities of both the systemic and local immune systems and in particular by reduced phagocytic activity of Kupffer cells [13]. A great deal of evidence indicates that patients with cirrhosis could present increased intestinal permeability [14]. This is related to structural changes that occur in the presence of portal hypertension and hypertensive enteropathy: an altered oxide-reductive state with consequent oxidative damage of the brush border membrane and the overproduction of nitric oxide resulting in tight junctions expansion and cytoskeleton destruction [15]. Altered intestinal permeability, together with bacterial overgrowth and immune dysfunction are associated with the migration of bacteria or their products from the gut to mesenteric lymph nodes or to other organs, a process known as bacterial translocation (BT). This phenomenon, in association with the presence of vascular shunts, is responsible for increased circulating levels of LPS. Blood concentration of bacterial endotoxin directly correlates with the severity of liver disease and participates in the initiation of a complex series of mechanisms that lead to the development of cirrhosis complications [16]. In particular, the main consequences of portal hypertension and BT are the occurrence of infections and HE. Bacterial infections are present in about 15%-47% of patients with liver cirrhosis and are especially related to Gram-negative bacteria. The most frequent are spontaneous bacterial peritonitis (SBP), urinary tract infections, pneumonia, pleural empyema and sepsis. It has been shown that patients with SBP have a higher prevalence of small intestinal bacterial overgrowth (SIBO) [17] and altered intestinal permeability [18] than patients without SBP. Ammonia and other toxic substances derived from the gut, in the presence of portal and systemic shunts as well as of reduced liver clearance capability, represent the pathogenic mechanisms of HE as described in the following paragraph. GUT MICROBIOTA AND HE HE is a reversible neuropsychiatric disorder associated with liver dysfunction after exclusion of other potential causes of brain disease [19] and is characterized by poor survival [20]. Main features are disturbances in cognitive function, personality and behaviour with a wide spectrum of neuropsychiatric abnormalities that range from mild impairment of cognitive function and consciousness to coma. There are two types of HE named overt and minimal HE (OHE, MHE). The first is present in 30%-45% of patients with cirrhosis and in 10%-50% of patients with transjugular intrahepatic portosystemic shunt [21]. OHE can be diagnosed clinically through a constellation of signs and symptoms by several scoring systems [22]. The most widely used are the West-Haven criteria (Table 1) which are based on neurological examination and specific questionnaires to detect mental status changes. In addition, OHE can be further divided into episodic or persistent depending on time course and clinical behaviour: episodic HE remains below the clinical detection level among different episodes, whereas 6694 December 14, 2012 Volume 18 Issue 46

16 Garcovich M et al. Hepatic encephalopathy and gut microbiota Table 1 West-Haven criteria for hepatic encephalopathy Stage Consciousness Intellect and behaviour Neurological findings 0 Normal Normal Normal examination 1 1 Trivial lack of awareness Impaired attention span; altered sleep; euphoria Mild asterixis 2 Lethargic Disoriented; inappropriate behaviour depression Asterixis; slurred speech 3 Somnolent but arousable Gross disorientation; bizarre behaviour Muscular rigidity/clonus hyper-reflexia 4 Coma Coma Decerebrate posturing 1 If impaired psychomotor testing, then minimal hepatic encephalopathy. Brain Blood-brain barrier Systemic circulation Portal-systemic shunts Liver Portal venous flow Gut microflora Ammonia Glutamine Methionine Nitrogen Serotonin GABA Figure 1 Pathophysiology of hepatic encephalopathy in liver cirrhosis. GABA: γ-aminobutyric acid. persistent HE is always clinically evident [23]. Prevalence for MHE ranges from 30% up to 80%, probably because there is still no accepted gold standard for diagnosis of MHE and the majority of the screening tests are time-consuming and cumbersome to perform [23,24]. It is characterized by alterations of psychometric and neurophysiological tests in otherwise asymptomatic patients [25]. This condition is considered a preclinical stage of OHE and is associated with poor quality of life, high risk of traffic violations and accidents and increased progression to OHE [26-28]. The diagnosis of MHE aims to detect attention deficits and processing speed and is based on different batteries of tests. Among them, the portal systemic encephalopathy (PSE) syndrome test, that includes number connection test A (NCT A), number connection test B (NCT B), line tracing test errors and time, serial dotting test and digit symbol test, has been validated in different countries and is recommended by the Working Group on HE [29]. Although the pathogenesis of this disorder is not well understood, a key role is played by circulating gut derived toxins of the nitrogenous compounds with a resultant neuroglial injury in the setting of a systemic inflammatory milieu (Figure 1). In particular, the currently accepted hypothesis is that endogenous neurotoxic substances escape from catabolism by the liver, due both to the impaired function of the cirrhotic liver and to the presence of portal-systemic shunts. These substances circulate in the systemic blood flow, reach the brain through the blood-brain barrier and results in different severity of cerebral impairment [30]. Several factors have been shown to precipitate HE, including gastrointestinal bleeding, electrolyte imbalances, infection, and medications such as sedatives and diuretics. They work by increasing the underlying inflammatory milieu, increasing toxins production or reducing the threshold for mental status decline, or a combination of 6695 December 14, 2012 Volume 18 Issue 46

17 Garcovich M et al. Hepatic encephalopathy and gut microbiota the above. Ammonia, mercaptans, phenols, short and medium-chain fatty acids and benzodiazepine-like compounds have all been found to be elevated in cirrhotic patients with HE. The majority of these toxic substances are produced in the intestine by the bacterial flora, and are absorbed into the portal venous flow. Thus, gut microflora contribute to the pro-inflammatory state of cirrhosis even in the absence of overt infection [31]. In addition, cirrhotic patients have a substantial alteration of the gut microecology with a high prevalence of SIBO and delayed oro-cecal transit time (OCTT) [17,32-36]. The latter has a multifactorial ethiology. First, it could be due to the presence of autonomic neuropathy that has been demonstrated as an independent predictor of reduced intestinal motility in this kind of patients [37]. Moreover, patients with autonomic dysfunction were found to have a higher incidence of new onset of HE [38]. Second, a delayed OCTT may be associated with metabolic alterations that occur in patients with portal hypertension and portal-systemic venous shunting. Finally, SIBO itself may leads to delayed OCTT since it has been shown a significant improvement of intestinal motility after antibiotic therapy [36]. There might be also a link between the cognitive impairment in cirrhosis with inflammation and specific bacterial taxa. For example, recent evidence showed how interleukin (IL)-23 system and innate immune response were highly correlated with several bacterial families in patients with cirrhosis and HE, and how there was a direct correlation between cognition, Porphyromonadaceae and Alcaligeneceae families [39]. Based on these considerations, the manipulation of gut flora could be useful in the treatment of HE. MODULATION OF GUT MICROBIOTA FOR THE MANAGEMENT OF HE Treatment goals and options are dependent on the stage and acuity of HE [23]. Once the diagnosis of OHE is confirmed, an extensive search for potential precipitating factors should be instituted along with treatment of OHE. As previously described, the leading causes are gastrointestinal bleeding, sepsis, dehydration resulting from diuretics, diarrhoea or vomiting, transjugular intra hepatic porto systemic shunting, constipation and the use of sedative and narcotic drugs. Their treatment can reverse OHE in most cases. However, when it is not possible to identify a precipitating factor despite an exhaustive search, specific treatment for OHE should be instituted. Most therapies for HE focus on treating episodes as they occur and are directed at reducing the nitrogenous load in the gut, an approach that is consistent with the hypothesis that this disorder results from the systemic accumulation of gut-derived neurotoxins in patients with impaired liver function and portosystemic shunting [40]. Therefore, the majority of therapeutic options currently in use are directed towards the gut. Prebiotics are nondigestible food ingredients that act by directly stimulating the growth of bacterial strains potentially beneficial to the host like Bifidobacteria and Lactobacilli, thereby indirectly reducing the influence of potentially more harmful resident flora (i.e. urease-producing species). They come in the form of indigestible fibers and have shown benefit for the management of HE, particularly MHE, both as prebiotics and when used in combination with probiotics (in which case they are termed synbiotics) [41,42]. Probiotics are living non pathogenic microorganisms that are thought to exert an effect in HE by reducing intestinal ammonia production by enterocyte glutaminase and reduce BT, modulate gut permeability and modulate proinflammatory responses. Furthermore, probiotics bypass the small bowel and get fermented by colonic bacteria to form lactic, acetic, and butyric acids, and gas (mainly hydrogen); any resultant prokinetic effect may increase the expulsion of ammoniagenic bacteria [43]. Probiotics have been studied for the treatment of HE and have shown some benefit, mostly in the setting of minimal disease [24,44,45]. The bacterial species that appear to be most successful include Lactobacilli and Bifidobacteria. Probiotics may also improve overall liver function, perhaps by reducing translocation and subsequent endotoxemia and by ameliorating the hyperdynamic circulation [41]. To quantify unambiguously the beneficial and harmful effects of any probiotics at any dosage, a recent meta-analysis identified seven randomized trials for the treatment of acute or chronic HE. The authors of this Cochrane review assessed a range of outcomes including death, recovery, adverse events, and quality of life. There was no benefit of probiotics shown for any of the primary outcomes including mortality. On the other hand, there was a significant difference in secondary outcomes such as lowering of plasma ammonia concentration compared with no treatment. The authors concluded that this finding is of questionable importance, not recommending the use of probiotics for patients with HE until further randomized clinical trials are undertaken [46]. Non-absorbable disaccharides, such as lactulose and lactilol, have traditionally been considered the first-line drug therapy for lowering the production and absorption of ammonia [47]. These substances are metabolized by the intestinal bacteria to acetic and lactic acid. The consequent acidification of the colonic contents creates a hostile environment for the survival of intestinal bacteria involved in the production of ammonia and facilitates the conversion of NH3 to non-absorbable NH4 +. Moreover, their cathartic effects cause an increased in faecal nitrogen excretion [48]. The non-absorbable disaccharides have been used for decades with anecdotal and clinical trial experience [49]. However, side effects of lactulose therapy, including cramping, diarrhoea and flatulence, result in frequent noncompliance [40]. Overdosage may also result in severe diarrhoea, electrolyte disturbances and hypovolaemia that, if severe enough, may itself precipitate encephalopathy symptoms. In spite of their anedoctal usefulness, in the last years the true efficacy of the disaccharides for this indication has been questioned. As the use of lactulose pre dated randomized controlled trials, a comprehensive meta-analysis 6696 December 14, 2012 Volume 18 Issue 46

18 Garcovich M et al. Hepatic encephalopathy and gut microbiota endorsed by the Cochrane Collaboration did not find any significant difference in outcomes in patients treated with and without lactulose [50]. Based on a critical analysis of available published literature, Als-Nielsen et al [50] concluded that the evidence in favour of utilizing nonabsorbable disaccharides in HE did not meet the current minimum criteria for adequacy. In fact, although in some cases the administration of lactulose was associated with improvement in mental status, it is difficult to assess the reason for improvement since precipitating factors were simultaneously being corrected. On the other hand newer clinical studies suggest benefits with lactulose conferring improved neuropsychometric and quality of life scores [47]. Also, two recently published meta-analysis on the clinical efficacy and safety of lactulose in patients with MHE, provide substantial evidence for the beneficial effects of non-absorbable disaccharides [51,52]. Therefore, at the present time, there is a lack of sufficient evidence to completely dismiss the use of non-absorbable disaccharides for the treatment of HE, while compliance and cost effectiveness should be carefully balanced against clinical outcomes. Antimicrobial agents have long been utilized as an alternative treatment option for patients intolerant or unresponsive to non-absorbable disaccharides due to their ability to inhibit ammonia production by intestinal bacteria. Neomycin is the most commonly used antimicrobial for HE, but despite the legitimate theoretical rationale for its use, there is a paucity of clinical data to support this practice [48,53]. Moreover, the occurrence of serious adverse effects such as nephrotoxicity and ototoxicity limit their use to relatively short periods of time. Other antimicrobials, including metronidazole and vancomycin, have been studied to a more limited extent than neomycin [48]. However, long-term use of metronidazole has been associated with neurotoxicity in patients with cirrhosis, whereas the risk for enteric bacteria resistance preclude the routine use of vancomycin for HE. Thus, with the potential for serious adverse effects and the lack of demonstrated clinical benefit, the routine management of HE with conventional antibiotics should be questioned. Rifaximin is a poorly absorbed synthetic antibiotic with a broad spectrum of antibacterial activity, against aerobic and anaerobic Gram positive and Gramnegative organisms [54]. As a derivative of rifamycin, it similarly works by blocking bacterial RNA synthesis. However, rifaximin has an additional pyridoimidazole ring that allows for high concentrations in the gastrointestinal tract and minimal systemic drug absorption. Due to its low rate of systemic bioavailability, the safety profile of rifaximin appears to be superior to that of systemic antibiotics, particularly for patients with liver disease, making it suitable for long-term use. Moreover, the risk of bacterial resistance appears to be lower with rifaximin than with systemic antibiotics because bacteria outside the gastrointestinal tract are not exposed to appreciable selective pressure [55]. The safety and efficacy profiles of rifaximin as treatment of overt HE have been extensively explored in several clinical trials [48]. The results of controlled double-bind studies demonstrated that rifaximin was more effective than non-absorbable disaccharides in the treatment of acute HE: mental state, electroencephalogram irregularities and PSE-index were all significantly improved in the rifaximin group [56-59]. While rifaximin was well tolerated, adverse events, including flatulence, diarrhoea, nausea and anorexia, were reported by patients in the disaccharides group. A recent study found a reduced hospitalization rate during rifaximin therapy compared with that of lactulose [60]. Once again, the HE grade was significantly lower and patients compliance was significantly higher in the rifaximin group. Further data suggest that rifaximin is at least as effective as neomycin in decreasing plasma ammonia levels and in improving the clinical symptoms related to HE with fewer clinically significant adverse events during a 21 d treatment period [61]. Based on these findings, the Cochrane review recommends the use of rifaximin in the treatment of acute HE [50]. Until recently, there has not been any conclusive evidence to support routine use of pharmacological prophylaxis to prevent future recurrence in patients who have recovered from an acute episode of HE. However, the prevention of episodes of HE is an important goal in the treatment of patients with liver disease, especially since symptoms of overt encephalopathy are debilitating and decrease the ability for self-care, leading to frequent hospitalizations, and a poor quality of life. A recent clinical trial has been conducted to evaluate the efficacy of rifaximin as secondary prophylaxis of overt HE [62]. This double blind, placebo controlled, multicentre trial randomized 299 patients with a recent history of recurrent, overt HE to receive either rifaximin or placebo for a period of 6 months. The majority of the patients in both groups were also maintained on concomitant lactulose therapy. During the study period, an acute episode of HE occurred in a significantly lower percentage of patients in the rifaximin group (22.1%) than in the placebo group (45.9%), with a hazard ratio (HR) of Furthermore, there was a significantly reduced risk of hospitalization in the rifaximin group when compared with placebo: 13.6% vs 22.6% of patients respectively with a corresponding HR of No significant difference in the incidence of adverse events was found between the two groups. The authors concluded that the addition of rifaximin to a standard lactulose regimen may offer advantages in terms of decreasing risk of both acute HE episodes as well as hospitalizations when compared with lactulose alone. Overall, this pivotal study expands previously reported findings of the efficacy of rifaximin in the treatment of overt HE and demonstrates a clinically relevant benefit of rifaximin as pharmacological prophylaxis of HE. The protective effect of rifaximin was confirmed by an extension trial performed on the same patients to assess the efficacy of rifaximin in maintaining remission over time [63]. The results of this preliminary study support a possible longterm protective pharmacological effect. In addition, an ancillary analysis of data from the same trial to assess the effect of rifaximin on health-related quality of life 6697 December 14, 2012 Volume 18 Issue 46

19 Garcovich M et al. Hepatic encephalopathy and gut microbiota (measured via the Chronic Liver Disease Questionnaire) provided evidence that rifaximin improves perception of daily well being and quality of life outcomes in all domains adversely affected by underlying liver diseases [64]. Therefore at this time, both lactulose and rifaximin can be used to prevent recurrent episodes of OHE. Ambivalence remains as concerning the treatment of MHE. It is clear that MHE has a major impact on quality of life and should be promptly diagnosed and treated. However, for many years, various treatment modalities including lactulose, probiotics and dietary manipulation, have been studied with unconvincing evidence. Limits on the use of antibiotics have been exceeded by the results of the randomised ischaemic mitral evaluation trial [65], which showed unequivocally that rifaximin improves psychometric test performance scores as early as after 2 wk of treatment. The same patients experienced, also, a significant improvement in health related quality of life, which was strongly correlated with the improvement in cognitive functions. The authors speculated that there are two possible mechanisms by which rifaximin could lead to an improvement in MHE: first by reducing the ammoniaproducing bacteria in the gut [61], second by decreasing BT and inflammation. The results of the RIME trial represent an important step in the establishment of rifaximin as an effective and safe treatment for MHE and are in agreement with the findings of a more recent study which demonstrates improved driving skills following rifaximin treatment in patients with MHE [66]. In this double-blind placebo controlled trial, patients randomized to rifaximin had a significant reduction in the number of total driving errors, and specifically speeding tickets and illegal turns, on a driving simulator, compared with those on placebo. Driving requires balance and integration of different cognitive functions, such as attention, adequate reaction time, visuo-motor coordination and can be considered a practical interpretation of the cognitive domains affected in MHE [67]. Since patients on rifaximin presented increased plasmatic levels of the anti-inflammatory cytokine IL-10, the authors speculated that rifaximin could act by regulating local intestinal immunity and inflammation. The same authors provide in another paper a cost effective analysis based on a Markov model of progression from cirrhosis without MHE, to MHE and to OHE focused on motor vehicle crashes as an objective endpoint. The results of this analysis indicate that diagnosis of MHE followed by lactulose therapy could result in substantial societal cost savings by preventing major motor vehicle crashes among MHE patients. In contrast, because of its high monthly cost, treatment with rifaximin is unlikely to generate overall cost savings unless the rifaximin monthly cost is substantially reduced [68]. CONCLUSION Different chronic liver diseases are associated with alterations of the intestinal microbiota, gut barrier dysfunction and translocation of bacteria or bacteria-derived antigens into the systemic circulation. The same processes, in turn, exacerbate liver disease leading to enhanced tissue damage and development of complications such as systemic infections, phosphate buffered saline, hepato renal syndrome and portal-systemic encephalopathy. Modulation of the gut microbiota may represent a good therapeutic target for the prevention and treatment of different complications associated with liver cirrhosis. In particular, due to its low rate of systemic bioavailability and a good tolerability profile, the non-absorbable antibiotic rifaximin could be an ideal antimicrobial for selective targeting at the gastrointestinal tract in this kind of patients. As reviewed, at present this antibiotic is recommended for the treatment of acute HE. Furthermore, recent literature appears to support a favourable benefit-risk ratio for rifaximin both in the prevention of overt HE and in the treatment of minimal HE. However, additional high-quality clinical trials are needed to more clearly define the effectiveness of longterm or periodic treatment with rifaximin on gut microbiota modulation in cirrhotic patients with HE. REFERENCES 1 Gill SR, Pop M, Deboy RT, Eckburg PB, Turnbaugh PJ, Samuel BS, Gordon JI, Relman DA, Fraser-Liggett CM, Nelson KE. Metagenomic analysis of the human distal gut microbiome. Science 2006; 312: Norman K, Pirlich M. Gastrointestinal tract in liver disease: which organ is sick? 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20 Garcovich M et al. Hepatic encephalopathy and gut microbiota of chronic liver diseases: from the patient to the molecule. Hepatology 2006; 43: S121-S Lin RS, Lee FY, Lee SD, Tsai YT, Lin HC, Lu RH, Hsu WC, Huang CC, Wang SS, Lo KJ. Endotoxemia in patients with chronic liver diseases: relationship to severity of liver diseases, presence of esophageal varices, and hyperdynamic circulation. J Hepatol 1995; 22: Gunnarsdottir SA, Sadik R, Shev S, Simrén M, Sjövall H, Stotzer PO, Abrahamsson H, Olsson R, Björnsson ES. Small intestinal motility disturbances and bacterial overgrowth in patients with liver cirrhosis and portal hypertension. Am J Gastroenterol 2003; 98: Scarpellini E, Valenza V, Gabrielli M, Lauritano EC, Perotti G, Merra G, Dal Lago A, Ojetti V, Ainora ME, Santoro M, Ghirlanda G, Gasbarrini A. Intestinal permeability in cirrhotic patients with and without spontaneous bacterial peritonitis: is the ring closed? 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Dig Dis Sci 2004; 49: Bajaj JS, Ridlon JM, Hylemon PB, Thacker LR, Heuman DM, Smith S, Sikaroodi M, Gillevet PM. Linkage of gut microbiome with cognition in hepatic encephalopathy. Am J Physiol Gastrointest Liver Physiol 2012; 302: G168-G Bass NM. Review article: the current pharmacological therapies for hepatic encephalopathy. Aliment Pharmacol Ther 2007; 25: Liu Q, Duan ZP, Ha DK, Bengmark S, Kurtovic J, Riordan SM. Synbiotic modulation of gut flora: effect on minimal hepatic encephalopathy in patients with cirrhosis. Hepatology 2004; 39: Malaguarnera M, Gargante MP, Malaguarnera G, Salmeri M, Mastrojeni S, Rampello L, Pennisi G, Li Volti G, Galvano F. Bifidobacterium combined with fructo-oligosaccharide versus lactulose in the treatment of patients with hepatic encephalopathy. Eur J Gastroenterol Hepatol 2010; 22: Wright G, Chattree A, Jalan R. Management of hepatic encephalopathy. Int J Hepatol 2011; 2011: Malaguarnera M, Greco F, Barone G, Gargante MP, Malaguarnera M, Toscano MA. Bifidobacterium longum with fructo-oligosaccharide (FOS) treatment in minimal hepatic encephalopathy: a randomized, double-blind, placebocontrolled study. Dig Dis Sci 2007; 52: Bajaj JS, Saeian K, Christensen KM, Hafeezullah M, Varma RR, Franco J, Pleuss JA, Krakower G, Hoffmann RG, Binion DG. Probiotic yogurt for the treatment of minimal hepatic encephalopathy. Am J Gastroenterol 2008; 103: McGee RG, Bakens A, Wiley K, Riordan SM, Webster AC. Probiotics for patients with hepatic encephalopathy. Cochrane Database Syst Rev 2011; (11): CD Prasad S, Dhiman RK, Duseja A, Chawla YK, Sharma A, Agarwal R. Lactulose improves cognitive functions and health-related quality of life in patients with cirrhosis who have minimal hepatic encephalopathy. Hepatology 2007; 45: Phongsamran PV, Kim JW, Cupo Abbott J, Rosenblatt A. Pharmacotherapy for hepatic encephalopathy. Drugs 2010; 70: Sharma BC, Sharma P, Agrawal A, Sarin SK. 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21 Garcovich M et al. Hepatic encephalopathy and gut microbiota Rev 2004; (2): CD Luo M, Li L, Lu CZ, Cao WK. Clinical eficacy and safety of lactulose for minimal hepatic encephalopathy: a metaanalysis. Eur J Gastroenterol Hepatol 2011; 23: Shukla S, Shukla A, Mehboob S, Guha S. Meta-analysis: the effects of gut flora modulation using prebiotics, probiotics and synbiotics on minimal hepatic encephalopathy. Aliment Pharmacol Ther 2011; 33: Strauss E, Tramote R, Silva EP, Caly WR, Honain NZ, Maffei RA, de Sá MF. Double-blind randomized clinical trial comparing neomycin and placebo in the treatment of exogenous hepatic encephalopathy. Hepatogastroenterology 1992; 39: Scarpignato C, Pelosini I. Rifaximin, a poorly absorbed antibiotic: pharmacology and clinical potential. Chemotherapy 2005; 51 Suppl 1: Debbia EA, Maioli E, Roveta S, Marchese A. Effects of rifaximin on bacterial virulence mechanisms at supra- and subinhibitory concentrations. J Chemother 2008; 20: Alcorn J. Review: rifaximin is equally or more effective than other antibiotics and lactulose for hepatic encephalopathy. ACP J Club 2008; 149: Fera G, Agostinacchio F, Nigro M. Rifaximin in the treatment of hepatic encephalopathy. Eur J Clin Res 1993; 4: Mas A, Rods J, Sunyer L, Rodrigo L, Planas R, Vargas V, Castells L, Rodrguez-Martnez D, Fernndez-Rodrguez C, Coll I, Pardo A; Spanish Association for the Study of the Liver Hepatic Encephalopathy Cooperative Group. Comparison of rifaximin and lactitol in the treatment of acute hepatic encephalopathy: results of a randomized, doubleblind, doubledummy, controlled clinical trial. J Hepatol 2003; 38: Lawrence KR, Klee JA. Rifaximin for the treatment of hepatic encephalopathy. Pharmacotherapy 2008; 28: Leevy CB, Phillips JA. Hospitalizations during the use of rifaximin versus lactulose for the treatment of hepatic encephalopathy. Dig Dis Sci 2007; 52: Pedretti G, Calzetti C, Missale G, Fiaccadori F. Rifaximin versus neomycin on hyperammoniemia in chronic portal systemic encephalopathy of cirrhotics. A double-blind, randomized trial. Ital J Gastroenterol 1991; 23: Bass NM, Mullen KD, Sanyal A, Poordad F, Neff G, Leevy CB, Sigal S, Sheikh MY, Beavers K, Frederick T, Teperman L, Hillebrand D, Huang S, Merchant K, Shaw A, Bortey E, Forbes WP. Rifaximin treatment in hepatic encephalopathy. N Engl J Med 2010; 362: Poordad F, Bass N, Sanyal AJ, Sheikh MY, Mullen KD, Sigal S, Frederick T, Brown RS, Joshi S, Merchant K, Huang S, Shaw AL, Bortey E, Forbes WP. The protective effect of rifaximin (1100 mg daily) from hepatic encephalopathy observed in a double-blind placebo controlled study is substantiated and durable over the long term. Hepatology 2009; 50: 448A-449A 64 Sanyal A, Younossi ZM, Bass NM, Mullen KD, Poordad F, Brown RS, Vemuru RP, Mazen Jamal M, Huang S, Merchant K, Bortey E, Forbes WP. Randomised clinical trial: rifaximin improves HRQL in cirrhotic patients with hepatic encephalopathy a double-blind placebo-controlled study. Aliment Pharmacol Ther 2011; 34: Sidhu SS, Goyal O, Mishra BP, Sood A, Chhina RS, Soni RK. Rifaximin improves psychometric performance and health-related quality of life in patients with minimal hepatic encephalopathy (the RIME Trial). Am J Gastroenterol 2011; 106: Bajaj JS, Heuman DM, Wade JB, Gibson DP, Saeian K, Wegelin JA, Hafeezullah M, Bell DE, Sterling RK, Stravitz RT, Fuchs M, Luketic V, Sanyal AJ. Rifaximin improves driving simulator performance in a randomized trial of patients with minimal hepatic encephalopathy. Gastroenterology 2011; 140: e1 67 Bajaj JS, Saeian K, Schubert CM, Hafeezullah M, Franco J, Varma RR, Gibson DP, Hoffmann RG, Stravitz RT, Heuman DM, Sterling RK, Shiffman M, Topaz A, Boyett S, Bell D, Sanyal AJ. Minimal hepatic encephalopathy is associated with motor vehicle crashes: the reality beyond the driving test. Hepatology 2009; 50: Bajaj JS, Pinkerton SD, Sanyal AJ, Heuman DM. Diagnosis and treatment of minimal hepatic encephalopathy to prevent motor vehicle accidents: a cost-effectiveness analysis. Hepatology 2012; 55: S-Editor Shi ZF L-Editor A E-Editor Xiong L 6700 December 14, 2012 Volume 18 Issue 46

22 Online Submissions: doi: /wjg.v18.i World J Gastroenterol 2012 December 14; 18(46): ISSN (print) ISSN (online) 2012 Baishideng. All rights reserved. FIELD OF VISION Glycogenotic hepatocellular carcinoma with glycogen-groundglass hepatocytes: A heuristically highly relevant phenotype Peter Bannasch Peter Bannasch, German Cancer Research Center, Im Neuenheimer Feld 280, D Heidelberg, Germany Author contributions: Bannasch P contributed to this paper. Correspondence to: Dr. Peter Bannasch, Professor, German Cancer Research Center, Im Neuenheimer Feld 280, D Heidelberg, Germany. [email protected] Telephone: Fax: Received: August 9, 2012 Revised: September 21, 2012 Accepted: November 14, 2012 Published online: December 14, 2012 Abstract Glycogenotic hepatocellular carcinoma (HCC) with glycogen-ground-glass hepatocytes has recently been described as an allegedly novel variant of HCC, but neither the historical background nor the heuristic relevance of this observation were put in perspective. In the present contribution, the most important findings in animal models and human beings related to the emergence and further evolution of excessively glycogen storing (glycogenotic) hepatocytes with and without ground glass features during neoplastic development have been summarized. Glycogenotic HCCs with glycogen-ground-glass hepatocytes represent highly differentiated neoplasms which contain subpopulations of cells phenotypically resembling those of certain types of preneoplastic hepatic foci and benign hepatocellular neoplasms. It is questionable whether the occurrence of glycogen-ground-glass hepatocytes in a glycogenotic HCC justifies its classification as a specific entity. The typical appearance of ground-glass hepatocytes is due to a hypertrophy of the smooth endoplasmic reticulum, which is usually associated with an excessive storage of glycogen and frequently also with an expression of the hepatitis B surface antigen. Sequential studies in animal models and observations in humans indicate that glycogen-ground-glass hepatocytes are a facultative, integral part of a characteristic cellular sequence commencing with focal hepatic glycogenosis potentially progressing to benign and malignant neoplasms. During this process highly differentiated glycogenotic cells including ground-glass hepatocytes are gradually transformed via various intermediate stages into poorly differentiated glycogen-poor, basophilic (ribosome-rich) cancer cells. Histochemical, microbiochemical, and molecular biochemical studies on focal hepatic glycogenosis and advanced preneoplastic and neoplastic lesions in tissue sections and laser-dissected specimens in rat and mouse models have provided compelling evidence for an early insulinomimetic effect of oncogenic agents, which is followed by a fundamental metabolic switch from gluconeogenesis towards the pentose-phosphate pathway and the Warburg type of glycolysis during progression from preneoplastic hepatic glycogenosis to the highly proliferative malignant phenotype Baishideng. All rights reserved. Key words: Acquired focal hepatic glycogenosis; Inborn hepatic glycogenosis; Hepatic preneoplasia; Hepatic neoplasia; Early metabolic aberrations; Progressionlinked metabolic switch Peer reviewers: Markus Peck-Radosavljevic, Professor, Department of Internal Medicine III, Division of Gastroenterology and Hepatology, University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria; Dr. Maria Cristina Carrillo, Instituto de Fisiologia Experimental, Departamento de Ciencias Fisiologicas, Suipacha 570, Rosario 2000, Argentina Bannasch P. Glycogenotic hepatocellular carcinoma with glycogen-ground-glass hepatocytes: A heuristically highly relevant phenotype. World J Gastroenterol 2012; 18(46): Available from: URL: v18/i46/6701.htm DOI: i INVITED COMMENTARY ON HOT ARTICLES The glycogenotic hepatocellular carcinoma (glycogenotic 6701 December 14, 2012 Volume 18 Issue 46

23 Bannasch P. Glycogenotic HCC with glycogen-ground-glass hepatocytes HCC) with glycogen-ground-glass hepatocytes has recently been described as a novel variant of HCC by Callea et al [1]. Relating the excessive storage of glycogen (glycogenosis) to a complete absence of glucose-6- phosphatase activity as measured by a microbiochemical approach, the authors referred to the long known finding of a focal hepatic glycogenosis (FHG) as an early event in experimental hepatocarcinogenesis [2], continuing however, ground-glass cells have not been reported in FHG. In contrast to this statement, the characteristic hepatocellular phenotype addressed was not only explicitly described for the first time in preneoplastic and neoplastic lesions in rodents [3,4] and in humans [5,6] (Figure 1) decades ago but has also been proven to be heuristically highly relevant in numerous publications since then. Glycogenotic ground-glass hepatocytes (GGH) predominate in subpopulations of many focal precancerous hepatocellular lesions, particularly in preneoplastic FHG and benign hepatocellular neoplasms such as hepatocellular adenomas and focal nodular hyperplasia, but may also occur in more or less extended subpopulations of HCC as observed in various species, including non-human primates and human beings [7,8]. However, glycogen-ggh hardly ever account for whole neoplasms. This also applies to the glycogenotic HCC depicted by Callea et al [1] in which the GGH are mixed with clear (glycogenotic) cells without ground-glass features. It is, hence, questionable whether glycogenotic HCC with glycogen- GGH should be considered a specific entity as proposed by Callea et al [1]. Extensive investigations in models of chemical, viral, and hormonal hepatocarcinogenesis and some observations in humans suggest that FHG with and without GGH indicates a critical early metabolic aberration in the pathogenesis of benign and malignant hepatocellular neoplasms [7,8]. Animal models of hepatocarcinogenesis The observations in humans were preceded by several seminal findings in animal models of hepatocarcinogenesis as repeatedly reviewed [2,9]. In their pioneering electron microscopic investigations in rats continuously exposed to 3-methyl-dimethylaminoazobenzene, Porter et al [10] detected a hypertrophy of the smooth endoplasmic reticulum in many hepatocytes, and addressed its light microscopic counterpart as hyaline degeneration [11]. The authors related this characteristic subcellular change to a decreased rather than an increased storage of glycogen and felt that only cells which, through mutation, loose the normal tendency to differentiate for glycogenesis will survive and so will be selected out for continued growth and differentiation [10]. However, investigations in other models of rat hepatocarcinogenesis, employing both continuous or limited (stop model) exposure to N-nitrosomorpholine at various dose levels and time schedules [3,4], or ethionine for up to approximately 20 wk [12] revealed that hypertrophy of the smooth endoplasmic reticulum is often associated with an excessive storage of glycogen, the smooth membranes forming either a typical network or peculiar lamellar complexes which often A B C Figure 1 Light micrographs of portions from human hepatocellular neoplasms with and without glycogenosis. A: Clear-cell hepatocellular adenoma consisting predominantly of glycogenotic cells. In some cells (arrows) there is a reduction of glycogen and focal increase in cytoplasmic basophilia; B: Highly differentiated hepatocellular carcinoma (HCC) composed of a mixed population of clear (glycogenotic) cells, acidophilic cells (ground-glass hepatocytes, arrows), and some glycogen-poor, basophilic cells; C: Poorly differentiated, glycogen-free, basophilic HCC. All: Hematoxylin and eosin stain, x 460, from Bannasch et al [6]. show a close spatial relationship with glycogen particles (Figure 2) but may also be free of glycogen forming fingerprints [4]. Steiner et al [12] designated the complexes of the smooth endoplasmic reticulum associated with glycogen as glycogen-bodies, and speculated that these formations indicate a resistance to the carcinogen, reflecting a reactivation of the glycogen-storing ability after an early loss of glycogen in response to toxicity. In contrast to both of these considerations, many studies on experimental hepatocarcinogenesis in different species revealed that FHG composed of glycogenotic clear and/or acidophilic cells, the latter showing a pronounced hypertrophy of the smooth endoplasmic reticulum (corresponding to glycogenotic GGH), regularly occur in a multi-centric fashion in early stages of neoplastic development induced in small rodents by a variety of chemicals [3,4,9,13]. More recently, typical glycogenotic GGH were also found after chronic infection of woodchucks with hepadnaviridae [14-16], in hepatitis B virus (HBV)-transgenic mice [17], and during hormonal hepatocarcinogenesis in 6702 December 14, 2012 Volume 18 Issue 46

24 Bannasch P. Glycogenotic HCC with glycogen-ground-glass hepatocytes RER N P M Figure 2 Portion of a glycogenotic acidophilic hepatocyte (corresponding to glycogen-ground-glass hepatocyte) induced in rat liver by N-nitrosomorpholine. Note abundant a- and b-glycogen particles (G) in close spatial relationship with large network complexes of proliferated smooth endoplasmic reticulum (SER). Rough endoplasmic reticulum (RER), mitochondria (M), peroxisomes (P), and nucleus (N). Inset: Group of acidophilic hepatocytes as seen under the light microscope. Transmission electron microscopy, lead citrate. Bar: 1 mm. diabetic animals after intrahepatic transplantation of pancreatic islets in rats and mice [18,19]. As demonstrated particularly in stop models of hepatocarcinogenesis in rats exposed to N-nitrosomorpholine or thioacetamide, glycogenotic hepatocytes with and without hypertrophy of the smooth endoplasmic reticulum may persist for weeks and months after withdrawal of the carcinogen, and may persist as almost entire populations of hepatocellular adenomas ( hyperplastic nodules, neoplastic nodules ) and in subpopulations of highly differentiated HCCs [4,20,21]. These observations are neither compatible with the idea of a preferential cellular survival by loss of differentiation for glycogenesis [10] nor with the notion of a resistance of these cells to carcinogen [12,22,23]. The functional significance of the persistent hypertrophy of the smooth endoplasmic reticulum has remained obscure, but many findings suggest that the organelle represents a metabolic compartment whose increase is a facultative consequence of the disturbed carbohydrate metabolism characterizing the acquired hepatocellular glycogenosis [4,24]. Independent of the observation of an acquired FHG produced in rat liver by N-nitrosomorpholine [5], Gössner et al [25] described a focal reduction in the activity of glucose-6-phosphatase in rats exposed to N-nitrosodiethylamine. A causal relationship between this enzyme deficiency and the accumulation of glycogen in preneoplastic FHG and hyperplastic liver nodules has been suggested by a number of authors [3,4,26,27]. This conclusion G G SER appears to be supported by the well known high risk of children suffering from an inborn hepatic glycogenosis type Ⅰ, especially type Ⅰa (von Gierke), due to a genetically fixed deficiency of the glucose-6-phosphatase, to develop hepatocellular adenomas and carcinomas when passing through adolescence [2,9,28]. This interpretation is in line with the recent finding that the targeted deletion of liver glucose-6-phosphatase in a knock-out mouse model results in hepatic glycogenosis and steatosis, and eventually also in multiple hepatocellular adenomas in all animals beyond 18 mo of age [29]. It is important to realize, however, that correlative cytochemical studies in rodent models of hepatocarcinogenesis have shown that the focal decrease of glucose- 6-phosphatase activity in FHG is regularly combined with decrease or increase in the activity of many other enzymes [7,8,30], especially enzymes of the carbohydrate metabolism [2,24,31]. In addition, over-expression of the key enzyme of de novo fatty acid synthase has been described in FHG in the N-nitrosomorpholine stop-model [32]. In rats exposed to N-2-fluorenylacetamide, Williams et al [33] demonstrated that the preneoplastic glycogenotic clear cell foci are resistant to the storage of iron. For a long time the cause of these complex metabolic alterations in FHG remained elusive. More recently, however, histochemical, microbiochemical and molecular biochemical studies on FHG and advanced preneoplastic and neoplastic liver lesions in tissue sections and laser-dissected specimens obtained from small rodents exposed to chemical carcinogens or oncogenic viruses provided evidence for an early insulin-like (insulinomimetic) effect of these agents [7,24,34-36]. This notion has been corroborated by a number of studies on hormonal hepatocarcinogenesis induced in diabetic rats and mice by local hyperinsulinemia [18,19,32,37-39]. The phenotype of preneoplastic FHG is not stable, but undergoes dramatic changes during progression to the benign and/or malignant neoplastic phenotype [3,4]. Collectively, all types of specific focal hepatocellular lesions appearing during the preneoplastic phase in rodents have been termed foci of altered hepatocytes, and have been widely used as early indicators of neoplastic development in toxicologic pathology [40-42]. The characteristic sequence of cellular changes starting with FHG follows an ordered pattern, passing through intermediate or mixed cell foci composed of glycogenotic, intermediate, and glycogen-poor basophilic (ribosomerich) cell types, the latter corresponding to the typical cell type in poorly differentiated HCCs [2,4,24]. Detailed morphological analysis of intermediate cell types at the light- and electron microscopic level has suggested that the hypertrophied smooth endoplasmic reticulum is usually transformed into rough endoplasmic reticulum by the addition of ribosomes during this phenotypic conversion [4,9,20]. Frequently, the intermediate cells show an accumulation of neutral fat, often leading to a combination of glycogenosis and steatosis [2,20]. Evidence of this sequence of cellular changes was originally provided by light- and electron-microscopic studies in rats 6703 December 14, 2012 Volume 18 Issue 46

25 Bannasch P. Glycogenotic HCC with glycogen-ground-glass hepatocytes A C E Figure 3 Human focal (A, B) and nodular (C, D) hepatic glycogenosis, and more advanced mixed cell populations (E, F) with intrafocal small cell change. A, B: Hepatic vein. Perivenular glycogenolytic (clear cell) focus in an hepatocellular carcinoma (HCC)-bearing liver with hepatitis B virus (HBV)- associated cirrhosis, demonstrated in serial sections with hematoxylin and eosin (A) and periodic acid schiff (PAS) (B)-reaction counterstained with orange G and iron hematoxilin (Tri-PAS). Bar: 100 mm; C, D: Glycogenotic (clear cell) nodule in an HCC-free liver with HBV-associated cirrhosis demonstrated in serial sections with hematoxylin-eosin (C) and Tri-PAS (D). Bar: 100 mm; E, F: Hematoxylin and eosin (E), proliferating cell nuclear antigen (F) immunostaining. Increased cell proliferation (arrows) in a mixed cell focus with less pronounced glycogen storage (not shown) and with intrafocal small cell change in liver with cryptogenic cirrhosis, demonstrated in serial sections. Bar: 50 mm, from Su et al [68]. B D F exposed for the lifetime or for limited time periods to N-nitrosomorpholine [3,4]. And it has since been substantiated by a series of morphometric studies [43-45] and by similar observations in other rodent models of hepatocarcinogenesis elicited by several genotoxic and nongenotoxic chemicals [7,41], by local hyperinsulinemia [18], by hepadnaviridae [14,15], and by oncogenic transgenes [17,46]. Most recently, multiple FHG, more advanced types of foci of altered hepatocytes, hepatocellular adenomas and HCC indicative of such a sequence, including an intermediate steatosis, were also observed in a knock-out mouse model with a reduced expression of the mitochondrial protein frataxin, which is responsible for the inherited neurodegenerative disease Friedreich s ataxia in humans [47]. The conversion of the highly differentiated glycogenotic clear or acidophilic to the de-differentiated glycogen-poor, basophilic (ribosome-rich) phenotype is associated with a fundamental metabolic switch characterized by a reduction in gluconeogenesis, an activation of the pentose phosphate pathway and the Warburg type of glycolysis as detailed elsewhere [7,8,24], and by an ever increasing cell proliferation which is inversely related to the gradual reduction of the glycogen initially stored in excess [48]. Based on these observations, Kopp-Schneider et al [49] developed the so-called color-shift model of hepatocarcinogenesis considering epigenetic changes in parenchymal colonies rather than multiple successive genomic mutations in single cells as the main cause of neoplastic cell conversion induced in the liver by exogenous oncogenic agents. The importance of epigenetic events in chemical hepatocarcinogenesis has been discussed by several authors previously [7,50], and has been emphasized in recent years by Pogribny et al [51,52]. Human hepatocarcinogenesis In human pathology, the predominance of clear (glycogenotic) cells (Figure 1A and B) in a minor proportion of HCCs [6,53], comprising about 8% in 150 cases studied by Buchanan et al [54], and in many hepatocellular adenomas [6,53-56] is well known. A favorable prognosis of the clear-cell variant of HCC has been reported [57]. Sasaki et al [58] described two cases of clear-cell HCC associated with hypoglycemia and hypercholesterolemia, and postulated a disturbed glucose metabolism of the tumor tissue, directed to lipogenesis and/or glucogenesis. Acquired FHG has been considered a preneoplastic condition in humans [6]. This idea was supported by the fortuitous observation of FHG (clear-cell foci) in HCC-bearing livers of children suffering from different disorders [59,60], in women after long-term use of oral contraceptives [61], in about 12% of 95 males studied in a consecutive autopsy series in Finland [62], in patients with Crohn s disease treated over years with azathioprine which is apparently also responsible for associated HCC development [63-67], and in a variety of other chronic liver diseases prone to develop HCC [8,24,53,68,69]. Special cases are patients with genetic hemochromatosis endowed with a high risk of developing HCC, which frequently show FHG excluding iron similar to the iron-resistant FHG observed in experimental hepatocarcinogenesis in rodents [8,53,69-73]. Particularly relevant are systematic histochemical and histological investigations on the phenotype and proliferation kinetics of foci and nodules of altered hepatocytes in more than 150 explanted and resected human livers with and without HCC [68,74,75]. The results suggest that foci of altered hepatocytes are proliferative preneoplastic lesions, mixed cell foci (frequently with small cell change ) being more advanced than FHG (Figure 3), potentially transforming into nodules of altered hepatocytes, highly differentiated HCC containing glycogenotic clear and ground-glass hepatocytes (Figure 4), and eventually also glycogen-poor, basophilic HCC (Figure 1C) [68]. In keeping with these findings, clear-cell change, steatosis and small cell change have been considered histological features predicting malignant transformation in nonmalignant hepatocellular nodules [76]. Analysis of clonality and chromosomal aberrations in nodules of altered hepatocytes microdissected from cirrhotic livers revealed 6704 December 14, 2012 Volume 18 Issue 46

26 Bannasch P. Glycogenotic HCC with glycogen-ground-glass hepatocytes N M P P M P Figure 4 Portion of a human trabecular hepatocellular carcinoma with mixed populations, including clear (glycogenotic) cells (small upper white circle), acidophilic (glycogenotic) ground-glass cells (somewhat larger white circle at the bottom, left), transitions from ground-glass hepatocytes into basophilic cell populations (largest white circle in the middle, right), and basophilic cell populations in the middle and at the bottom right (not marked by circles). Hematoxylin and eosin. a loss of chromosomal inactivation mosaicism in three large regenerative nodules and in all (12) nodules of altered hepatocytes with small cell change, indicating their neoplastic nature [77]. Even among 60 nodules of altered hepatocytes without small cell change, almost 50% (29) were shown to be monoclonal, whereas FHG and 14 regenerative nodules were found to be polyclonal. Interestingly, Cai et al [78] using a similar approach to analyze focal nodular hyperplasia, the pathogenesis and neoplastic nature of which has been debated for decades [53], found that this lesion, as a whole, is polyclonal, but represents a cluster of nodules of altered hepatocytes, some of which are monoclonal harboring chromosomal aberrations as in hepatocellular adenomas. The lack of genomic alterations in fatty and clear-cell changes in HCC and precursor nodular lesions in cirrhotic livers emphasized by some authors [79] is in line with the polyclonal nature of many of these lesions [77], but in view of the increasing evidence for a decisive role of epigenetic events in the development and progression of human HCC [80] the findings by Laurent et al [79] do not argue against a preneoplastic nature for these cellular changes. A pronounced hypertrophy of the smooth endoplasmic reticulum was discovered in biopsies from cirrhotic livers and liver cell carcinomas at the light and electron microscopic level (Figure 1B) almost half a century ago, and related to aberrations of glycogen metabolism including glycogenosis from the very beginning [5,6]. The altered hepatocytes (Figures 1B and 4) were designated as acidophilic (or eosinophilic ). Later on, Popper et al [81,82] found frequent association of this phenomenon with the expression of the hepatitis B surface antigen (HBsAg) localized in the lumen of hypertrophied smooth endoplasmic reticulum (Figure 5), and coined the term ground glass hepatocyte which has become an important diagnostic entity in chronic liver diseases elicited by HBV and has dominated the literature since Figure 5 Portion of a ground-glass hepatocyte from a human liver with hepatitis B virus-associated cirrhosis, showing an accumulation of glycogen granules (long arrow) in the nucleus (N) and abundant smooth endoplasmic reticulum containing filamentous hepatitis B surface antigen (short arrows). M: Mitochondria: P: Peroxisome. Transmission electron microscopy, lead citrate. Bar: 1 mm. then [83]. Wills [84] described HBsAg-free ground glasslike hepatocytes exhibiting glycogen bodies in liver biopsies from an immunosuppressed (azathioprine, prednisone, clonidine, frusemide) renal transplant patient, similar to those observed in experimental chemical hepatocarcinogenesis [4,12]. Irrespective of the expression of HBsAg, glycogenotic hepatocytes showing hypertrophy of the smooth endoplasmic reticulum (corresponding to GGH) have often been observed in human FHG, hepatocellular adenomas, and HCC and considered preneoplastic or highly differentiated neoplastic phenotypes [6,53,68]. In 30 specimens of HBV-associated cirrhosis, GGH were identified in 17 of 25 showing HBsAg expression [85] (Figure 5). Without mentioning any particular relationship to glycogen, others described GGH containing pre-s mutants in chronic HBV infection, postulating that they represent preneoplastic lesions [83]. Wisell et al [86] depicted partially persisting glycogen pseudoground glass hepatocytes in 12 patients immunosuppressed for numerous indications, but detected neither viral particles nor hypertrophy of the smooth endoplasmic reticulum under the electron microscope. Similar observations were reported by Bejarano et al [87] but no efforts were made in either of these studies to directly compare GGH in serial sections of defined focal lesions at the light and electron microscopic level. Different types of altered hepatocytes, which superficially resemble glycogenotic GGH appearing during hepatocarcinogensis, have also been observed in several other diseases but will not be further discussed in this context [83,86,87]. An intriguing form of acquired hepatic glycogenosis was discovered by Mauriac et al [88] in a child with poorly controlled insulin-dependent diabetes type 1. The excessive storage of glycogen resulted in hepatomegaly and was associated with growth retardation, delayed puberty, and a cuchingoid face (named after Harvey Williams Cushing). Many additional cases resembling Mauriac s 6705 December 14, 2012 Volume 18 Issue 46

27 Bannasch P. Glycogenotic HCC with glycogen-ground-glass hepatocytes syndrome, especially with respect to hepatic glycogenosis, have subsequently been described [89-91]. Torbenson et al [91] emphasized that this glycogenic hepatopathy is an underrecognized complication of diabetes mellitus. In addition to children with insulin-dependent diabetes, hepatomegaly due to glycogen storage has also been recognized in adults afflicted by non-insulin-dependent diabetes type 2 with poor glycemic control [91-93]. To the best of my knowledge neither GGH nor a relationship of glycogenic hepatopathy to the evolution of HCC in patients suffering from diabetes mellitus has hitherto been described, but it might be timely to take a closer look into this possibility. The high risk of diffuse glycogenosis characterizing inborn hepatic glycogen storage diseases, particularly glycogen storage disease type Ⅰ due to glucose- 6-phosphatase deficiency, developing into hepatocellular adenomas potentially progressing to HCC has been well established [2,9,28,94] since the first description of a case by Mason et al [95]. In the meantime, hepatocellular neoplasms are now known to also be occasionally found in other types of glycogen storage disease, namely glycogenoses type Ⅲ (amylo-1,6-glucosidase deficiency), type Ⅳ (α-1,4-glucan:α-1,4-glucan-6-glycosyl transferase deficiency) and type Ⅵ (phosphorylase deficiency, Hers disease) [94,96-99]. 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30 Online Submissions: doi: /wjg.v18.i World J Gastroenterol 2012 December 14; 18(46): ISSN (print) ISSN (online) 2012 Baishideng. All rights reserved. Targeting late SV40 factor: Is the achilles heel of hepatocarcinogenesis revealed? FIELD OF VISION Amir Shlomai Amir Shlomai, Institute of Gastroenterology and Liver Diseases, Tel-Aviv Sourasky Medical Center and Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv 64239, Israel Author contributions: Shlomai A collected the material and wrote the manuscript Correspondence to: Dr. Amir Shlomai, MD, PhD, Institute of Gastroenterology and Liver Diseases, Tel-Aviv Sourasky Medical Center and the Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv 64239, Israel. [email protected] Telephone: Fax: Received: May 7, 2012 Revised: May 29, 2012 Accepted: June 7, 2012 Published online: December 14, 2012 Abstract Hepatocellular carcinoma (HCC) is a dreadful cancer and a major cause of death among patients with chronic liver disease and cirrhosis. The apparent alterations in a diversity of intracellular pathways found in HCC has set the rational for developing moleculardirected drugs that simultaneously inhibit multiple pathways, such as the multi-kinase inhibitor Sorafenib. However, recently this concept has been challenged by showing that HCC is heavily dependent on a single oncogene designated late SV-40 factor (LSF), a transcription factor that is over-expressed in liver cancer cells and that its expression is strongly correlated with tumor grade and aggressiveness. Furthermore, using an intensive screening for drugs that inhibit LSF activity, Grant et al have found a molecule designated factor quinolinone inhibitor 1 that can specifically block the ability of LSF to bind its target promoters, resulting in a massive death of HCC cells both in vitro and in vivo. The innovative findings of HCC representing oncogene addiction to LSF and the ability of a single molecule to block the activity of this oncogene resulting in tumor abolishment are encouraging and provide us with the hope that the Achilles heel of HCC has been found Baishideng. All rights reserved. Key words: Oncogene addiction; Hepatocellular carcinoma; Late SV40 factor; Transcription factor; Multikinase inhibit Peer reviewers: Mara Massimi, PhD, Department of Basic and Applied Biology, University of L Aquila, Via Vetoio-Coppito, L Aquila, Italy; Dr. Matthias Ocker, MD, Professor, Institute for Surgical Research, Philipps-University Marburg, Baldingerstrasse, Marburg, Germany Shlomai A. Targeting Late SV40 factor: Is the achilles heel of hepatocarcinogenesis revealed? World J Gastroenterol 2012; 18(46): Available from: URL: com/ /full/v18/i46/6709.htm DOI: org/ /wjg.v18.i INTRODUCTION Hepatocellular carcinoma (HCC) is the fifth most common cancer in men and the seventh in women, and its incidence has been sharply rising during the last two decades [1]. Whereas patients with localized disease can benefit from curative therapeutics modalities, such as liver resection, liver transplantation or radiofrequency ablation, until recently little could be offered to patients with advanced disease, whose survival was often measured in a few months [2]. MULTIPLE PATHWAYS ARE DISTORTED IN HEPATOCELLULAR CARCINOMA Disruption of a variety of cellular pathways, as well as mutations in tumor suppressors and oncogenes have been described in patients with HCC [3] ; mutations in the genes encoding for TP53 and β-catenin, amplifications of the vascular endothelial growth factor (VEGF) and Cyclin D1 (CCDN1) genes, silencing of E-Cadherin tumor suppressor and activation of the myelocytomatosis viral oncogene are a partial list. Indeed, the more effort 6709 December 14, 2012 Volume 18 Issue 46

31 Shlomai A. Targeting LSF in hepatocellular carcinoma invested in elucidating molecular mechanisms underlying HCC, the clearer it became that the complexity and diversity of such mechanisms make the development of potential gene-targeted drugs a highly complicated task. SORAFENIB-THE FIRST APPROVED MOLECULAR-DIRECTED DRUG FOR HEPATOCELLULAR CARCINOMA In 2008, Llovet et al [4] have published the results of a phase 3 multi-center placebo control trial showing that the multi-kinase inhibitor Sorafenib prolongs survival in patients with advanced HCC. The rational for using a multi-kinase inhibitor that simultaneously blocks Raf, VEGF, platelet-derived growth factor and c-kit signaling is to cover as much diverse signaling pathways involved in hepatic carcinogenesis as possible. However, although the introduction of this first molecular-directed drug is a major breakthrough in the field of HCC, the results are still far from optimal as reflected by the only modest improvement in life expectancy of 2.8 mo on average in the price of often-severe adverse events [5,6]. LATE SV40 FACTOR-AN ESSENTIAL ONCOGENE IN HEPATOCELLULAR CARCINOMA Recently, Yoo et al [7] has recognized the Late SV40 factor (LSF) transcription factor as a central oncogene in HCC. LSF, which is induced in the liver via inflammatory cytokines, has a major role in DNA synthesis by transcriptionally activating the rate-limiting enzyme in pyramidine synthesis, thymidylate synthase, as well as other target genes important for DNA synthesis and cell survival. LSF inhibition results in constraining DNA synthesis, which ultimately leads to cell death. Interestingly, Sarker s group has found that LSF protein is significantly overexpressed in HCC cells as compared to normal human liver cells. Furthermore, the degree of LSF expression revealed a significant correlation with the stage and grade of the tumor. Most importantly, whereas over expression of LSF pushed the tumor towards a more aggressive phenotype, LSF inhibition resulted in abrogation of tumor growth and its metastatic potential, both in-vitro and in-vivo. Interestingly, among the various genes induced by LSF following its binding to their promoters, SPP1 gene encoding for the osteopontin (OPN) protein has been found to be induces to the greatest extant. Indeed, the importance of OPN in promoting hepatic carcinogenesis was recently emphasized by a study showing that OPN is much more sensitive than the traditional alpha-fetoprotein as a marker for early HCC [8]. Whatsoever, Sarker s work as well as a later work by Fan et al [9] strongly indicated that LSF is an essential oncoprotein required for the maintenance and propagation of liver cancer, making it a potentially ideal target for HCC treatment. Furthermore, the merit of these findings lies in the fact that although multiple genes and signaling pathways are impaired in HCC, the cancerous liver cells are heavily dependent on a single oncogenic protein, the transcription factor LSF, for their survival. This phenomenon, designated oncogene addiction, has been recognized in various cancers in the last few years, making the oncogene to which a particular cancer is addicted to an ideal target for anti-cancer therapy [10,11]. FACTOR QUINOLINONE INHIBITOR 1 SPECIFICALLY INHIBITS LATE SV40 FACTOR ACTIVITY RESULTING IN ABOLISHMENT OF HEPATOCELLULAR CARCINOMA However, how can one translate experimental abolishment of LSF achieved mainly by dominant negative constructs or knockdown strategies to a drug that inhibits LSF function in-vivo and that can be easily delivered to the liver? A study recently published in the PNAS provided an unexpected and exciting solution to this problem [12]. By screening for small molecules that could block the interaction of LSF to its DNA binding sites along the genome, Grant et al [12] have revealed a small molecule named factor quinolinone inhibitor 1 (FQI1) that inhibits LSF DNA binding activity both in-vitro and in-vivo. Functionally, treating HCC cells with FQI1 results in a massive apoptosis of HCC cells whereas normal hepatocytes remain intact. FQI1 treatment results in a robust activity in-vivo, as well, reflected by inhibition of tumor growth in mouse HCC xenografts. Importantly, no toxicity was observed in FQI1 treated animals, as evaluated by animals general well being and by careful examination of various non-hepatic tissues that remained intact following treatment. Noteworthy is the observation that tumors from FQI1-treated animals expressed LSF at similar levels to those of control mice, whereas the expression of a central LSF target gene, OPN, as well the proliferative activity of the tumor were dramatically reduced. This observation reflects the inhibition of LSF activity as a transcription factor by blocking its binding to DNA, rather than reducing its level following FQI1 treatment. Furthermore, the close correlation between the concentrations of FQI1 required for inhibition of LSF transactivation and those required for proliferation inhibition strongly suggest that FQI1 specifically targets LSF and does not share a general non-specific anti proliferative activity. FQI1 AS AN EMERGING ANTI HEPATOCELLULAR CARCINOMA DRUG- PROMISES AND CHALLENGES The originality and the importance of the aforementioned studies are dual. First, the identification of a single oncogene serving as a cellular transcription factor to 6710 December 14, 2012 Volume 18 Issue 46

32 Shlomai A. Targeting LSF in hepatocellular carcinoma which HCC is addicted and completely dependent on. This finding may completely change the current concept of using drugs inhibiting multiple alternated cellular targets [5], to a strategy that specifically inhibits a particular target that is essential for HCC maintenance and propagation. Second, in contrast to what was formally considered as an almost impossible target for drug therapy, the efficiency of FQI1 strongly indicates that targeting the activity rather than the level of a transcription factor is an effective and specific mechanism for an anti-cancer drug. Further studies should address potential caveats and open question remaining before implementing those findings to an efficient anti-hcc drug. The bioavailability of FQI1 in human subjects following treatment should be carefully checked and the long-term consequences in terms of adverse effects should be monitored. The potential for tumor resistance due to mutations in the LSF DNA binding domain is certainly there and should be taken into consideration. In this regard, the combination of the current molecular-targeted drug, Sorafenib, with LSF inhibitors has the potential to minimize the risk of cancer cells escaping their oncogene addiction to LSF. Last but not least, the validity of LSF role in HCC should be ascertained for the various etiologies of HCC, including viral, metabolic and toxic. In summary, the introduction of a small molecule that specifically inhibits the activity of an oncogene on which HCC heavily depends seems as stimulating news to the field. Time will tell whether, similar to the Greek mythology, LSF represents the Achilles heel of HCC, an innovation that can ultimately lead to defeating this deadly cancer. REFERENCES 1 El-Serag HB. Hepatocellular carcinoma. N Engl J Med 2011; 365: Forner A, Llovet JM, Bruix J. Hepatocellular carcinoma. Lancet 2012; 379: Farazi PA, DePinho RA. Hepatocellular carcinoma pathogenesis: from genes to environment. Nat Rev Cancer 2006; 6: Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, de Oliveira AC, Santoro A, Raoul JL, Forner A, Schwartz M, Porta C, Zeuzem S, Bolondi L, Greten TF, Galle PR, Seitz JF, Borbath I, Häussinger D, Giannaris T, Shan M, Moscovici M, Voliotis D, Bruix J. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 2008; 359: Chan SL, Yeo W. Targeted therapy of hepatocellular carcinoma: present and future. J Gastroenterol Hepatol 2012; 27: Song T, Zhang W, Wu Q, Kong D, Ma W. A single center experience of sorafenib in advanced hepatocellular carcinoma patients: evaluation of prognostic factors. Eur J Gastroenterol Hepatol 2011; 23: Yoo BK, Emdad L, Gredler R, Fuller C, Dumur CI, Jones KH, Jackson-Cook C, Su ZZ, Chen D, Saxena UH, Hansen U, Fisher PB, Sarkar D. Transcription factor Late SV40 Factor (LSF) functions as an oncogene in hepatocellular carcinoma. Proc Natl Acad Sci USA 2010; 107: Shang S, Plymoth A, Ge S, Feng Z, Rosen HR, Sangrajrang S, Hainaut P, Marrero JA, Beretta L. Identification of osteopontin as a novel marker for early hepatocellular carcinoma. Hepatology 2012; 55: Fan RH, Li J, Wu N, Chen PS. Late SV40 factor: a key mediator of Notch signaling in human hepatocarcinogenesis. World J Gastroenterol 2011; 17: Weinstein IB. Cancer. Addiction to oncogenes--the Achilles heal of cancer. Science 2002; 297: Weinstein IB, Joe AK. Mechanisms of disease: Oncogene addiction--a rationale for molecular targeting in cancer therapy. Nat Clin Pract Oncol 2006; 3: Grant TJ, Bishop JA, Christadore LM, Barot G, Chin HG, Woodson S, Kavouris J, Siddiq A, Gredler R, Shen XN, Sherman J, Meehan T, Fitzgerald K, Pradhan S, Briggs LA, Andrews WH, Sarkar D, Schaus SE, Hansen U. Antiproliferative small-molecule inhibitors of transcription factor LSF reveal oncogene addiction to LSF in hepatocellular carcinoma. Proc Natl Acad Sci USA 2012; 109: S- Editor Shi ZF L- Editor Cant MR E- Editor Xiong L 6711 December 14, 2012 Volume 18 Issue 46

33 Online Submissions: doi: /wjg.v18.i World J Gastroenterol 2012 December 14; 18(46): ISSN (print) ISSN (online) 2012 Baishideng. All rights reserved. TOPIC HIGHLIGHT Marco Giuseppe Patti, MD, Professor, Series Editor Thinking in three's: Changing surgical patient safety practices in the complex modern operating room Verna C Gibbs Verna C Gibbs, Department of Surgery, San Francisco Veterans Affairs Medical Center, San Francisco, CA 94121, United States Author contributions: Gibbs VC designed project, analyzed data and wrote the paper. Correspondence to: Verna C Gibbs, MD, Department of Surgery, San Francisco Veterans Affairs Medical Center, SFVAMC (112) 4150 Clement Str., San Francisco, CA 94121, United States. [email protected] Telephone: Fax: Received: April 17, 2012 Revised: September 23, 2012 Accepted: September 29, 2012 Published online: December 14, 2012 Abstract The three surgical patient safety events, wrong site surgery, retained surgical items (RSI) and surgical fires are rare occurrences and thus their effects on the complex modern operating room (OR) are difficult to study. The likelihood of occurrence and the magnitude of risk for each of these surgical safety events are undefined. Many providers may never have a personal experience with one of these events and training and education on these topics are sparse. These circumstances lead to faulty thinking that a provider won t ever have an event or if one does occur the provider will intuitively know what to do. Surgeons are not preoccupied with failure and tend to usually consider good outcomes, which leads them to ignore or diminish the importance of implementing and following simple safety practices. These circumstances contribute to the persistent low level occurrence of these three events and to the difficulty in generating sufficient interest to resource solutions. Individual facilities rarely have the time or talent to understand these events and develop lasting solutions. More often than not, even the most well meaning internal review results in a new line to a policy and some rigorous enforcement mandate. This approach routinely fails and is another reason why these problems are so persistent. Vigilance actions alone have been unsuccessful so hospitals now have to take a systematic approach to implementing safer processes and providing the resources for surgeons and other stakeholders to optimize the OR environment. This article discusses standardized processes of care for mitigation of injury or outright prevention of wrong site surgery, RSI and surgical fires in an action-oriented framework illustrating the strategic elements important in each event and focusing on the responsibilities for each of the three major OR agents-anesthesiologists, surgeons and nurses. A Surgical Patient Safety Checklist is discussed that incorporates the necessary elements to bring these team members together and influence the emergence of a safer OR Baishideng. All rights reserved. Key words: Complex adaptive systems; Wrong site surgery; Retained surgical items; Retained foreign objects; Retained foreign bodies; Surgical patient safety; Surgical fires; Safety checklist Peer reviewer: Hon-Yi Shi, PhD, Associate Professor, Graduate Institute of Healthcare Administration, Kaohsiung Medical University, 100, Shih-Chuan 1st Road, San Ming District, Kaohsiung 807, Taiwan, China Gibbs VC. Thinking in three's: Changing surgical patient safety practices in the complex modern operating room. World J Gastroenterol 2012; 18(46): Available from: URL: DOI: INTRODUCTION Operating rooms (ORs) used to be just complicated places, but the modern OR has changed. No place epitomizes the complexity of health care delivery better than the OR where there is the routine interface of heterogeneous, variously trained personnel using high technology 6712 December 14, 2012 Volume 18 Issue 46

34 Gibbs VC. Safety in the modern operating room equipment while providing service to an unconscious, anesthetized patient. In fact it is most helpful to think of the modern OR as a complex adaptive system that consists of (1) heterogeneous interdependent decision making agents; (2) who interact frequently with each other; and (3) develop a characteristic called emergence which arises when the whole actually begins to perform better than the sum of its parts [1]. That is through the learning and interdependence of the heterogeneous agents a larger, higher functioning system is created. Anyone who has worked in a highly functioning OR over time can understand how this entity can evolve but also how rare and difficult it is to achieve. Today it is expected that the delivery of surgical care will be (1) knowledge-driven (safe, effective); (2) patientcentered; and (3) system-based (timely, efficient and equitable) [2]. Hospitals and the surgeons who operate within them are also learning that if the care is not of high value, that is high quality surgical outcomes at low total costs of care, there will be low or no reimbursement for their effort [3]. Thus begins another step in the journey to stimulate hospitals and surgeons to accelerate improvement in the quality of surgical care and increase efforts to prevent errors and treatment complications. Multiple stakeholders have begun to reengineer or design, adopt and implement new OR practices and refined communication methods in order to reduce errors and waste. The specific surgical patient safety events are uncommon but the effort needed for prevention requires innovative solutions and illustrate the difficulty in changing OR culture (e.g., shared customary beliefs, values and behaviors). The three main preventable surgical patient safety events which occur within the OR are (1) the surgical wrongs; (2) retained surgical items (RSI); and (3) surgical fires. The incidence of all three combined may affect at most 5000 patients a year (approximately 2000 wrong surgery events, 2000 RSI, 500 surgical fires) in the United States. National sentinel event disclosure is disparate and depends on state-based and regulatory agency voluntary reporting. Data on close-calls, which are likely to be more frequent, remain sketchy. No matter the total number of events, we do know that the number is greater than zero and therefore these are still not never events. It has been tempting to use a person approach (e.g., the forgetfulness, inattention, ignorance, carelessness of individuals) in the analysis of why these events occur but we know that a system approach (e.g., analysis of the conditions under which individuals work and how defenses failed) that incorporates human factors (e.g., the interaction of human abilities, expectations and limitations) while harder to design and implement is likely to be more successful [4]. These safety problems are reflective of the culture of safety in the OR rather than on patient or case characteristics. The people who work in the OR make it safe and it is important to know that there are subtle behaviors that aren t well quantified but are highly valued that arise from the development of expertise. Expertise is the result of knowledge and experience. The behaviors are intuitive and have been recognized as characteristics of safe people [5]. These feelings and intuitions are very important to nurture and develop but do have a quality of mystery to them but it is highly desirable for an organization to value expertise and develop safe people. When hospitals address these safety issues and there is culture change there may be positive consequences which decrease or prevent other more common perioperative occurrences such as surgical infections and venous thromboembolic events. ORs have not yet been characterized as highly reliable organizations (akin to nuclear reactors or aircraft carriers) so there is much room for improvement. The current projects developed to help hospitals take action to prevent wrong site surgery, RSI and surgical fires focus on specific OR practice and communication problems. Unsafe OR practices are usually highly variable and efforts to standardize multi-stakeholder processes of care and develop specific communication modes are shared core elements in prevention strategies. Practice change and communication improvements are often viewed disparagingly as soft approaches which lack rigorous data and experimental validation however making sense of the rare and unexpected requires different tools and approaches [6]. Increasingly, event reporting and analysis of close calls have informed failure modes and collective learning on how to manage unsafe acts which is just as important as preventing them. To change OR culture requires change in the practices of the people who are engaged in the work. To change their practices you have to change the relationships with whom they work and the environment in which they work. Finally, a surgical patient safety checklist which is a simple, widely applicable, inexpensive, unifying tool, can be used by all OR personnel to enforce the individual safe practices which have programmatically been developed and enhance inter-personnel communication [7]. We describe how one can be used to teach and enforce safer practices and address serious and avoidable surgical complications [8]. WRONG SURGERY The three types of wrong surgery are (1) wrong patient surgery, where the performance of an operation or procedure is carried out on a person other than the one for whom the procedure was intended; (2) wrong procedure surgery, is the performance of a procedure other than that intended or indicated for a specific patient; and (3) wrong site surgery where a planned procedure is performed at the wrong place, part, level, side or site [9]. Wrong site surgery is a type of wrong procedure surgery and is the most common occurrence of the three [10]. All three of these events are usually the result of mistakes in correctly applying identification procedures. The Joint Commission (TJC) has been at the fore December 14, 2012 Volume 18 Issue 46

35 Gibbs VC. Safety in the modern operating room front of the wrong site surgery issue since 1998 when the first Sentinel Event Alert newsletter on the subject was printed. Subsequently TJC published a follow-up alert in 2001, in 2003 the first Wrong-Site Surgery Summit was held and in 2004 the Universal Protocol was launched. In spite of all of these efforts, it has most recently been estimated that wrong site, wrong procedure and wrong patient cases still occur more than 40 times a week in the United States [11]. The essential elements of the Universal Protocol mandate that OR teams must (1) complete a preoperative verification process; (2) mark the operative site; and (3) take a time out immediately before starting the procedure. The verification process is to ensure that the correct patient has been consented and is present for the operation and all relevant documents and studies are available before the procedure is performed. The most important elements are that the three major primary operative sources: the history and physical, the consent, and the official OR schedule are in exact agreement. If there are discrepancies the patient is considered the primary source for rectification. Patient verification and identification practices are usually performed by nursing and anesthesia personnel with requirements that at least one of these stakeholders must remain in constant attendance with the patient while being brought into the OR once patient identification has been performed. The site marking is within the domain of the surgeon and must be performed before the patient is brought into the OR and should be visible while the patient is prepped and draped. The mark must be visible within the operative field and referenced during the time out. Recent alcohol based prep solutions frequently diminish the site mark and practices must be established to use different inks or have a process in place whereby the person who performed the prep can refresh the diminished mark with a sterile surgical marker to ensure that it will be able to be seen before an incision is made. The time out is truly a team based activity called by the surgeon, monitored and orchestrated by the circulating nurse with responses from anesthesia and any other providers. The time out is usually performed just before the incision is made and all team members have to stop all activities. At the time out the three questions are answered: (1) Who is this patient? (2) What operation is to be performed? and (3) Is this the correct site mark, using secondary confirmatory evidence present on an armband or on the operative consent and unanimity from all stakeholders is required. Many facilities have checklists and scripted procedures with hard stops (e.g., the scalpel isn t passed until the time out is completed) to enforce compliance. All questions are supposed to be asked in a non-intimidating environment where all staff feel equal in their contribution to safe patient care. These conditions have been challenging to meet. While the Universal Protocol as a directive was comprehensive and widely mandated the implementation and effectiveness of the protocol was variable [12]. Review of wrong site surgery cases reveal that multiple errors occurred along the OR journey and multiple defenses failed which might have caught the mistakes [5]. Inadequate standardization of practices and poor training and enforcement of practices provide the latent factors which exist that sets OR personnel up for failure. More than just the directive to use the Universal Protocol appears to be needed and hospitals need guidance on how to effectively make the process work. To meet this goal, in July 2009 TJC started an eight hospital demonstration project using a Robust Process Improvement method that is a fact based, systematic and data-driven problem solving methodology [13]. Project teams are developed for observational audits looking to discover specific risk points and contributing factors that lead to close calls and wrong site surgery events. The timeframe begins at the time of scheduling a surgical procedure and ends with confirmation that the intended operation was correctly performed. Preliminary results from TJC project have shown that in 39% of cases, errors were introduced in the verification step that increased the risk of a wrong site surgery event [11]. Usually these errors involved inadequate information about the patient and scheduling confusion. Identifying this failure mode lead to the development of standardized ways of collecting and having the information accessible. Site marking and time out practices have been contributory but were not as frequent a source of errors as the initial verification process [5]. Further refinements to improving this practice will be available at TJC Targeted Solutions Toolkit which will provide a step-bystep process to measure performance [13]. RETAINED SURGICAL ITEMS A RSI occurs when surgical material, usually a sponge, needle, instrument or miscellaneous small item or device fragment is inadvertently left inside a patient. Retained surgical item is the preferred term rather than retained foreign body or object since bullets, shrapnel or ingested objects can be retained but aren t the result of surgical error. An RSI is a surgical patient safety problem and results from problems in unreliable surgical OR practices and problems with communication between OR stakeholders [14]. Reports of RSI have relied on mandatory state and voluntary regulatory agency reporting requirements which vary as does the definition of what is considered retention. The National Quality Forum s (NQF) definition is most frequently referenced and in 2011 all NQF Serious Reportable Events were reviewed which included the issue of surgical retention. The new directive excludes reporting of unremovable items intentionally left at the judgment of a surgeon and most importantly addressed the definition of when an item is considered retained after surgery. Many states construed after surgery to mean closure of the incision, which meant even in cases where the sponge or surgical item was 6714 December 14, 2012 Volume 18 Issue 46

36 Gibbs VC. Safety in the modern operating room discovered to be missing while the patient was on the OR table, and was removed in the OR without delay or harm, if the wound had been closed these cases were reported as a RSI. With the new 2011 definition, this area has been corrected and the operation ends after all incisions or procedural access routes have been closed in their entirety, devices have been removed and if relevant, final surgical counts have concluded and the patient has been taken from the operating/procedure room [15]. In 2011 an item is considered to be retained only if found after the patient is out of the OR or procedure room. This change alone may lead to decreased numbers of RSI cases. The most common RSI is the cotton gauze surgical sponge which has been found in the abdomen/pelvis, chest and increasingly in the vagina. Retained sponges occur after any type of surgical procedure and the risk of retention is unrelated to the number of sponges used during an operation. Sponges have been retained when only 10 sponges were used and small biopsy or skin incision made. This finding has lead to policy changes which should require that surgical sponges must be accounted for in all cases in which surgical sponges are used and an incision is made [16]. There should be no determinates for case exclusion because of size of the incision or length of the case. Traditional means of accounting for sponges has relied on the longstanding practice of counting. Observational audits and focused reviews of cases of retained sponges has shown that the practice of counting sponges is highly variable between ORs and even within rooms in the same OR suite. This variation leads to sources of error and with increased demands of the OR environment, frequent change of shift between nursing personnel and multiple diversions the common practice of counting has proven unreliable. Examination of cases of retained sponges have often revealed that the sponges were counted during the procedure but no one knows where the error occurred or how the sponge was retained. In fact, overall about 80% of retained sponge cases occur in the setting of a correct count [17]. That is at the end of the case the counts were called correct. In about 20% of cases of retention there was a known incorrect count at the end of the case, something was missing, yet the patient still got out of the OR without finding the sponge. The missing item is subsequently discovered sometime later. This occurs because of knowledge and communication problems usually at the interface with radiology, the taking and interpretation of xrays or with inadequate follow-up at the next level of care to determine if a sponge has been retained. Because of these observations about the counting of sponges a revised manual sponge management practice was developed by the NoThing Left Behind Surgical Patient Safety Project [17]. The practice is called Sponge ACCOUNTing that uses the adjunct of plastic bluebacked hanging sponge holders and wall mounted dry erase boards posted in each OR on which to record in a standardized manner the surgical counts. Sponge ACCOUNTing was developed to standardize and improve the transparency of the practice of sponge management. The guiding principles have outlined three important steps to ensure that all sponges used in the procedure have been accounted for with important unique actions for each OR stakeholder during the case and a team based show me step at the debriefing or end of the case. The nursing essence is (1) that all sponges are only used in groups of 10 and each plastic sponge holder contains 10 pockets; (2) at the natural pause point which occurs at the time wound closure begins, it is the job of the surgeon to perform a methodical wound exam (MWE), not just a swish or sweep of the wound, but to do the best job possible to look for and remove any sponges (or other items not intended to remain in the patient) and give them to nursing staff; and (3) the last step is at the end of the case when the skin is closed and all of the sponges (the used and unused sponges) must be off the field and in the pockets of the plastic hanging sponge holders. Since there are 10 pockets per holder and sponges are managed only in multiples of ten there should be one holder for each 10 sponges and there should be no empty holder pockets at the end of the case. The team based activity is for the surgeon to say show me the holders so they can see that there are no empty pockets or for the nurse to say to the surgeon here let me show you, there are no empty pockets. Who shows who doesn t matter, it s that there is a visual confirmation that all sponges have been accounted for. There are now three technological adjuncts for sponge management (1) a device which counts 2D matrix labeled sponges; (2) a device which detects radiofrequency tagged sponges; and (3) devices which can count and detect RadioFrequency IDentification (RFID) chip embedded sponges. The essential components of each device are a distinct type of detection element attached to a surgical sponge and a distinct compatible electronic readout system. The computer assisted sponge counting system consists of two-dimensional matrix labeled sponges and a scanning device that can read the labels [18]. The matrix label is scanned in with a handheld or table mounted scanner as the sponges are put on the sterile field and then each sponge is scanned out when the sponges are removed from the table. The matrix labels are embedded onto surgical sponges of various sizes and each sponge has a unique identifier that enables the scanner to count different types of sponges. The sponges are counted maintaining line of sight for each sponge. In order to account for all sponges at the final count, the sponges must be removed from the patient and individually passed under the scanner. The scanner has no capacity to read-through the patient and detect the presence of a matrix labeled sponge. In the event of a missing sponge an X-ray is used to determine if it is in the patient. This system is in place in many hospitals and most recently the experience from the Mayo Clinic has been reported [19]. After 18 mo of use the device accurately 6715 December 14, 2012 Volume 18 Issue 46

37 Gibbs VC. Safety in the modern operating room counted all sponges in all cases throughout the hospital and there were no cases of retained sponges. The radiofrequency detection system consists of sponges that have a small passive radiofrequency tag sewn into a pocket on each sponge and a handheld wand or mat which contain the antennae and detection system [20]. The tag is 4 mm 12 mm and is recognized as only a yes or no signal. The tag is detected when the handheld wand or mat is activated and the computer console presents a visual and audible signal that a sponge has been detected. The system does not distinguish between sponge types or number of sponges. The signal readout will be the same intensity if there are one or five sponges. In the event of a missing sponge the mat can be activated to determine if the sponge is in the patient or the wand can be used to wand the patient or scan the trash to find the sponge. This system does not count sponges so at this time is usually used in conjunction with a manual counting practice such as Sponge AC- COUNTing. Some hospitals have adopted mandatory wanding protocols to be used for specific types of cases, others require wanding in lieu of using xray to find a missing sponge. There is an early trial from the University of North Carolina in the assessment and safety of this device and after 24 mo they have not had any retained sponges [21]. RFID systems have a unique RFID chip sewn into each type of sponge and a separate computer console with a scanning bucket into which used sponges are placed [22,23]. Each sponge has a specific RFID chip and thus sponges of different types pooled together can be distinguished and counted. Unopened packages of sponges are placed on a front panel of the console to be electronically counted-in and the sponges are then opened and placed on the sterile field. Used sponges can be put directly into the bucket or into plastic-bag lined kick buckets and the entire plastic bag full of sponges then placed into the scanning bucket. The sponges will all be individually counted-out. If there is a missing sponge it can be detected with a wand. All of these devices are undergoing clinical assessments in different hospitals around the world. With the continued improvement in the prevention of retained sponges, reports of retained miscellaneous small items and unretrieved device fragments are increasing [17,24]. The preventive strategy for these types of items are not applicable only to the OR since retained guidewires, sheaths and catheters are found after interventional vascular, cardiac and radiological procedures performed in various sites throughout a hospital. In addition various types of providers now must develop standardized processes to account for all of the items and parts of devices at the conclusion of invasive procedures. This is new territory for interventional radiologists and cardiologists and practices originating in the OR can be shared with these other clinical groups to help speed accountability. In the OR, small miscellaneous items or broken parts or pieces of tools and material used during an operation when found to be missing may require a clinical decision to leave the item behind because the risk of removal may cause greater harm than leaving the object behind. If a small item or un-retrieved device fragment is intentionally left behind, the patient must be informed. Leaving fragments and small objects in patients can be prevented because often human factors are involved in why the devices or objects break or fracture. Identifying early when an item is missing or broken means best practices in the OR will involve the coordination of actions between the surgeon and the surgical technologists who must become the content experts on the equipment. If something is missing or broken the scrub person has to recognize the defect and speak up so actions to find the missing part can be undertaken. Retained whole instruments are exceedingly rare and previous reports that indicated instruments as the second most common item most certainly included small devices and fragments in this category rather than the separate class in which they are now considered. Inside or out of the OR, standardized practices have to be established that are transparent, widely applicable and simple to perform so multiple providers at the completion of every case, delivery or invasive procedure can be sure there is NoThing Left Behind. SURGICAL FIRES OR fires occur near or around a patient but a surgical fire is a fire that occurs in or on a patient and includes airway fires [25]. Surgical fires are the rarest of the three surgical patient safety events with an estimate from a 2009 study suggesting that between fires a year occurred in the United States [26]. This national estimate was based on data from the Pennsylvania safety event reporting system which is the best available evidence. Surgical fires are dangerous, lethal and preventable but are so infrequent that most surgeons are painfully unaware of what preventive measures need to be taken. A surgical fire requires the classic constellation of three components, the fire triad : (1) an oxidizer; (2) an ignition source; and (3) fuel. The breadth of elements and circumstances that exist in each of these components are underappreciated and make it difficult to easily standardize or develop simple practices [27,28]. For example, possible fuels sources can include almost anything on the patient or in a surgical field: tracheal tubes, sponges, drapes, gauze, alcohol or other volatile compound containing solutions, oxygen masks, nasal cannulae, hair, fat, dressings, gowns, gastrointestinal tract gases, suction catheters, cable coverings, gloves and packaging materials. The ignition sources can be from almost any energy generating device and all differentially function or have the potential to cause injury based upon the oxidizer enriched atmosphere. In 2007 the American Society of Anesthesiologists published a practice advisory for the prevention and management of OR fires which provides a complex algorithm that outlines a risk assessment approach and action plans for surgical providers [25]. In 2009, further 6716 December 14, 2012 Volume 18 Issue 46

38 Gibbs VC. Safety in the modern operating room recommendations were promulgated for oxidizer management and communication guidelines to follow for prevention [26]. These efforts are thorough but the end results are still highly complex and detailed and because of the rarity of surgical fires it remains difficult to engage providers. Otolaryngology surgeons are the most familiar with surgical fire prevention measures because they frequently operate with ignition sources near the airway, within a highly oxidized atmosphere however all types of surgeons need to learn and adopt safer surgical fire preventive strategies because all types of cases actually provide risky conditions. To this end a provider oriented accountability approach for surgical fire management has been developed which assigns responsibility for action to specific stakeholders. This protocol teaches providers to know their role in preventing fires and respond quickly should a fire occur [29,30]. The roles for prevention are based on the domains of control for each of the elements in the fire triad. Anesthesiologists have the control and direct ability to act on the management of oxidizers which includes oxygen and nitrous oxide. Nurses have control and direct ability to monitor the fuel sources and provide immediate remedy to extinguish flames with saline. The Surgeon has control over the ignition source and is responsible for assessing the environment before starting or using electrocautery. Surgical fire prevention requires constant situational awareness because of the changing nature throughout an operation of the availability of different fuels, oxidizer status and ignition sources. Information exchange between the anesthesiologist, nurse and surgeon are key. If a fire breaks out rapid responses from all three stakeholders are important and some suggest fire drills should be conducted for high risk cases so team members are adequately primed to respond appropriately. If there is the risk of an airway fire the anesthesiologist must keep the surgeon aware of oxidizer concentrations and atmospheric conditions and be ready to stop all airway gases and remove the tracheal tube. The surgeon must have thoughtful control and use of cutting devices while nursing personnel in the scrub position must be ready with saline and wet sponges. All of these coordinated actions will have to take place in seconds in order to save the patient from extensive injury. To this end, surgical fire case-specific risk assessment strategies are useful activities and allow all providers to be primed at the time and ready to go rather than relying on memory or remote training. Surgical fire risk assessment can be incorporated into part of the operative briefing and safety checklist so each case can be assessed and necessary steps taken before the case starts to make sure all team members know what to do and are on the same page [8]. SURGICAL PATIENT SAFETY OR CHECK- LIST Common to the prevention of surgical patient safety events and mitigation of harm is the need to have all team members prepared to address events as they occur. The nature of preparedness involves having knowledge and the appropriate skills and tools available. Simulation training and drills are ways in which many industries maintain preparedness. The use of checklists as an adjunct to the pre-existing skill set of team members who come together has also been important to make sure all necessary tools and practices are performed correctly. In 2009 a surgery safety checklist was first introduced which was beyond what was traditionally used by nursing services to make sure the patient was prepared for surgery. This 19 item checklist was divided into three parts: (1) sign in activities; (2) time-out activities; and (3) signout activities which covered the recommended practices the World Health Organization (WHO) had developed to ensure the intraoperative safety of surgical patients worldwide [7]. The elements were simple and were intended to enhance inter-professional communication and prevent surgical events that may result from poor teamwork and inadequate surgical operative preparation. This checklist was adopted and disseminated by the Institute for Healthcare Improvement and has been in place in more than 3900 hospitals around the world [31]. Each facility is encouraged to develop a surgical safety checklist that will conform to their site-specific conditions but at the minimum the original 19 points are strongly encouraged to remain. Many surgical subspecialties e.g., ophthalmology have customized the original WHO safety checklist to their needs and recent work has been started on the development of crisis checklists for the operating room which aim to guide providers to perform the correct actions in intraoperative crises [32]. Implementation of some form of a surgical safety checklist has been widespread and the benefits of checklist use continue to be evaluated. There is no one surgical safety checklist that is the best but there are many which provide the necessary information for all stakeholders to come together and make sure the correct patient is being operated on, the correct operation is known and the operating team is properly prepared to perform that operation safely. A surgical patient safety checklist should help surgical providers address the preventative solutions to prevent the wrongs, RSI and surgical fires at a minimum [33]. An example of a surgical patient safety checklist that covers this territory is included in the reference section [34]. The checklist is used three times during a patient s operation (1) as an aid during a pre-operative briefing that is conducted when the patient is first brought to the OR; (2) during the time-out; and (3) as a reporting tool during the de-briefing that is held at the end of the case to discuss what problems occurred and also what went well. During the pre-operative briefing patient elements are reviewed, such as procedure verification and imaging, antibiotic administration, venous thromboembolism prophylaxis management, beta-blocker use, allergy history and blood availability but more importantly the surgical team puts together a surgical fire risk assessment score December 14, 2012 Volume 18 Issue 46

39 Gibbs VC. Safety in the modern operating room If the fire risk is considered high, based on a simple 3 point scoring system, then each of the three stakeholders in fire prevention go over what they are supposed to do as outlined on the back of the checklist. In this way the circulating nurse and surgical technologist, the surgeon and the anesthesiologist discuss what maneuvers and actions will be taken during that specific high risk case. During the time-out the three essential questions are answered using the checklist as a memory jogger and confirming primary source information in the computer or on the patient s armband. These actions prevent the occurrence of a wrong site surgery. At the de-briefing, all surgical items are accounted for, pathology specimens are reviewed to determine that they are correct and labeled correctly and any equipment or instrument problems are noted with specific referral units noted who are to take action for remediation thus preventing RSI. CONCLUSION The three surgical patient safety events, wrong site surgery, RSI and surgical fires can be prevented and even if prevention strategies fail mitigation of patient harm can be accomplished with application of consistent safety practices and effective communication. Effective communication includes the use of a comprehensive surgical patient safety checklist which brings all surgical providers together for at least a few moments to have a shared mental model for the patient s surgical care. The three major stakeholders working together in our complex OR environment are nurses, surgeons and anesthesiologists who face ever increasing work demands and knowledge requirements. It is not humanly possible to master all the knowledge domains for all safe practices for these three events, let alone all the other things that are necessary for the performance of an uncomplicated procedure. However, thought modules, checklists and assignment of areas of expertise and responsibility can be helpful in getting humans to work effectively and efficiently together. Of the three surgical patient safety events, it is not a stretch to see that there are knowledge domains which can drive improved performance and the three domains of expertise are possessed by each of the three major stakeholders. The content experts for wrong site surgery are surgeons, for RSI it is nurses and for surgical fires it is anesthesiologists. Prevention of wrong site surgery is highly dependent on preoperative practices of patient identification and patient preparation in which the surgeon s knowledge and preparation of the primary documents and materials falls within their care domain. It is the surgeon who has determined what operation is to be performed, has marked the operative site and has seen the patient at some time prior to their arrival in the OR. During the surgeon activated time-out confirmatory actions are conducted in concert with the use of a checklist to become the primary means of wrong site surgery preventive actions. The prevention of RSI strongly depends on actions under the control of the scrub person and circulating nurse who track and account for all the surgical material which is used. The use of a reliable sponge management practice in concert with a MWE is essential to prevent retained surgical sponges. The nurse also provides the interface with other stakeholders such as radiology should intra-operative x-rays be needed. For prevention of surgical fires it is the anesthesiologist who monitors oxygen status and has the situational awareness to alert and direct actions of the other stakeholders in the event of a fire. This is not to say that the other stakeholders don t have roles and responsibilities but when providers of care come together to perform a surgical case there is a team leader. The role of team leader is actually not fixed and immutable. It will have to change as the circumstances of the operation change and the team leader interestingly enough should be the person who is the content expert of that situation as it arises. It is not possible for everyone to know everything about all things. It is possible for the content experts of each of their domains to be present, willing and able to share their knowledge and communicate effectively with other personnel. Rather than thinking one person has to know it all and do everything, thinking in threes can help all providers put a vast amount of information into some useable context where it will be up to the three major stakeholders to work together consistently in every case to prevent the three major surgical patient safety events. REFERENCES 1 Plsek PE, Greenhalgh T. Complexity science: The challenge of complexity in health care. BMJ 2001; 323: Institute of Medicine Crossing the Quality Chasm: A new health system for the 21st century. Washington DC: National Academies Press, 2001: Porter ME. What is value in health care? N Engl J Med 2010; 363: Reason J. Human error: models and management. BMJ 2000; 320: Clarke JR, Johnston J, Blanco M, Martindell DP. Wrong-site surgery: can we prevent it? Adv Surg 2008; 42: Sargut G, McGrath RG. Learning to live with complexity. Harv Bus Rev 2011; 89: 68-76, Haynes AB, Weiser TG, Berry WR, Lipsitz SR, Breizat AH, Dellinger EP, Herbosa T, Joseph S, Kibatala PL, Lapitan MC, Merry AF, Moorthy K, Reznick RK, Taylor B, Gawande AA. A surgical safety checklist to reduce morbidity and mortality in a global population. N Engl J Med 2009; 360: Murphy J. A new effort to promote fire safety in the OR. Available from: URL: http: // TIPS/Docs/TIPS_ NovDec10WWW.pdf 9 Gibbs VC. Patient safety practices in the operating room: correct-site surgery and nothing left behind. Surg Clin North Am 2005; 85: , xiii 10 Neily J, Mills PD, Eldridge N, Dunn EJ, Samples C, Turner JR, Revere A, DePalma RG, Bagian JP. Incorrect surgical procedures within and outside of the operating room. Arch Surg 2009; 144: Crane M. Wrong-site surgery occurs 40 times a week. Available from: URL: http: // _print 12 Michaels RK, Makary MA, Dahab Y, Frassica FJ, Heitmiller E, Rowen LC, Crotreau R, Brem H, Pronovost PJ. Achieving 6718 December 14, 2012 Volume 18 Issue 46

40 Gibbs VC. Safety in the modern operating room the National Quality Forum s Never Events : prevention of wrong site, wrong procedure, and wrong patient operations. Ann Surg 2007; 245: Joint Commission Center for Transforming Healthcare. Further reducing the risk of wrong site surgery. Available from: URL: org/projects/detail.aspx?project=2 14 Gibbs VC. Retained surgical items and minimally invasive surgery. World J Surg 2011; 35: National Quality Forum. NQF updated serious reportable events. Available from: URL: org/project_details.aspx?id=10286#t=2&s=&p= Gibbs VC, Coakley FD, Reines HD. Preventable errors in the operating room: retained foreign bodies after surgery-- Part I. Curr Probl Surg 2007; 44: Gibbs VC. NoThing Left Behind : A National Surgical Patient-Safety Project to Prevent Retained Surgical Items. Available from: URL: 18 Surgicount Medical website. Available from: URL: http: // 19 Cima RR, Kollengode A, Clark J, Pool S, Weisbrod C, Amstutz GJ, Deschamps C. Using a data-matrix-coded sponge counting system across a surgical practice: impact after 18 months. Jt Comm J Qual Patient Saf 2011; 37: RF Surgical website. Available from: URL: http: //www. RFsurg.com 21 Rupp CC, Kagarise MJ, Nelson SM, Deal AM, Phillips S, Chadwick J, Petty T, Meyer AA, Kim HJ. Effectiveness of a radiofrequency detection system as an adjunct to manual counting protocols for tracking surgical sponges: A prospective trial of 2,285 patients. J Am Coll Surg 2012; 215: Clearcount Medical website. Available from: URL: http: // 23 Orlocate website. Available from: URL: http: // 24 Schultz DG. FDA Public Health Notification: Unretrieved device fragments. Rockville, MD: Center for Devices and Radiological Health, US Food and Drug Administration. Available from: URL: ucm htm 25 Caplan RA, Barker SJ, Connis RT, Cowles C, de Richemond AL, Ehrenwerth J, Nickinovich DG, Pritchard D, Roberson D, Wolf GL. Practice advisory for the prevention and management of operating room fires. Anesthesiology 2008; 108: ; quiz ECRI Institute. New clinical guide to surgical fire prevention: patients can catch fire here s how to keep them safer. Health Devices 2009; 38: Roy S, Smith LP. What does it take to start an oropharyngeal fire? Oxygen requirements to start fires in the operating room. Int J Pediatr Otorhinolaryngol 2011; 75: Hart SR, Yajnik A, Ashford J, Springer R, Harvey S. Operating room fire safety. Ochsner J 2011; 11: Watson DS. New recommendations for prevention of surgical fires. AORN J 2010; 91: Fire Safety Tool Kit. Available from: URL: http: //www. aorn.org/practiceresources/toolkits/firesafetytoolkit 31 WHO Surgical safety checklist. Available from: URL: 32 Ziewacz JE, Arriaga AF, Bader AM, Berry WR, Edmondson L, Wong JM, Lipsitz SR, Hepner DL, Peyre S, Nelson S, Boorman DJ, Smink DS, Ashley SW, Gawande AA. Crisis checklists for the operating room: development and pilot testing. J Am Coll Surg 2011; 213: e10 33 Zahiri HR, Stromberg J, Skupsky H, Knepp EK, Folstein M, Silverman R, Singh D. Prevention of 3 never events in the operating room: fires, gossypiboma, and wrong-site surgery. Surg Innov 2011; 18: SFVAMC Surgery Patient Safety Checklist. Available from: URL: TIPS_%20NovDec10WWW.pdf S- Editor Gou SX L- Editor A E- Editor Xiong L 6719 December 14, 2012 Volume 18 Issue 46

41 Online Submissions: doi: /wjg.v18.i World J Gastroenterol 2012 December 14; 18(46): ISSN (print) ISSN (online) 2012 Baishideng. All rights reserved. TOPIC HIGHLIGHT Marco Giuseppe Patti, MD, Professor, Series Editor Modern treatment of gastric gastrointestinal stromal tumors Kevin K Roggin, Mitchell C Posner Kevin K Roggin, Mitchell C Posner, Department of Surgery, Pritzker School of Medicine, University of Chicago, IL 60637, United States Author contributions: Roggin KK performed literature review and analyzed studies, and wrote the manuscript; Posner MC assisted with the analysis and edited the manuscript. Correspondence to: Kevin K Roggin, MD, FACS, Associate Professor, Department of Surgery, Pritzker School of Medicine, University of Chicago, 5841 South Maryland Ave, MC 5094 G-216, Chicago, IL 60637, United States. [email protected] Telephone: Fax: Received: April 11, 2012 Revised: June 26, 2012 Accepted: June 28, 2012 Published online: December 14, 2012 Abstract Gastrointestinal stromal tumors (GIST) are rare mesenchymal smooth muscle sarcomas that can arise anywhere within the gastrointestinal tract. Sporadic mutations within the tyrosine kinase receptors of the interstitial cells of Cajal have been identified as the key molecular step in GIST carcinogenesis. Although many patients are asymptomatic, the most common associated symptoms include: abdominal pain, dyspepsia, gastric outlet obstruction, and anorexia. Rarely, GIST can perforate causing life-threatening hemoperitoneum. Most are ultimately diagnosed on cross-sectional imaging studies (i.e., computed tomography and/or magnetic resonance imaging in combination with upper endoscopy. Endoscopic ultrasonographic localization of these tumors within the smooth muscle layer and acquisition of neoplastic spindle cells harboring mutations in the c-kit gene is pathognomonic. Curative treatment requires a complete gross resection of the tumor. Both open and minimally invasive operations have been shown to reduce recurrence rates and improve long-term survival. While there is considerable debate over whether GIST can be benign neoplasms, we believe that all GIST have malignant potential, but vary in their propensity to recur after resection and metastasize to distant organ sites. Prognostic factors include location, size (i.e., > 5 cm), grade (> 5-10 mitoses per 50 high power fields and specific mutational events that are still being defined. Adjuvant therapy with tyrosine kinase inhibitors, such as imatinib mesylate, has been shown to reduce the risk of recurrence after one year of therapy. Treatment of locally-advanced or borderline resectable gastric GIST with neoadjuvant imatinib has been shown to induce regression in a minority of patients and stabilization in the majority of cases. This treatment strategy potentially reduces the need for more extensive surgical resections and increases the number of patients eligible for curative therapy. The modern surgical treatment of gastric GIST combines the novel use of targeted therapy and aggressive minimally invasive surgical procedures to provide effective treatment for this lethal, but rare gastrointestinal malignancy Baishideng. All rights reserved. Key words: Gastrointestinal stromal tumors; Laparoscopic resections of gastrointestinal stromal tumors; Imatinib mesylate; Gastrectomy Peer reviewer: Yasuhiro Kodera, Professor, Department of Surgery II, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya , Japan Roggin KK, Posner MC. Modern treatment of gastric gastrointestinal stromal tumors. World J Gastroenterol 2012; 18(46): Available from: URL: com/ /full/v18/i46/6720.htm DOI: org/ /wjg.v18.i INTRODUCTION Gastrointestinal stromal tumors (GIST) are rare mesenchymal tumors that occur throughout the smooth muscle layer of the gastrointestinal (GI) tract [1]. GIST represent less than 1% of all GI tract malignancies. The most common location of these tumors is the stomach (70%), small bowel (20%-30%), small intestine and colon/rec December 14, 2012 Volume 18 Issue 46

42 Roggin KK et al. Modern treatment of gastric GIST tum (10%). Uncommonly, they can arise within the greater omentum, esophagus, appendix, and gallbladder. Most cases are sporadic, and affect men slightly more frequently than women (54% vs 46%) [2]. The annual incidence in the United States has remained stable at 3000 to 4000 cases per year. The world-wide age-adjusted annual incidence rates range from 6.8 to 14.5 cases per million and vary between countries of origin. The median age at diagnosis is 58 years of age, but GIST have been reported in newborns and adolescents [3]. GIST can range in size from several millimeters to over 30 cm in diameter. Tumor diameter appears to significantly influence biologic behavior, as small GIST may remain indolent for many years and large, massive GIST have higher rates of recurrence and associated metastases [2]. Historically, smooth muscle sarcomas were classified as leiomyosarcomas [4]. The development of malignant GIST requires the transformation of the interstitial cells of Cajal, pacemakers of the GI tract, to a malignant phenotype through activating or gain of function mutations in the c-kit proto-oncogene [5]. GIST are spindle cell neoplasms that usually retain the ultrastructural characteristics of smooth muscle cells, but have immunohistochemical staining for c-kit, CD-34, smooth muscle actin, desmin and S-100 [6]. Approximately 70% of GIST are spindle cell type neoplasms; the minority are epithelioid (20%) or mixed cell type (10%) [7]. Hirota et al [5] first described the novel mutation in the KIT tyrosine kinase receptor gene in Since this landmark discovery, most leiomyosarcomas have been reclassified as GIST. KIT is located on chromosome 4q11-q12 and functions as a transmembrane receptor for its ligand, stem cell factor. In the non-cancerous state, this ligand binds to the extracellular portion of the receptor to induce homodimerization and downstream activation of its cell-signaling pathways [8]. Wild-type c-kit normally regulates cellular differentiation, growth, and survival. Approximately 80%-90% of GIST harbor mutations in the KIT genome. Mutations in the platelet-derived growth factor receptor (PDGFRα) occur in 5%-10% of c-kit-wild type GIST. DOG1 mutations may help identify GIST with wild type c-kit and PDGFR [9]. Mutated KIT receptors induce ligand-independent, unregulated activation of the downstream cell signaling pathways which collectively results in a loss of normal cell adhesion, differentiation, and proliferation to promote tumorigenesis. Exon 11 mutations in the KIT gene cause constitutively activated receptors leading to unregulated autophosphorylation of the intracytoplasmic tyrosine kinases [10]. KIT mutations in exons 9, 13, 17 are less common and have been associated with more aggressive tumor behavior. Biologically, gastric GIST tumors grow locally within the stomach (intra- or extraluminal expansion) and eventually obtain the capability to metastasize via hematogenous routes to the solid viscera (liver, small bowel, lungs) and peritoneal cavity. Tumors can also spread along the smooth muscle planes within the stomach or can rupture into the peritoneal cavity causing sarcomatosis. Complete surgical resection is thought to be the only curative treatment for GIST. The recent use of cytostatic agents, such as imatinib mesylate, in patients with metastatic disease has been associated with durable recurrencefree survival [11]. This important observation suggests that overall survival may not be the most important endpoint to consider when making treatment decisions. Radical gastrectomy is seldom required for extirpation of these tumors [3]. In contradistinction to gastric adenocarcinoma, where it is essential to obtain at least fivecentimeter proximal and distal margins, GIST tumors can be effectively treated by a complete gross resection of the tumor [1]. Given the infrequency of lymphatic metastases, regional lymphadenectomy is not indicated. Minimally-invasive operations are now frequently used to treat gastric GIST. Retrospective series suggest that these techniques may reduce perioperative stress and are associated with lower rates of postoperative complications, shorter hospital stays and equivalent recurrence rates (Table 1) [12-16]. EPIDEMIOLOGY AND DIAGNOSIS Miettinen et al [3] published the largest retrospective series of gastric GIST that reviews 1869 cases seen at the Armed Forces Institute of Pathology from The vast majority of cases occurred in patients over 40 years of age and the median tumor diameter was 6 cm (range: cm). Most gastric GIST had spindle-cell or epithelioid differentiation.over 90% of these neoplasms had mutations in the c-kit gene; PDGFR mutations were more frequently identified in epitheliod tumors. The metastatic potential of these tumors strongly correlated with their size and rate of mitotic activity. Unfavorable prognostic factors included proximal tumors (gastric cardia and gastroesophageal junction), the presence of coagulative necrosis, ulceration, and invasion deep to the mucosal layer. Most GIST are incidentally diagnosed during evaluations for nonspecific GI symptoms, such as pain, nausea and vomiting, and weight loss [2]. Tumor hemorrhage commonly occurs when large tumors develop an ischemic, punctate ulcer (Figure 1A). Usually, the bleeding can be temporized using endoscopic sclerotherapy or electrosurgical coagulation techniques. Seldom is it necessary to take patients urgently for surgical resection with intractable hemorrhage (Figure 1B and C). Often these patients can be stabilized with medical and endoscopic therapy and have elective operations to extirpate these tumors. Intraperitoneal tumor rupture with hemoperitoneum and tumor dissemination is a difficult clinical problem that is associated with a significant risk of intraperitoneal sarcomatosis. Computed tomography (CT) scanning is the most widely used and effective staging modality [2]. Multiplerow detector can localize the tumor within the stomach and remains a very sensitive technique to detect distant metastasis (at least 1 mm in diameter) within the liver or lungs; small volume intraperitoneal disease is often only detected on diagnostic laparoscopy and is respon December 14, 2012 Volume 18 Issue 46

43 Roggin KK et al. Modern treatment of gastric GIST A B1 C1 B2 C2 Figure 1 Clinical images of complicated gastrointestinal stromal tumors. A: Large intraluminal gastric gastrointestinal stromal tumors (GIST) with punctate central ulceration. The bleeding ulcer was treated endoscopically with sclerotherapy and electrocautery (cauterized tissue; white oval). The patient had an interval resection electively without additional hemorrhage from the tumor; B: Acute presentation of a patient with a ruptured gastric GIST with hemoperitoneum. These images represent contrast-enhanced computed tomography (CT) scan from a patient with a large extraluminal gastric GIST along the greater curvature of the stomach. B1 demonstrates axial CT images of the bi-lobed tumor with irregular borders (arrows); B2 shows additional axial images at the caudal extent of gastric tumor with layering of blood in the splenic recess (oval). He was diagnosed with hemoperitoneum and was resuscitated with packed red blood cells, fresh frozen plasma, and platelets; the patient was on antiplatelet therapy at the time of admission. He stabilized and had an upper endoscopy/ultrasonography for tissue diagnosis and to plan definitive treatment; C: Ruptured gastric GIST following conservative management. Contrast-enhanced CT images following a six-week period of conservative management of the patient with ruptured gastric GIST. C1 demonstrates the more organized bi-lobed tumor with distinct borders (arrows); C2 shows coronal images of the organized hemorrhagic component within the splenic recess after a period of observation (oval). Ultimately this patient had an interval open subtotal gastrectomy for a high-grade GIST. A B Figure 2 Endoscopy ultrasound images with fine needle aspiration biopsy. A: A 3 cm 3 cm submucosal intraluminal mass within the gastric cardia; B: This Endoscopy ultrasound image shows the fine needle aspiration biopsy needle (horizontal white line in upper right corner of image) puncturing the submucosal gastric gastrointestinal stromal tumors. sible for the reported 10%-15% of false negative rate with dynamic CT. Magnetic resonance imaging (MRI) is an acceptable alternative to CT for patients with renal dysfunction or in whom the risk of cumulative ionizing radiation may be prohibitive. Positron emission tomography (PET) remains an experimental test that may be useful in confirming distant metastatic disease and determining the response to neoadjuvant targeted therapy. PET scans usually indicate tumor responsiveness to imatinib mesylate within days to weeks of induction therapy. Upper endoscopy (EGD) with ultrasonography (EUS) is an essential diagnostic modality to acquire tissue for diagnosis, usually by fine needle aspiration (FNA) or core-needle biopsy (Figure 2). In addition, EUS is accu December 14, 2012 Volume 18 Issue 46

44 Roggin KK et al. Modern treatment of gastric GIST Table 1 Summary of large-series (> 35 cases) of minimally-invasive resections for gastric gastrointestinal stromal tumor Ref. Location MIS/ GIST Proximal tumors n (%) Size (cm) Operative time (min) Surgical treatment of gastric GIST is the only known cu- Complications n (%) Conversion to open surgery n (%) LOS (d) R0 resection rate Intermediate/ high risk GIST n (%) Recurrence rate n (%) Median F/U (mo) (range) Sasaki et al [16] Japan 45 1 /37 6 (13) 3.2 ( ) 100 (30-240) 1 (2) 1 (2) NR (24) 0 74 (1-81) Sexton et al [14] Germany 112/61 7 (11) 3.8 (± 1.8) (± 67.3) 10 (16.4) 1 (2) 3.9 (± 2.2) (25) 3 (5) 15 (0-103) Wilhelm et al [15] Germany 93/ (39) 2.6 ( ) (7.5) 6 (6.5) (13) 0 40 (2-99) Otani et al [13] Japan (60) 3.6 ( ) 141/188 4 NR (28) 2 (3) 53 Novitsky et al [12] NC (34) 4.4 (± 2.0) 135 (± 56) 4 (8) (± 1.6) (28) 4 (8) 36 (4-84) Total (for GIST) (38) 8 (3) 63 (23) 9 (3) 1 Forty-five laparoscopic operations and 37 confirmed gastrointestinal stromal tumors (GIST); 2 no positive margins, but one patient had a laparoscopic resection in the setting of distant metastatic disease; 3 ninty-three consecutive patients, including 62 GIST; there was 1 laparascopic-assisted endoscopic resection, 55 laparoscopic wedge resections, and 34 transgastric resections; 4 the mean operative time was 141 for laparoscopic operations and 188 min for laparoscopy-assisted operations. NC: North Carolina; n: Number of patients in each series; MIS: Minimally invasive operations; Proximal tumors: GIST at gastroesophageal junction or within gastric cardia; Size: Median pathologic tumor size; Complications: Surgical morbidity; LOS: Length of hospital stay; NR: Not reported; R0 resection: Gross and microscopically-negative margins; F/U: Follow-up. A B Figure 3 Gastroesophageal junction gastrointestinal stromal tumors. A: An axial computed tomography image of a gastric gastrointestinal stromal tumor (white oval) located along the posterior wall of the gastroesophageal junction (GEJ); B: Coronal images of the tumor (white arrow) show its proximity to the GEJ. rate in determining the depth of penetration and origin of these neoplasms and also allows one to potentially consider a hybrid endoscopy/laparoscopic resection [17]. The published National Comprehensive Cancer Network (NCCN) guidelines outline the recommended principles of tissue sampling for GIST ( org). Since most GIST are soft, fragile, well-encapsulated tumors, indiscriminate biopsies increase the risk of tumoral hemorrhage and rupture. This is associated with higher rates of tumor recurrence and/or intraperitoneal dissemination. The decision to perform a preoperative or pretreatment biopsy should be individualized and only performed when the results of the sampling would definitively influence the choice of treatment [18]. Biopsy is mandatory for all locally-advanced gastric GIST that will be treated with pre-resection neoadjuvant targeted therapy. Careful review of the acquired tissue by experienced GI histopathologists and use of comprehensive immunohistochemical staining for c-kit and other markers is essential to confirm the diagnosis. Given the accuracy and real time localization of these tumors, EUS-guided biopsy is generally preferable to CT- or ultrasound-guided FNA biopsy techniques [2,19]. EGD/EUS can identify the key anatomic relationships of the tumor to the gastric wall layers. GIST at the gastroesophageal junction (Figure 3), pylorus and along the posterior wall of the stomach represent unique surgical challenges and influence the required operation. EGD can also effectively be used to treat tumor hemorrhage and avoid the need for urgent gastric operations. EUS can determine the depth of penetration through the layers of the gastric wall and potentially identify tumors that can be extirpated using endoscopic resection techniques. Only intragastric GIST that arise from the superficial circular muscular layer or muscularis mucosa can be removed with endoscopic enucleation [20]. These procedures are technically demanding and require considerable experience and skill. Some of these cases take place in the endoscopy suites, but are often coordinated with surgical specialists to assist with the management of hemorrhage or gastric perforation. Often these resections are best performed in surgical operating rooms using laparoscopic-assisted techniques with an experienced surgeon present for the operation. SURGICAL TREATMENT 6723 December 14, 2012 Volume 18 Issue 46

45 Roggin KK et al. Modern treatment of gastric GIST A Figure 4 Locally-advanced gastric gastrointestinal stromal tumors. A: Representative contrast-enhanced computed tomography images show a large, proximal gastric gastrointestinal stromal tumors that invades into the splenic hilum (oval); B: On the coronal images the arrow indicates a heterogeneous mass invading into the spleen with areas of viable tumor and necrotic areas represented by calcifications. rative therapy [1]. It is essential to completely remove the entire tumor without violating the capsule of the mass. Tumor spillage or hemorrhage is associated with high locoregional recurrence rates and/or development of peritoneal sarcomatosis [18]. Given the rarity of lymphatic dissemination, regional lymphadenectomy is not routinely performed. Since these tumors originate from the muscular layer of the gastric wall, enucleation is an option, but may be associated with higher recurrence rates unless the intramuscular pedicle can be clearly identified. Standard operations include both open and minimally invasive operations. Wedge or a full-thickness partial gastrectomy is an effective strategy for tumors that are located along the lesser or greater curvature of the stomach [21]. Posteriorly-based gastric GIST often require transgastric resections through an anterior longitudinal gastrotomy; the tumor is everted and its pedicle divided with a linear stapling device [22]. Anatomic gastrectomy (i.e., subtotal or total gastrectomy) is reserved for large tumors that involve a significant portion of the stomach. Endoscopic-assisted, laparoscopic gastric resections are cutting-edge operations that combine precise intraoperative localization of these tumors with gastric-volume preservation techniques. NCCN guidelines suggest that small (< 1 cm) gastric GIST without high-risk endoscopic ultrasonographic features (i.e., irregular borders, cystic spaces, ulceration, echogenic foci and heterogeneity) may be followed with close endoscopic surveillance at 6-12 mo intervals ( In the absence of biopsy-proven metastatic disease, patients with an acceptable performance status and GIST confined to the stomach should undergo complete surgical resection. In patients with marginally resectable tumors or in cases that GIST are potentially resectable, but the need for concomitant en bloc organ resection or total gastrectomy is likely, consideration should be given to neoadjuvant treatment with imatinib mesylate to cytoreduce or downstage tumors so that a less morbid or less extensive operation can be considered in the future (Figure 4) [23-25]. B Multiple single institutions highlight the increased use of laparoscopic or minimally-invasive operations for gastric GIST [12-16]. Resection techniques include: (1) laparoscopic transgastric resections; (2) laparoscopic full-thickness or wedge resections; (3) laparoscopic extramucosal enucleation; and (4) combined laparoscopic, endoscopic resections [26]. The five largest published reports of laparoscopy resections for gastric GIST are summarized in Table 1 [12-16]. Most of these retrospective series include non-gist, benign submucosal tumors (leiomyomas). Although a formal meta-analysis was not performed given the small number of patients, general trends are evident. It appears that minimally-invasive operations for gastric GIST have been successfully used to treat patients with large tumors in difficult locations (i.e., proximal stomach and gastroesophageal junction). The data also suggest reasonable operative times, acceptable complication rates, and few conversions to open operations. Since none of the series had strict criteria for postoperative discharge to home, the reported postoperative length of stay is difficult to interpret, but was shorter than historic controls for open operations. Importantly, despite nearly one-third of the patients having intermediate to high-risk lesions, nearly 100% were completely removed and did not recur after 1-4 years of follow-up [12-16]. We urge caution in broadly extrapolating these results to all patients with gastric GIST; most series had relatively short follow-up, involved a considerable selection bias, and most operations were performed by surgeons with considerable experience with these techniques [27]. Our institutional experience with laparoscopic resection of GIST suggest that these techniques are both feasible and effective treatment for tumors less than eight centimeters in diameter. We advocate using a multidisciplinary approach with combined surgical oncology and minimally-invasive specialists to estimate the biologic behavior and determine the optimal method of resection. Cutting-edge modifications include the use of robot-assisted laparoscopic resections [28], natural orifice surgery [29], gasless laparoscopic resections [30], single-port techniques [31,32], and novel methods of removing posteriorly based tumors [26]. One report described an experimental transgastric technique that utilized the retractable, metal-rimmed EndoCatch bags to elevate posterior wall GIST to facilitate laparoscopic stapled transection of the tumor pedicle [33]. OUTCOMES AFTER SURGICAL RESECTION OF GASTRIC GIST Since GISTs are rare neoplasms that demonstrate a spectrum of biologic behavior, outcomes following surgical resection are difficult to ascertain. Recurrence free survival appears dependent on tumor size, location, and mitotic rate [6,34]. Prior to the use of imatinib mesylate as an adjuvant treatment following complete resection of gastric GIST, several large, retrospective reports suggest 6724 December 14, 2012 Volume 18 Issue 46

46 Roggin KK et al. Modern treatment of gastric GIST local recurrence rates as high as 40% and five-year survival rates as ranging between 40%-90% [1,35-38]. Dematteo et al [1] published a series of 200 patients with GIST in 2000; more than half of these patients had gastric GIST. In the 93 patients with primary GIST, 80 (86%) had a complete resection with a median disease-specific survival of 54%. Fujimoto et al [36] reported a series of 140 patients that had curative operations for gastric GIST. The five- and ten-year overall survival rates for the 129 patients with curative operations were 93% and 88%, respectively. Independent predictors of poor prognosis included male patients [hazard ratio (HR) = 0.469, P = 0.013], tumor size greater than or equal to 10 cm (HR = 20.98, P = 0.001), a mitotic index of 10+ (HR = 45.95, P < 0001), and epithelioid cell histologic component (HR = 5.32, P = 0.014). Models to estimate the risk of recurrence have been created from large, retrospective data set of patients with verified GIST [6,34]. Size (> 10 cm) and mitotic rates greater than five per 50 high-powered fields are the most significant variables that predict malignant behavior. Conventional chemo- and radiation therapy are historically ineffective adjuvant treatments for GIST and do not significantly improve survival in patients with recurrent, metastatic or unresectable primary tumors [18]. The evolution of targeted therapy has dramatically altered outcomes for patients with advanced GIST. Imatinib mesylate is an orally bioavailable, selective molecular inhibitor of cellular tyrosine kinases. First used to treat Philadelphia chromosome-positive chronic myelogenous leukemia, imatinib inhibits tyrosine receptor kinases such as PDGFR and KIT [39]. The Federal Drug Administration (FDA) approved imatinib mesylate for use in patients with metastatic GIST in The American College of Surgeons Oncology Group (ACOSOG) phase Ⅱ non-randomized Z9000 trial examined the use of adjuvant imatinib for one-year following complete resection of high-risk GIST (> 10 cm tumors or ruptured GIST). Imatinib-use was associated with decreased recurrence rates (vs historic controls) [40]. The ACOSOG Z9001 was a randomized, double-blind, placebo-controlled, multicenter trial that conclusively showed a statistically significant reduction in the risk of recurrence with oneyear of adjuvant imatinib mesylate therapy (400 mg daily dose; HR = 0.35, range: , P < ) [41]. Seven hundred and thirteen patients with completely resected c-kit positive GIST (greater than 3 cm) were randomized in an intention to treat analysis. At a median followup of 19.7 mo, the study was halted when it became evident that only 30 (8%) of patients in the imatinib group and 70 (20%) in the placebo arm had recurrent disease identified. Further maturation of this data is necessary to determine whether the adjuvant treatment improves overall survival in treated patients. The FDA approved imatinib mesylate in 2008 as adjuvant therapy following complete resection of GIST for all patients without restrictions on time (to initiate therapy) or histopathologic criteria. The European Medicines Agency approved adjuvant imatinib in 2009 for adult patients with resected c-kit-positive GIST at significant risk of relapse of disease. At least one year of postoperative imatinib mesylate therapy (400 mg daily) is now considered the standard of care for tumors greater than 3 cm with high-risk features (> 5-10 mitoses/50 high power field) per the results of ACOSOG Z9001 [2]. Several postoperative models of risk assessment have been used to estimate the likelihood of recurrence for patients who do not meet the aforementioned criteria [6,34]. The optimal duration of imatinib and long-term survival benefit remains the subject of several ongoing randomized, controlled international cooperative group trials and industry-sponsored studies. Current protocols include the recently completed EORTC trial that randomized 900 patients with completed resected intermediate- and high-risk GIST to receive either two years of adjuvant imatinib mesylate vs observation. The primary endpoint of the EORTC trial was overall survival, so the final results will require approximately ten years for complete analysis. The Scandinavian Sarcoma Group phase Ⅱ trial, (SSGXVII; one vs three years of adjuvant imatinib mesylate) and the non-randomized Novartis Pharmaceutical Trial (NCT ; five years of adjuvant imatinib) were both designed to test extended use of adjuvant imatinib mesylate following complete resection. Patients at a higher risk of recurrence may justify indefinite use of adjuvant therapy. Three recent cooperative group trials using imatinib in patients with locally-advanced, unresectable or metastatic GIST have suggested that the KIT mutation genotype may have prognostic value to estimate the duration of response and optimal dose of imatinib mesylate [10,42,43]. Patients in these trials with exon 11-mutations had better treatment outcomes (improved tumor response, progression-free survival, and overall survival) when compared to patients with KIT exon 9-mutants and wild-type patients. At the American Society of Clinical Oncology annual meeting in 2010, it was reported that only deletions (all types) in the KIT exon 11 gene was associated with an increased risk of recurrence [44]. Heinrich et al [43] also reported that GIST with KIT exon 9-mutations had higher tumor response rates to neoadjuvant imatinib mesylate with daily doses of 800 mg (vs 400 mg). POST-OPERATIVE SURVEILLANCE NCCN guidelines suggest that following complete resection of gastric GIST; patients should be followed with comprehensive history and physical examinations every 3-6 mo for 5 years, then annually ( Abdominal/pelvic contrast enhanced CT scans were recommended every 3-6 mo for at least three to five years postoperatively. Given the risk of renal insufficiency with iodinated contrast and the cumulative ionizing radiation exposure with frequent CT scans, we believe that less intensive surveillance programs should be advocated. MRI remains an acceptable alternative for suitable patients and avoids the deleterious radiation exposure that is associated with serial CT scans. It is reasonable to 6725 December 14, 2012 Volume 18 Issue 46

47 Roggin KK et al. Modern treatment of gastric GIST A B Table 2 Summary of retrospective single-institutional experience with surgical resection of metastatic gastrointestinal stromal tumor after treatment with imatinib mesylate n (%) A: mm Figure 5 Neoadjuvant treatment of a locally-advanced gastrointestinal stromal tumors with imatinib mesylate. A: This woman presented with abdominal pain and fullness. A computed tomography (CT) scan identified a massive (> 30 cm), homogeneous tumor in the gastric fundus that was exophytic and extending caudally towards the pelvic inlet; B: After tissue diagnosis confirmed a gastric gastrointestinal stromal tumor (GIST), the patient was treated with six months of low-dose imatinib mesylate (400 mg/d) until a maximal response was achieved. The coronal views of this interval CT scan demonstrated a much smaller, well-encapsulated, homogenous tumor (solid white arrowhead). She had a radical resection of the gastric GIST and was free of disease until 24 mo when she developed a metastatic lesion in the left lateral segment of the liver. Following complete metastectomy, she was treated with several targeted tyrosine kinase inhibitors until she ultimately succumbed from her metastatic disease 19 mo from her second operation and 43 mo from her initial operation. consider an EGD at one-year after resection to rule out a local or anastomotic recurrence. Less frequent surveillance programs have been suggested for small (< 2 cm), low-risk tumors. Patients on investigational adjuvant protocols routinely are scanned more frequently to determine the efficacy of treatment. NEOADJUVANT TREATMENT OF LOCALLY-ADVANCED GASTRIC GIST Locally-advanced unresectable or borderline-resectable gastric GIST are often treated with neoadjuvant imatinib mesylate therapy prior to surgical resection (Figure 5) [45]. Theoretically, the use of preoperative imatinib may downstage or substantially cytoreduce GIST preoperatively and diminish the need for concomitant, en bloc organ resections. Over the past five years, there have been several small, single-institution; retrospective reports documenting outcomes following neoadjuvant treatment of borderline or locally-advanced GIST (Table 2) [25,44,46-51]. Approximately 75% of these highly selected patients with unresectable GIST were subsequently treated with R0/R1 resections. The duration of neoadjuvant therapy and best method of detecting maximal treatment effect have been the subject of two recent phase Ⅱ trials [52,53]. The RTOG 0132/ACRIN 6665 cooperative group trial prospectively administered neoadjuvant imatinib mesylate (600 mg/d) for eight weeks to patients with both potentially resectable (n = 30) and recurrent/metastatic GIST (n = 22) [53]. The majority of patients had disease stabilization; only 12% had a partial tumor response to therapy. Patients were Ref. Number of patients R0/R1 resections Sym et al [47] (62) DeMatteo et al [25] (80) Gronchi et al [48] (82) Raut et al [49] (83) Rutkowski et al [51] (92) Bonvalot et al [46] (68) Andtbacka et al [50] (48) Totals (74) R0/R1 resections: Complete gross removal of the gastrointestinal stromal tumor with/without negative microscopic margins. resected with minimal morbidity and given an additional two years of adjuvant therapy. The patients without metastatic disease had estimated two-year progressionfree and overall-survival rates of 83% and 93%, respectively. McAuliffe et al [52] randomized 19 patients with locally-advanced GIST to receive nanoneoadjuvant imatinib therapy; subjects were given 600 mg/d for 3 d, 5 d or 7 d prior to surgical resection. Seventeen of 19 patients had a subsequent resection without significant morbidity and were given two years of adjuvant therapy. Approximately 30% of these patients had an objective radiologic response to imatinib (CT/PET) and 12% of the resected tumors had an increase in apoptosis by terminal deoxynucleotidyl transferase- mediated dutpbiotin nick end labeling assay. These studies provide the proof of principle that neoadjuvant imatinib mesylate may be a safe and effective method of treating patients with locally-advanced GIST. CONCLUSION Gastric GIST are rare neoplasms that have traditionally required complete surgical resection to achieve cure. Both traditional and minimally invasive gastric resections can be used to remove these tumors with minimal morbidity and excellent perioperative outcomes. The revolutionary use of specific, molecularly-targeted therapies, such as imatinib mesylate, reduces the frequency of disease recurrence when used as an adjuvant following complete resection. Neoadjuvant treatment with these agents appears to stabilize disease in the majority of patients and may reduce the extent of surgical resection required for subsequent complete tumor removal. Importantly, tyrosine kinase inhibitors likely extend the progression-free survival of most patients with GIST. The optimal sequencing of therapies and incorporation of predictive genomic data highlight future challenges in this disease. ACKNOWLEDGMENTS The authors would like to thank Roberta Carden for proofreading and editing this manuscript December 14, 2012 Volume 18 Issue 46

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49 Roggin KK et al. Modern treatment of gastric GIST 34 Dematteo RP, Gold JS, Saran L, Gönen M, Liau KH, Maki RG, Singer S, Besmer P, Brennan MF, Antonescu CR. Tumor mitotic rate, size, and location independently predict recurrence after resection of primary gastrointestinal stromal tumor (GIST). Cancer 2008; 112: Crosby JA, Catton CN, Davis A, Couture J, O Sullivan B, Kandel R, Swallow CJ. Malignant gastrointestinal stromal tumors of the small intestine: a review of 50 cases from a prospective database. Ann Surg Oncol 2001; 8: Fujimoto Y, Nakanishi Y, Yoshimura K, Shimoda T. Clinicopathologic study of primary malignant gastrointestinal stromal tumor of the stomach, with special reference to prognostic factors: analysis of results in 140 surgically resected patients. Gastric Cancer 2003; 6: Pierie JP, Choudry U, Muzikansky A, Yeap BY, Souba WW, Ott MJ. The effect of surgery and grade on outcome of gastrointestinal stromal tumors. 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Phase II trial of neoadjuvant/adjuvant imatinib mesylate (IM) for advanced primary and metastatic/recurrent operable gastrointestinal stromal tumor (GIST): early results of RTOG 0132/ACRIN J Surg Oncol 2009; 99: S- Editor Gou SX L- Editor A E- Editor Xiong L 6728 December 14, 2012 Volume 18 Issue 46

50 Online Submissions: doi: /wjg.v18.i World J Gastroenterol 2012 December 14; 18(46): ISSN (print) ISSN (online) 2012 Baishideng. All rights reserved. Marco Giuseppe Patti, MD, Professor, Series Editor TOPIC HIGHLIGHT Adult to adult living related liver transplantation: Where do we currently stand? Erica M Carlisle, Giuliano Testa Erica M Carlisle, Giuliano Testa, Department of Surgery, Baylor University Medical Center, Dallas, TX 75246, United States Author contributions: Carlisle EM and Testa G both participated in the research, planning, and editing of the of the manuscript; Carlisle EM was the primary writer of the manuscript. Correspondence to: Giuliano Testa, MD, Professor of Surgery, Department of Surgery, Baylor University Medical Center, 3410 Worth Street, Dallas, TX 75246, United States. [email protected] Telephone: Fax: Received: April 18, 2012 Revised: August 3, 2012 Accepted: August 14, 2012 Published online: December 14, 2012 University of Heidelberg, Im Neuenheimer Feld 110, Heidelberg, Germany; Salvatore Gruttadauria, MD, Assistant Professor, Abdominal Transplant Surgery, ISMETT, Via E. Tricomi, Palermo, Italy; Mitsuo Shimada, Professor, Department of Digestive and Pediatric Surgery, Tokushima University, Kuramoto , Tokushima , Japan Carlisle EM, Testa G. Adult to adult living related liver transplantation: Where do we currently stand? World J Gastroenterol 2012; 18(46): Available from: URL: DOI: org/ /wjg.v18.i Abstract Adult to adult living donor liver transplantation (AALD- LT) was first preformed in the United States in The procedure was rapidly integrated into clinical practice, but in 2002, possibly due to the first widely publicized donor death, the number of living liver donors plummeted. The number of donors has since reached a steady plateau far below its initial peak. In this review we evaluate the current climate of AALDLT. Specifically, we focus on several issues key to the success of AALDLT: determining the optimal indications for AALDLT, balancing graft size and donor safety, assuring adequate outflow, minimizing biliary complications, and maintaining ethical practices. We conclude by offering suggestions for the future of AALDLT in United States transplantation centers Baishideng. All rights reserved. Key words: Adult to adult living donor liver transplantation; Outflow; Graft size; Liver failure; Ethics; Biliary complications Peer reviewers: Thilo Hackert, MD, Department of Surgery, INTRODUCTION At the time of its initial introduction into clinical practice, many believed that adult to adult living donor liver transplantation (AALDLT) would be a panacea for the severe shortage of cadaveric donors that resulted in extensive times on the waiting list and high patient mortality. This belief was illustrated by multiple studies from the late 1990s that suggested that AALDLT was safe and claimed that it would significantly decrease mortality on the transplantation waiting list [1,2]. The first United States AALDLT was performed in 1997, and over the next 3-5 years AALDLT was vigorously embraced by many United States transplantation surgeons. This enthusiasm was obvious in the documented increase from one to 38 United States AALDLT centers and from one to 266 United States AALDLT procedures between 1997 and 2000 [3]. However, despite rapid integration of this novel procedure into clinical practice, multiple questions remained unanswered. Little was known regarding the proper indications for AALDLT. Further, there was relatively limited data on how graft size impacted recipient and donor safety. Additionally, techniques to assure adequate outflow and minimize biliary complications 6729 December 14, 2012 Volume 18 Issue 46

51 Carlisle EM et al. Liver transplantation were only in their infancy. Finally, the multitude of ethical issues surrounding AALDLT had not been rigorously addressed. In 2002, possibly due to the first widely publicized death of a living donor, the number of living liver donors plummeted. The current number of annual living liver donors has now reached a relatively static plateau at about 250 donors per year, which is far below the initial peak of 524 donors in 2001(Figure 1) [4]. While there is a current climate of concern in the United States regarding the safety and appropriateness of AALDLT, multiple other countries (mainly Asian countries, Turkey and Egypt) have experienced a continued rise in AALDLT. Presumably, this contrast is the result of societal norms and logistic difficulties that impede cadaveric organ donation in these areas. Review of data from current United States and Asian transplantation centers along with critical review of the literature from United States surgeons who enthusiastically embraced AALDLT in its early years and then altered their practice as increasing risk became apparent, should be undertaken to help us determine how and if AALDLT can safely be reinstated as a more widely utilized procedure in United States transplantation centers. In this review, we draw upon these studies to address the current status of AALDLT. Specifically, we explore issues related to both the technical/scientific aspects of AALDLT as well as the ethical issues that surround AALDLT. We then offer suggestions for the future of AALDLT in the United States. OPTIMAL INDICATIONS FOR ADULT TO ADULT LIVING DONOR LIVER TRANSPLANTATION HAVE NOT BEEN ESTABLISHED Extensive efforts have been dedicated to determining the optimal indications for AALDLT. Review of the recent literature identifies two central issues: the appropriateness of AALDLT for patients with acute liver failure (ALF) and whether the Model for End-stage Liver Disease (MELD) score can be utilized to determine the suitability of AALDLT for a given patient. Here, we briefly highlight recent literature regarding these controversial topics. In many Asian centers where socio-cultural norms drastically limit utilization of cadaveric liver donation, AALDLT is being increasingly relied upon to offer expedient transplantation to patients with ALF (Table 1) [5]. Despite relatively widespread use of AALDLT for ALF in many Asian countries, issues regarding graft size and donor safety have prevented widespread acceptance of AALDLT for ALF in the United States. This reluctance to utilize AALDLT in the setting of ALF presumably increases the risk of death due to failure to quickly receive a graft for these critically ill patients. For example, we have found that even when patients with ALF are Number of living liver donors t /yr Figure 1 Number of United States living liver donors per year [4]. managed in large-volume, Western liver transplantation centers, the transplantation rate ranges from 41%-72%, and the median waiting time for a deceased donor graft is d [6]. These statistics clearly indicate the impressive need for more readily available liver grafts. The issue of AALDLT for ALF also sparks an ethical debate among the transplant community regarding issues such as how to assure adequate informed donor consent in a relatively pressured setting or whether it is appropriate to ask donors to give an organ to a recipient who may not have a successful outcome. Based on our recent analysis of the current ethical issues with AALD- LT for ALF, we feel that with adherence to rigorous informed consent efforts mediated by a donor advocate and recognition that most donors rate the experience of donating positively even if the recipient has a poor outcome, AALDLT can be offered to patients with ALF in a way that maintains the highest ethical standards for both the donor and the recipient [5]. Thus, given the extreme need for more liver grafts and the precedent of successful AALDLT outcomes in many Asian centers, United States centers should consider broadening the indication for AALDLT to include ALF. In addition to efforts to determine whether AALD- LT is appropriate for ALF, extensive efforts have also been directed toward determining if the MELD score can help predict the suitability of AALDLT for a given patient. The MELD score, which is based upon creatinine, total bilirubin, and INR, was originally developed by Malinchoc et al [7] to predict 3 mo survival in patients undergoing elective transjugular intrahepatic portosystemic shunt procedures. The MELD score has since been shown to be a valuable predictor of pre-transplantation survival, and in 2002 it became a critical tool in assigning priority to patients on the United Network for Organ Sharing (UNOS) transplantation waiting list. At the inception of this policy, little data regarding the predictive value of the MELD score in AALDLT existed thus promoting general concern among transplantation physicians that a higher MELD score (i.e. a sicker patient) may correlate with poor outcomes after AALDLT December 14, 2012 Volume 18 Issue 46

52 Carlisle EM et al. Liver transplantation Table 1 Literature review of adult to adult living donor liver transplantation for acute liver failure [5] Ref. Year Location n Recipient survival (%) Lobe utilized Donor complications (%) Donor survival (%) Liu et al [38] 2002 China Right Nishizaki et al [39] 2002 Japan Left No comment 100 Wu et al [40] 2004 Taiwan, China Right Lee et al [14] 2007 South Korea Both Kilic et al [41] 2007 Turkey 6 83 Right Campsen et al [42] 2008 United States Right Ikegami et al [43] 2008 Japan Both Park et al [44] 2010 South Korea Both This concern was based primarily upon the relatively poor early outcomes of AALDLT for critically ill patients. This issue was addressed in work by Testa et al [8] that investigated the appropriateness of AALDLT for patients with decompensated end-stage liver disease. The authors highlighted several early studies of AALDLT that described a 1 year survival of about 50% for critically ill patients which was far inferior to the 77%-80% 1 year survival reported for patients with a more favorable clinical status. The authors also cited their own 1 year survival for AALDLT in patients with a MELD score > 30 as 43%, which although relatively low, was considered successful given the high likelihood of patient death before a cadaveric donor graft could be allocated to them [8]. The relatively low survival rates reported in these studies led to a general consensus that critically ill patients should not be candidates for AALDLT. However, Testa el al advocated that rather than abandoning the procedure for these patients, the outcomes of AALDLT in the critically ill could be improved by gaining further experience in appropriate donor selection and working to overcome the technical difficulties of the operation [8]. Recent data from various transplantation centers, especially those in Korea, that heeded this recommendation and continued working to overcome the technical issues that initially contributed to poor outcomes now demonstrate survival in critically ill patients that closely parallels survival of patients who are less ill. Additionally, Hwang et al [9] and Bhangui et al [10] have recently demonstrated the success of AALDLT for patients with hepatocellular carcinoma. These improvements in outcome across a variety of disease etiologies and recipient clinical status suggest that AALDLT is an appropriate procedure for even the most critically ill patients. To further understand which critically ill patients would benefit most from AALDLT, several studies have since emerged to specifically investigate the predictive validity of the MELD score in AALDLT. For example, in a retrospective review of 62 AALDLT recipients by Hayashi et al [11], MELD score failed to predict 1-year patient or graft survival. To further analyze the predictive capacity the MELD score and 23 other preoperative factors Morioka et al [12] conducted a retrospective review of 335 cases of AALDLT in Japan between 1994 and The authors concluded that lack of pre-transplant encephalopathy, MELD score 30 (including points for persistent ascites and low serum sodium), and donor age < 50 were the key factors for obtaining successful outcomes with AALDLT. This work was contrasted by Durand et al [13] who conducted a retrospective review of 331 DDLT and 128 AALDLT cases to develop statistical models to determine the most efficacious means of organ allocation. This group determined that AALDLT is most advantageous when performed in patients at high risk of death. More simply, these authors demonstrated that the most critically ill patients will derive the most robust statistical benefit from AALDLT [13]. The studies reviewed here offer slightly conflicting suggestions regarding which patients will benefit most from AALDLT. Further work must be performed to more clearly delineate the indications for the procedure and to establish a meaningful selection criteria for potential recipients. However, it is our belief that current data generally suggests that the indications for AALDLT should be broadened. Reflection on the drastic improvements in survival for critically ill patients (including those with ALF) in many Asian centers illustrates how diligent refinement of surgical technique and increased procedural experience can result in drastically improved outcomes of AALDLT for even the most critically ill patients. Perhaps United States transplantation centers can call upon the wealth of knowledge generated by these centers to successfully incorporate AALDLT into the routine treatment of United States patients. DELICATE BALANCE BETWEEN GRAFT SIZE AND DONOR SAFETY In addition to determining the optimal indications for AALDLT, the transplantation community must advise on the delicate balance of assuring adequate graft size while maintaining donor safety. It is well accepted that AALDLT must be performed in careful balance between recipient needs and donor safety. For example, if acquiring adequate graft volume for the recipient equates to unacceptable risk for the donor, the transplantation must not be performed. Currently, a graft weight to body weight ratio of > 0.8% and a graft size of at least 40% of the standard liver volume is accepted as a minimum requirement for donation [14]. It has been advocated by some that use of a right lobe graft as compared to a 6731 December 14, 2012 Volume 18 Issue 46

53 Carlisle EM et al. Liver transplantation left lateral lobe as is used in pediatric living donor liver transplantation (LDLT) may allow for larger graft size while still maintaining reasonable donor risk. While it is presumed that the increased graft size places donors at a higher risk following a right hepatectomy, no controlled studies substantiate this concern. Further, presumption that donor morbidity or mortality is directly and solely related to the extent of the liver resection is not reasonable given the numerous other factors that may contribute to poor donor outcomes. The Korean experience demonstrates that with increased operative experience, strict donor selection, and an institutional focus on AALDLT, complications and mishaps can be drastically minimized [15]. Numerous studies have also been conducted in an effort to determine the safest selection criteria for donors. Authors at a prominent United States transplantation center reviewed the screening of 66 potential donors for 15 eventual AALDLT procedures [16]. The group relied upon 3D helical imaging including hepatic lobe volume renderings, vascular anatomy, virtual resection planes, preoperative arteriography, and medical/psychological examination, and found that even with robust preoperative donor screening in an experienced hepatobiliary center, morbidity occurred in 67% of donors [16]. A slightly more recent study of 893 AALDLT cases between 1994 and 2005 in Korea demonstrates how modification of graft size and careful donor selection can result in marked reduction in donor morbidity [15]. Specifically, until 2001, this group reported an AALDLT donor complication rate of 6.7% which was predominantly due to complications in right lobe liver donors. In 2002, authors changed their donor selection procedure such that liver resection exceeding 65% of total liver volume was avoided except in young donors with no evidence of hepatic steatosis. This change resulted in a reduction of donor morbidity to 1.3% and prompted authors to conclude that a majority of major living donor complications are avoidable through strict selection of living donor/graft type (with cautious selection of the donor right liver if it appears to be larger than 65% total liver volume), intensive postoperative surveillance, and prompt feedback regarding surgical technique. Perhaps most interestingly the authors commented that the experience that they gained from implementation of AALDLT has actually optimized all hepatobiliary surgery practices at their institution [15]. There is also an interesting body of work evaluating the accuracy of preoperative assessment of graft volume via 3D-CT volumetry in AALDLT. Both Hiroshige et al [17] and Kayashima et al [18] have demonstrated that 3D-CT volumetry may overestimate graft volume by as much as 13%, especially in donors under 30 yr old. Authors suggest that this may be due to graft dehydration secondary to University of Wisconsin solution. Studies such as these illustrate the difficulty in assuring accurate pre-operative assessment of graft volume. Given the obvious importance of assuring adequate graft size, future work to further optimize pre-operative assessment of graft volume is certainly warranted. Clearly, the transplantation community will benefit from further study regarding how to safely balance donor safety and graft size; however, studies such as those discussed here suggest that we are beginning to develop a more robust understanding of how to address this critical issue. We suggest that implementation of strict policies regarding acceptable graft size is the best method for avoiding complications related to small for size grafts or resection of an unsafe donor graft volume. However, surgical technique alone, even that delivered by expert surgeons, cannot substitute for the institutional organization at all levels of patient care that is mandatory to minimize complications. Any institution willing to offer AALDLT to its patients must invest in AALDLT. Specifically, in order to replicate the results of the best transplantation centers in the world, United States centers should focus on how the entirety of the operative experience, ranging from pre-operative donor evaluation to the number of capable transplant surgeons to the coordinated management of postoperative care, can be structured to provide the highest levels of success. ISSUE OF OUTFLOW The right lobe of the liver is increasingly being utilized to assure adequate graft volume in AALDLT. However, use of the right lobe brings with it a heightened risk of impaired graft outflow. Specifically, the right lobe graft carries an increased risk of early post-operative congestion of the paramedian segments 5 and 8 due to interruption of the venous drainage of the middle hepatic vein (MHV). Post-operative congestion has been shown to lead to congestive necrosis which has been reported to incite early post-operative graft failure and recipient death [19-21]. Harvest of an extended right lobe graft with inclusion of the entire donor MHV is an effective means of preventing outflow obstruction: however, the larger graft size may increase the donor s risk of morbidity and mortality. The surgeon s decision to resect or not resect the donor MHV is thus a complicated one that rests upon several key factors. Donor residual liver volume, the importance of MHV in right lobe drainage, the ratio of graft weight to recipient body weight, and the MELD score must all be carefully evaluated prior to determining whether resection of the MHV is appropriate. Generally, it seems prudent to suggest that if the donor residual liver volume is marginal (ratio < 0.6) or the rest volume is < 30%, the MHV should remain with the donor and segments 8 and 5 should be re-anastomosed. Several procedural modifications have been proposed to generate improved outflow in cases where the MVH must be interrupted. For example, in a review of 74 AALDLT patients, Malago et al [22] observed that rapid regeneration of the graft in the first 10 post-operative days 6732 December 14, 2012 Volume 18 Issue 46

54 Carlisle EM et al. Liver transplantation resulted in medial displacement of the graft such that kinking, torsion, and compression/occlusion of the outflow tract resulted. By using a cadaveric iliac vein graft to create an interposition conduit that allowed drainage of all intrahepatic veins (diameter > 5 mm) draining segments 5 and 8 into the MHV, the authors devised a modification of the outflow tract that enlarged the caval orifice and assured better outflow [22]. Successful use of this cadaveric vein outflow reconstruction procedure in patients undergoing AALDLT was also demonstrated by Dong et al [19]. Continued efforts to develop procedural modifications that assure successful outcomes in recipients of AALDLT while maximizing donor safety are imperative. Additionally, it is good practice to remember that every donor-recipient pair presents a unique set of anatomical challenges, however, these challenges should not be considered separately. Favorable outcomes are a result of the safest and most feasible combination of donor and recipient need. Any compromise at the expense of either may result in serious complications and poorer outcomes. Procedural flexibility is ultimately the best policy to protect the donor and assure prompt, successful graft functioning in the recipient. BILIARY COMPLICATIONS In addition to issues of impaired outflow, reliance on right lobe grafts for AALDLT has brought with it an increased rate of biliary complications. Overall, since the introduction of AALDLT with right lobe grafts, biliary complications have been the leading cause of post operative complication and re-operation [23-27]. The reported incidence of biliary complication in AALDLT ranges from 15%-60% [25,28-31], which is substantially greater than that of cadaveric full-liver transplantation 5%-15% [29,32,33], and pediatric LDLT with the left lateral lobe of 4%-6% [34]. Presumably, anatomic differences between the left and right hepatic biliary systems are responsible for this differential complication rate. Specifically, reliance upon the left hepatic duct usually allows a relatively easy dissection due to straightforward ductal anatomy. However, use of the right lobe hepatic system is markedly complicated by the numerous anatomic variations of the right duct. Multiple technical modifications for biliary reconstruction have been published over the past decade (use of recipients right and left hepatic ducts, end-side reconstruction of donor ducts to native common hepatic bile duct, donor ductoplasty, stents, T-tubes, etc.), however none has provided a standardized, replicable method that consistently decreases the incidence of biliary complications [24,30]. Although, biliary complications result in high recipient morbidity and occasionally mortality, early detection and prompt treatment offers a favorable rate of recovery in these patients. Perhaps most importantly, a high rate of success in non-operative management of biliary complications has been consistently demonstrated in the literature. For example, in a review of 429 patients that underwent liver transplantation, the success rates for treatment of biliary complications by endoscopic retrograde cholangiopancreatography and percutaneous transhepatic radiologic procedures were 100% and 78% respectively [35]. Given the consistently high rate of biliary complications despite over a decade of efforts to perfect the biliary anastomosis, assurance of robust nonoperative treatment modalities is imperative. Thus while continued refinement of technique is critical, we have limited expectation of an easy fix for biliary complications in AALDLT. It is generally clear that while most of the other technical obstacles to AALDLT have found favorable solutions, we are still far from having developed a biliary anastomosis technique that will provide consistently positive results. While relying on continued advancement in minimally invasive procedures to remedy these frequent complications is currently an acceptable compromise, it is imperative that we continue significant efforts to construct a biliary anastomosis with equivalent or lower complication rates than that of deceased donor liver transplant. ETHICAL ISSUES The ethical issues surrounding AALDLT are complex and warrant thoughtful discussion prior to widespread implementation of the procedure. Overall, the majority of regulations on liver transplantation are guided by a simple reality: liver grafts are provided by a public supply, and the supply is insufficient to meet current societal demands. The introduction of AALDLT presumably alters this climate by increasing the available supply of grafts. This increase in supply offers the opportunity of transplantation to patients who previously had relatively minimal chances of obtaining a graft given that their clinical status was not critical enough to assure high priority listing on the transplantation waiting list or because the etiology of their disease precluded them from being a transplant candidate (hepatocellular carcinoma, polycystic liver disease, etc. While introduction of AALDLT clearly expands the opportunity for many patients awaiting transplantation it carries with it multiple ethical questions. Overall, the issue of whether AALDLT is ethically appropriate requires careful assessment of the risks and benefits to the individual donor-recipient pair. The benefits to the potential recipient are relatively obvious. AALDLT offers potential recipients decreased time on the waiting list or, in some cases, the opportunity to completely bypass the waiting list. Further, with AALDLT potential recipients are able to undergo transplantation as an elective rather than urgent surgery thereby allowing increased control of variables such as graft ischemic time. However, given the healthy status of the donor, the age old vow of physicians, primum non nocere (first do no harm), mandates that the risks and 6733 December 14, 2012 Volume 18 Issue 46

55 Carlisle EM et al. Liver transplantation benefits to the donor be the primary focus of a discussion of transplantation ethics. In our brief review of the literature, it is evident that donors experience a relatively high risk of morbidity and a realistic risk of mortality in even the most well-qualified transplantation centers with the most experienced transplantation surgeons. The risk of short and long-term medical morbidity is compounded by the financial burdens that may result due to ongoing medical bills and time lost from work. Currently, no system is in place to assure that donors are not excessively burdened by such issues. However, one must remember that while this risk may be of high concern for United States donors, it may be less of an issue in European countries where public health insurance typically covers all donor expenses. While substantial health and financial risk is obvious for the donor, great benefits may also be incurred by consenting to living liver donation. The psychological benefit from this altruistic decision is one of the primary benefits to donors. A vast body of literature exists that consistently supports the finding that even in cases of poor recipient outcome, undergoing donation helps donors feel as if they have done everything possible to help save the life of their loved one [36]. Donation may also bring with it a decreased caregiver burden and increased participation of the recipient in subsequent household matters. Several other issues are central to a thorough discussion of the ethics of AALDLT. First, there is great concern among the transplantation community that critical recipient status (especially in the setting of ALF) or familial pressure may limit the ability of potential donors to fully engage in an informed consent discussion. Many worry that donors may feel pressured to donate, and they encourage a robust donor evaluation process with inclusion of a donor advocate who assures donors the opportunity to make their decision with minimal pressure from family or members of the health care team. Additionally, many worry that performing an AALDLT in a setting in which recipients may have a poor outcome (e.g., ALF) may not be appropriate. However, multiple authors have determined that even in settings in which recipients die or suffer significant morbidity, most donors rate the donation experience positively as they feel as if it has allowed them to do all they can to help save the life of their loved one [36]. Clearly, the ethical issues involved with AALDLT are complex and deserving of further discussion to assure the highest ethical standards are maintained when caring for the donor-recipient pair. HAVE WE IMPROVED SINCE 1997? Critical review of the success and failure of AALDLT over the past decade is imperative in assessing our current status. Improved transparency in outcomes reporting, development of uniform regulations, an increasing number of National Institutes of Health funded studies, and increasing procedural experience all suggest that our ability to safely and successfully perform AALDLT has improved since the initial introduction of the procedure in MOVING FORWARD Critical review of AALDLT suggests that the Western world both embraced and dismissed AALDLT too quickly [37]. A new framework for integration of AALDLT into United States medical practice that is rooted in clinical expertise, genuine need, and accurate reporting should be developed to create a new starting point for AALDLT in the United States. Focus upon the key factors of determining the appropriate indications, balancing graft size with donor safety, assuring adequate outflow, minimizing biliary complications, and mandating ethical rigor is imperative. Additionally, we offer several suggestions that we believe will assure thoughtful integration of AALDLT into United States practice. First, all AALDLT activity should be concentrated in centers with dedicated AALDLT staff and adequate field strength (capacity of surgical team to meet the technical demands of a relatively innovative procedure, proven success with all facets of hepatobiliary surgery, etc.). Next, we believe that proper training programs for staff along with validated criteria to demonstrate clinical competency should be developed. Further, incorporating knowledge from centers that are already world leaders in AALDLT to establish patient and graft survival that is superior to deceased donor liver transplantation must be the ultimate goal. Finally, working to improve the societal image of AALDLT is critical for assuring integration into current United States medical practice. It is our belief that careful attention to these key factors will help redefine the role of AALDLT in United States transplantation centers. And while this may not provide the panacea for long wait times and high recipient mortality that was originally assumed, we believe that robust integration of AALDLT into United States practice will offer significant improvements for patients awaiting liver transplantation. REFERENCES 1 Lo CM, Fan ST, Liu CL, Lo RJ, Lau GK, Wei WI, Li JH, Ng IO, Wong J. Extending the limit on the size of adult recipient in living donor liver transplantation using extended right lobe graft. Transplantation 1997; 63: Wachs ME, Bak TE, Karrer FM, Everson GT, Shrestha R, Trouillot TE, Mandell MS, Steinberg TG, Kam I. Adult living donor liver transplantation using a right hepatic lobe. 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The Hamburg liver transplant program. Clin Transpl 1997; Park JS, Kim MH, Lee SK, Seo DW, Lee SS, Han J, Min YI, Hwang S, Park KM, Lee YJ, Lee SG, Sung KB. Efficacy of endoscopic and percutaneous treatments for biliary complications after cadaveric and living donor liver transplantation. Gastrointest Endosc 2003; 57: Crowley-Matoka M, Siegler M, Cronin DC. Long-term quality of life issues among adult-to-pediatric living liver 6735 December 14, 2012 Volume 18 Issue 46

57 Carlisle EM et al. Liver transplantation donors: a qualitative exploration. Am J Transplant 2004; 4: Cronin DC, Millis JM, Siegler M. Transplantation of liver grafts from living donors into adults--too much, too soon. N Engl J Med 2001; 344: Liu CL, Fan ST, Lo CM, Yong BH, Fung AS, Wong J. Rightlobe live donor liver transplantation improves survival of patients with acute liver failure. Br J Surg 2002; 89: Nishizaki T, Hiroshige S, Ikegami T, Uchiyama H, Hashimoto K, Soejima Y, Shimada M. Living-donor liver transplantation for fulminant hepatic failure in adult patients with a left-lobe graft. Surgery 2002; 131: S182-S Wu YM, Ho MC, Hu RH, Ko WJ, Yang PM, Lai MY, Lee PH. Liver transplantation for acute hepatic failure. Transplant Proc 2004; 36: Kilic M, Aydin U, Noyan A, Arikan C, Aydogdu S, Akyildiz M, Karasu Z, Zeytunlu M, Alper M, Batur Y. Live donor liver transplantation for acute liver failure. Transplantation 2007; 84: Campsen J, Blei AT, Emond JC, Everhart JE, Freise CE, Lok AS, Saab S, Wisniewski KA, Trotter JF. Outcomes of living donor liver transplantation for acute liver failure: the adultto-adult living donor liver transplantation cohort study. Liver Transpl 2008; 14: Ikegami T, Taketomi A, Soejima Y, Yoshizumi T, Sanefuji K, Kayashima H, Shimada M, Maehara Y. Living donor liver transplantation for acute liver failure: a 10-year experience in a single center. J Am Coll Surg 2008; 206: Park SJ, Lim YS, Hwang S, Heo NY, Lee HC, Suh DJ, Yu E, Lee SG. Emergency adult-to-adult living-donor liver transplantation for acute liver failure in a hepatitis B virus endemic area. Hepatology 2010; 51: S- Editor Shi ZF L- Editor A E- Editor Zhang DN 6736 December 14, 2012 Volume 18 Issue 46

58 Online Submissions: doi: /wjg.v18.i World J Gastroenterol 2012 December 14; 18(46): ISSN (print) ISSN (online) 2012 Baishideng. All rights reserved. TOPIC HIGHLIGHT Marco Giuseppe Patti, MD, Professor, Series Editor Multidisciplinary approach for patients with esophageal cancer Victoria M Villaflor, Marco E Allaix, Bruce Minsky, Fernando A Herbella, Marco G Patti Victoria M Villaflor, Department of Medicine, Section Hematology/Oncology, University of Chicago Medicine, Chicago, IL 60637, United States Marco E Allaix, Fernando A Herbella, Marco G Patti, Department of Surgery, University of Chicago Medicine, Chicago, IL 60637, United States Bruce Minsky, Radiation Oncology, University of Chicago Medicine, Chicago, IL 60637, United States Author contributions: Villaflor VM, Allaix ME, Minsky B, Herbella FA and Patti MG designed and revised the manuscript; Villaflor VM wrote the manuscript. Correspondence to: Victoria M Villaflor, MD, Department of Medicine, Section Hematology/Oncology, University of Chicago, 5841 S Maryland MC2115, Chicago, IL 60637, United States. [email protected] Telephone: Fax: Received: April 18, 2012 Revised: November 19, 2012 Accepted: November 24, 2012 Published online: December 14, 2012 Abstract Patients with esophageal cancer have a poor prognosis because they often have no symptoms until their disease is advanced. There are no screening recommendations for patients unless they have Barrett s esophagitis or a significant family history of this disease. Often, esophageal cancer is not diagnosed until patients present with dysphagia, odynophagia, anemia or weight loss. When symptoms occur, the stage is often stage Ⅲ or greater. Treatment of patients with very early stage disease is fairly straight forward using only local treatment with surgical resection or endoscopic mucosal resection. The treatment of patients who have locally advanced esophageal cancer is more complex and controversial. Despite multiple trials, treatment recommendations are still unclear due to conflicting data. Sadly, much of our data is difficult to interpret due to many of the trials done have included very heterogeneous groups of patients both histologically as well as anatomically. Additionally, studies have been underpowered or stopped early due to poor accrual. In the United States, concurrent chemoradiotherapy prior to surgical resection has been accepted by many as standard of care in the locally advanced patient. Patients who have metastatic disease are treated palliatively. The aim of this article is to describe the multidisciplinary approach used by an established team at a single high volume center for esophageal cancer, and to review the literature which guides our treatment recommendations Baishideng. All rights reserved. Key words: Esophageal Cancer; Multimodality therapy; Multidisciplinary therapy; Chemoradiotherapy; Esophageal resection; Esophagectomy Peer reviewers: Toru Hiyama, MD, PhD, Health Service Center, Hiroshima University, Kagamiyama, Higashihiroshima , Japan; Dr. Yeng S Ang, MD, Department of Gastroenterology, Royal Albert Edward Infirmary, Wigan Lane, Wigan, Greater Manchester WN1 2NN, United Kingdom Villaflor VM, Allaix ME, Minsky B, Herbella FA, Patti MG. Multidisciplinary approach for patients with esophageal cancer. World J Gastroenterol 2012; 18(46): Available from: URL: i46/6737.htm DOI: INTRODUCTION Esophageal cancer is a growing epidemic with approximately new diagnosis and deaths annually worldwide [1,2]. Adenocarcinoma has increased in incidence while the incidence of squamous cell esophageal carcinoma has decreased in the Western world. This seems to be linked to gastroesophageal (GE) reflux disease and Barrett s esophagus [3-7]. The prognosis for these patients is generally poor because of the advanced stage at the time of presentation. The increase in use of pro December 14, 2012 Volume 18 Issue 46

59 Villaflor VM et al. Multidisciplinary approach for esophageal cancer ton-pump inhibitors over-the-counter has also decreased the impetus to seek physician assistance for reflux symptoms. Hence most of these patients will be diagnosed at a late stage, with approximately 50 percent of patients have advanced unresectable or metastatic cancer [7]. Most patients are not considered curable at diagnosis and are treated with chemotherapy and radiation, mostly with palliative intent. In patients who are fortunate enough to have potentially resectable disease, the data are not clear as to the best approach. Patients with very early disease may only require endoscopic mucosal resection or surgical resection. Others are treated with a combination of chemotherapy plus radiation (chemoradiation) plus surgery if they are deemed resectable. Ideally, we would like to have large randomized trials that were powered properly to support our treatment plans. As these studies do not exist in the esophageal cancer world, we are left to rely mainly on meta-analysis, small randomized trials, and historical reports to make decisions for our patients. These treatments require specialists from surgery, medical oncology, radiation oncology, and gastroenterology. It is imperative that these individuals work in a multidisciplinary fashion in order to deliver comprehensive care. The goal of this paper is to discuss the approach of an established multidisciplinary team in the treatment of patients with locoregionally advanced disease. EVALUATION OF THE PATIENT To obtain an adequate volume of tissue for diagnosis, a minimum of 7 core/pinch biopsy specimens in addition to brushings are recommended at the time of endoscopy. This approach improves the accuracy of diagnosis to 98%-100% [7]. In addition, it provides tissue for molecular marker analysis, as cancer therapy is beginning to focus on targeted therapies which may require tumor marker analysis. Staging studies should include a computed tomography of the chest and abdomen, a position emission tomography (PET) scan and endoscopic ultrasound by a specialized gastroenterologist trained and proficient in this technique. Biopsies should be obtained from suspicious lymph nodes if accessible. An esophagram is also helpful in determining the degree of esophageal stricture. With the seventh edition American Joint Committee on Cancer Staging System, it is imperative to determine the histology of the tumor and number of lymph nodes involved. In patients who have respiratory symptoms, a bronchoscopy should be done to evaluate for tracheoesophageal fistula formation. In patients with other pulmonary or abdominal findings on imaging studies, one may wish to pursue thoracoscopy or laparoscopy. In addition, it is important to assess the performance status, the nutritional status and the patient s comorbidities of prior to determining an appropriate treatment plan. TREATMENT APPROACHES Treatment of ct1-2n0 disease Surgery alone remains the standard of care for patients with local disease (ct1n0 and some ct2n0 tumors). At our institution and other high volume institutions, patients with T1aN0 tumors are treated with minimally invasive techniques such as endoscopic mucosal resection. There is limited experience with the use of radiation or chemoradiotherapy in the curative setting for patients with ct1n0 disease [8]. Thirty-four patients with either medically inoperable disease or refused surgery were treated with external beam alone (64 Gy) or external beam (52 Gy) plus 8 to 12 Gy with brachytherapy. The median follow-up was 61 mo, 5-year survival was 59%, 68% local relapse-free survival, and 80% cause-specific survival [8]. For most ct2n0 tumors, surgery alone may not be sufficient since approximately 50% of patients may have lymph node metastasis [9-12]. However, if the nodes are negative (pt2n0m0) there is no role for postoperative adjuvant chemotherapy or chemoradiation. Treatment of ct3-4 and/or N positive disease There remains much controversy in what is considered the current standard of care for patients with locally advanced esophageal cancer (ct3-4 and or N positive) [13-15]. Initially, surgical resection was the main modality for esophageal cancer treatment. Since the 1980 s, studies have evaluated the utility for perioperative chemotherapy, postoperative and more commonly preoperative chemoradiation to improve outcome. These studies have been criticized for a variety of insufficiencies including inadequate power, the type of chemotherapy regimen, the dosing of chemotherapy, the radiation dose and fraction size, radiation delivery schedules, number of patients enrolled, initial staging, multiple organ sites and histologic subtype. At our institution, we advocate the use of neoadjuvant chemoradiotherapy based on the following data. DO PATIENTS BENEFIT FROM NEOADJUVANT RADIATION? There have been five phase Ⅲ trials which evaluated neoadjuvant radiation in esophageal cancer. None of the studies have demonstrated an increase in overall survival or resectability of esophageal cancer patients treated with radiation alone [16-20]. Nygaard et al [19] reported a 3-year overall survival benefit only after adding patients who also received chemotherapy to the statistical analysis. A meta-analysis of neoadjuvant radiation revealed a trend toward improved 5-year overall survival but failed to show a statistically significant survival advantage [21]. Data do not support the use of radiation as a single modality in the neoadjuvant treatment of esophageal cancer (Table 1). The role of radiation alone should be limited to palliation. DO PATIENTS BENEFIT FROM PERIOPERATIVE CHEMOTHERAPY? Neoadjuvant or perioperative chemotherapy has also 6738 December 14, 2012 Volume 18 Issue 46

60 Villaflor VM et al. Multidisciplinary approach for esophageal cancer Table 1 Randomized trials of neoadjuvant radiation in esophageal cancer Ref. Histology n Rad dose (Gy) 2-yr survival, % been evaluated in patients with locally advanced gastric and GE cancer. At least five phase Ⅲ trials have compared cisplatin-based regimens to surgery alone in esophageal cancer and three studies showed a survival advantage (Table 2) [22-26]. The Medical Research Council trial is the largest of these studies as it randomized 802 patients with esophageal adenocarcinoma or squamous cell carcinoma. There was a 5-year overall survival advantage of approximately 6% [24]. The Magic trial randomized 503 patients, predominately gastric cancer patients, and demonstrates a 5-year overall survival advantage of 13% [22]. The French Cooperative Group study randomized 224 predominately gastric cancers with a survival advantage of 14% at 5 years [26]. All of these studies noted no evidence of increased morbidity or mortality in patients who received neoadjuvant chemotherapy. Many studies that evaluated perioperative chemotherapy have shown some overall survival benefit. This is evident in Gebski s meta-analysis that evaluated 1724 patients who received chemotherapy and surgery versus surgery alone in 8 trials. Of note was a 7% absolute benefit in 2 years survival (P = 0.014) in adenocarcinoma patients only [27]. These data are complicated because only 2 of the studies evaluate only esophageal cancer [23,24]. While we feel there is some benefit to perioperative chemotherapy, we do not advocate its use as the data suggest neoadjuvant chemoradiotherapy to be superior in esophageal cancer. DO PATIENTS BENEFIT FROM 5-yr survival, % Launois et al [18] SCC NR /5 NR Gignoux et al [17] SCC / Wang et al [20] NR Nygaard et al [19] SCC 50 - NR 9 (3 yr) (4 wk) NR 21 (3 yr) Arnott et al [16] SCC 86 - NR 17 AC (10 d) NR 9 SCC: Squamous cell carcinoma; NR: Not reported; AC: Adenocarcinoma. CONCURRENT CHEMORADIOTHERAPY? Chemotherapy combined with radiation enhances the effects of radiation by synergistically damaging the DNA following cell cycle synchronization [28,29]. Chemotherapy theoretically also reduces the risk of distant metastatic disease by eradication of micrometastases [30]. Chemoradiation is useful in both the neoadjuvant setting for all esophageal cancer patients or in the adjuvant setting for patients with GE junction tumors. Additionally, in patients who are not surgical candidates chemoradiation may be used as definitive treatment [31]. Ideally, concurrent chemoradiation should be done by a multidisciplinary group proficient in these procedures as many situations may result in a less favorable outcome. Situations which may occur include unnecessarily missed chemotherapy or radiation doses for complications which could be managed by groups more experienced in this technique. Additionally, the use of unconventional chemotherapy or radiation regimens or erroneous staging studies may also be problematic. Initially, concurrent chemoradiation was evaluated as definitive treatment for patients who were not surgical candidates in the Radiation Therapy Oncology Group (RTOG) trial [31]. In this study 134 patients were randomized to cisplatin combined with infusional fluorouracil and concurrent radiation or to radiation alone. The patients predominately had esophageal squamous cell carcinoma. Interim analysis revealed that a statistically significant survival advantage favoring concurrent chemoradiotherapy hence changing the treatment paradigm in inoperable locally advanced esophageal cancer. The 5-year overall survival was 27% vs 0% with radiation alone [31]. Despite the reduction in the risk of persistent disease or local recurrence with concurrent chemoradiotherapy compared to radiation alone, the incidence of locoregional failure was a dismal 47% [31]. Hence, in an effort to reduce locoregional failure, radiation dose was then addressed by the INT 0123 trial [32]. A total of 236 patients were randomized to high (68.4 Gy) or low (50.4 Gy) dose radiation all given with concurrent cisplatin and infusional fluorouracil per the RTOG regimen. An interim analysis failed to reveal a local control or survival benefit with high dose radiation hence, 50.4 Gy has become standard of care for both neoadjuvant and definitive radiotherapy [32]. Patients with esophageal cancer have unacceptably high locoregional failure rates of approximately 50% with chemoradiation and a dismal prognosis of 20%-25% at 5 years with surgery alone [23,33-35]. Based on the limited success of these two approaches, a number of studies evaluating the combination of chemoradiation and surgery were developed. DO PATIENTS BENEFIT FROM SURGERY? Surgery has been considered an essential part of the treatment of patients with esophageal carcinoma [36]. Past experiences showed that a nonsurgical approach was associated with mediocre survival results [37]. However, the better survival achieved with surgical therapy may have a high price. In 1980, Earlam et al [38] reviewed the literature and reported 29% mortality for esophagectomy. Today, some still quote these numbers as a justification for nonsurgical approach to esophageal cancer, stating if a patient with esophageal cancer may either die by the tumor or die by the knife. However, it is hard to believe that these numbers remain true after significant improvements in antibiotics, intensive care, surgical equipment, and technique. Additionally, developments in chemotherapy and radiotherapy have occurred as well December 14, 2012 Volume 18 Issue 46

61 Villaflor VM et al. Multidisciplinary approach for esophageal cancer What is the best surgical technique? There are 3 different basic approaches for esophagectomy: (1) transhiatal; (2) transthoracic; and (3) en bloc or radical. The transhiatal approach has a theoretical advantage of a decreased morbidity and mortality due to the avoidance of a thoracotomy (and thus, a decreased operative time and pain). Even though concern has been raised about lesser oncologic radicality, several studies compared the outcomes for transhiatal versus transthoracic esophagectomy. Two meta-analysis in 2 different decades (2001 and 2011) showed no differences in survival comparing these two approaches [39,40]. The transthoracic group; however, had significantly more respiratory complications, wound infections, and early postoperative mortality, whereas anastomotic leak, anastomotic stricture, and recurrent laryngeal nerve palsy rates were significantly higher in the transhiatal group. Population studies reached the same conclusions. Chang et al [41] evaluated a pool of 868 patients from the American Surveillance, Epidemiology, and End Results-Medicare linked database (1992 to 2002) with similar results. Additionally, Connors et al [42] consulted the registries of patients from the American Nationwide Inpatient Sample database and found similar outcomes for both procedures. The need for lymphadenectomy (radical esophagectomy) is an ongoing debate in esophageal surgery. Also, it is unclear if the patients that may benefit from these procedures are the ones with early cancer or locally advanced tumors. Moreover, the extent of the lymphadenectomy (1 field-thoracic, 2 fields- thoracic and abdominal, 3 fields-thoracic, abdominal and cervical) is a controversial topic. The lack of randomized trials addressing this issue increases the controversy. Although some studies showed better survival after en bloc esophagectomy others showed results similar to a transhiatal esophagectomy [43,44]. Morbidity and mortality for this procedure is not always reported; however, it seems to be high, especially in 3-field [45]. Minimally invasive surgery has the advantages of better cosmetic results, reduced operative stress, postoperative immobility, and pain. As far as we are aware, no randomized controlled trials have compared minimally invasive and open esophagectomy to date. Available data, including 3 recent meta-analysis suggests that minimally invasive esophagectomy is similar to conventional esophagectomy in terms of complications, oncologic radicality and survival [46-49]. Modern outcomes for mortality and survival Different studies from experienced centers show a rate of mortality close to 0 in patients with non-advanced or even advanced tumors [50-53]. The application of standardized protocols with a multidisciplinary team improved significantly the outcomes of esophagectomy. It is not easy to access the survival related to esophagectomy only since most series of surgery alone are related to initial cancer and most surgeons refer patients to neoadjuvant or adjuvant chemotherapy [54]. However, patients with potentially resectable tumor not referred for surgery have a lower survival rate [55]. Relationship between volume and outcomes Different papers repeatedly reported better outcomes for esophagectomy in high volume centers [42,56,57]. This better results may be attributable to surgeons experience, since a decrease in more than 50% in the index of complications following esophagectomy is observed when the operation is performed by surgeons experienced in more than 100 esophagectomies [58]. However, hospital volume is also important, since the preparedness of the multidisciplinary team and hospital services to attend esophagectomy patients is crucial to better outcomes. Even low volume hospitals with high nurse ratios, lung transplantation services, complex medical oncology services, bariatric surgery services, and positron emission tomography scanners have lower mortality rates compared with low-volume hospitals with none of these characteristics [59]. Very interestingly, survival was not linked to volume [60]. DO PATIENTS WHO UNDERGO SURGERY BENEFIT FROM NEOADJUVANT CHEMORADIATION? To date, there have been eleven randomized trials performed evaluating the utility of neoadjuvant chemoradiotherapy added to surgery (Table 3). These trials have incorporated a variety of chemotherapy regimens, doses and fraction sizes of radiation and timing of both chemotherapy and radiation. Of these studies only 3 have shown a benefit with concurrent chemoradiotherapy. The CALGB 9781 study randomly assigned patients to cisplatin, infusional fluorouracil with concurrent radiation and surgery or to surgery alone. The study was unable to adequately accrue due to patient and investigator bias favoring the neoadjuvant arm. Despite the lack of accrual in this study there was an impressive five-year overall survival of 39% with multimodality treatment and 16% with surgery alone (P = 0.002) [61]. The study performed by Walsh et al [62] randomized 113 patients with adenocarcinoma to cisplatin, infusional fluorouracil and radiotherapy followed by surgery or to surgery alone. The median overall survival was 16 mo with multimodality treatment and 11 mo with surgery alone (P = 0.01). Three-year survival of 32% with multimodality treatment and 6% with surgery alone (P = 0.01). This study has been criticized for inadequate radiation dose, inadequate fluorouracil dose, survival in the control arm lower than historical controls, and lack of adequate staging prior to chemoradiotherapy [62]. The CROSS trial has been reported in abstract form by van der Gaast et al [63]. A total of 363 patients with adenocarcinoma or squamous cell carcinoma were randomized to preoperative paclitaxel/carboplatin plus 41.4 Gy vs surgery alone December 14, 2012 Volume 18 Issue 46

62 Villaflor VM et al. Multidisciplinary approach for esophageal cancer Table 2 Randomized trials of peri-operative chemotherapy in gastric and esophageal cancer Ref. Histology Regimen n Resection pcr 5-yr survival % P value Median survival (mo) Kelson et al [23] SCC (45%) S % NA 23 (3 yr) NS 16.1 AC (55%) CF S CF % 2.50% 26 (3 yr) 14.9 Cunningham et al [22] AC - Gastric S % NA % GEJ ECF S ECF % 0% MRC et al [24] SCC (35%) S % NA AC (65%) CF S % NA Roth et al [25] AC/SCC S 20 NR NR 5 NS 9 BVC S Ychou et al [26] AC 75% GEJ S NA NR 25% Gastric CF S (CF) NR 38 NR CF: Cisplatin and 5-fluorouracil; SCC: Squamous cell carcinoma; ECF: Epirubicin, cisplatin and 5-fluorouracil; AC: Adenocarcinoma; NA: Not Applicable; GEF: Gastroesphageal junction/distal esophagus; NR: Not recorded; NS: Not significant; S: Surgery; BVC: Bleomycin, vindesine, cisplatin; pcr: Protein catabolic rate; GEJ: Gastroesophageal junction. Table 3 Randomized trials of neoadjuvant combined modality therapy for esophageal cancer Ref. Cell type n Total dose (Gy) 5-yr survival, % Median survival (mo) P value Criticism Nygaard et al [19] SCC 47 BP surg 11.5 (3 yr) 8 NS Unconventional chemotherapy and low dose RT 41 Surgery 9.5 (3 yr) 7 Bosset et al [34] SCC 143 P surg 19 NS Split course RT and unconventional chemo schedule 139 Surgery 9 19 Tepper et al [61] SCC (25%) 30 PF surg < Only 56 of 475 planned patients entered AC (75%) 26 Surgery Walsh et al [62] AC (100%) 58 PF surg 32 (3 yr) 16 < 0.05 Only 6% 5 yr survival benefit with surgery alone 55 Surgery 6 (3 yr) 11 Gaast et al [63] SCC (25%) 175 Carbo/tax surg 59 (3 yr) 49 < Only 41 Gy RT AC (75%) 188 Surgery 48 (3 yr) 26 Le Prise et al [85] SCC 41 PF + 20 (split) + surg 19 (3 yr) 10 NS Only some patients received split course radiotherapy 45 Surgery 14 (3 yr) 10 chemotherapy Apinop et al [86] SCC 35 PF surg NS Low dose RT 34 Surgery 10 7 Lee et al [87] SCC 51 PF (bid) +surg 49 (3 yr) 28 NS 1.2 Gy bid radiation 50 Surgery 51 (3 yr) 27 Urba et al [88] SCC (25%) 47 PF surg 30 (3 yr) 17 NS 15% survival benefit but not statistically significant AC (75%) 50 Surgery 16 (3 yr) 18 Burmeister et al [89] SCC (37%) 128 PF surg NS Only 35 by radiation delivered AC (62%) 128 Surgery Mariette et al [90] 97 PF surg NR TI-2 only, Hi postoperative mortality c CRT 98 Surgery 44 P: Cisplatin; BP: Bleomycin and cisplatin; NS: Not significant; PF: Cisplatin and 5-fluorouracil; AC: Adenocarcinoma; SCC: Squamous cell carcinoma; NR: Not reported; NS: Not significant; RT: Radiation; CRT: Chemoradiotherapy. With a median follow-up of 32 mo patients who received chemoradiation had a significant benefit in 3-year survival (59% vs 48%, P = 0.011). There was also an increase in (RO) resection rates 67% vs 92%, favoring chemoradiotherapy [63]. Given the contradictory and inconclusive results in many of the trials evaluating neoadjuvant multimodality treatment based on disparate study populations, differing histology, differing chemotherapy and radiotherapy doses and regimens, and small numbers of patients, data have been pooled in an effort to synthesize the data into larger numbers to discover if a survival benefit exists [64]. The first meta-analysis published by Urschel et al [65], included nine randomized controlled trials and 1116 patients. A trend toward 3-year survival improvement favoring neoadjuvant chemoradiotherapy was noted with the most pronounced effect with concurrent chemoradiotherapy as compared to a sequential approach. There was a decreased risk of local-regional recurrence but concerning trend toward increased treatment mortality with multimodality treatment. There was no difference in the risk of distant recurrence. Fiorica et al [66] noted an improvement in patients who received neoadjuvant chemoradiotherapy. Gebski et al [27] also evaluated neoadjuvant chemotherapy and chemoradiotherapy compared to surgery alone in a meta-analysis. This recent metaanalysis evaluated 1209 patients with both adenocarcinoma and squamous cell carcinoma of the esophagus in 6741 December 14, 2012 Volume 18 Issue 46

63 Villaflor VM et al. Multidisciplinary approach for esophageal cancer ten trials. A statistically significant benefit was noted with neoadjuvant chemoradiotherapy compared to surgery alone with a 19% decrease in the risk of death corresponding to a 13% absolute difference in 2 year survival. An absolute survival benefit of 7% was noted for neoadjuvant chemotherapy as compared to surgery alone. The Preoperative Chemotherapy or Radiochemotherapy in Esophagogastric Adenocarcinoma Trial (POET) attempted to determine in a prospective, randomized fashion if neoadjuvant concurrent chemoradiotherapy is more beneficial then perioperative chemotherapy [67]. There was a trend toward improved pathologic complete response with neoadjuvant chemoradiotherapy but the study was closed early due to lack of accrual [67]. Given these data, we plan neoadjuvant chemoradiotherapy in our eligible patients. DO PATIENTS WHO UNDERGO CHEMORADIATION BENEFIT FROM SURGERY? Two randomized trials examine whether surgery is necessary after chemoradiation. In the FFCD 9102 trial, 445 patients with clinically resectable T3-4N0-1M0 squamous cell carcinoma of the esophagus received initial chemoradiation [68]. Patients initially received 2 cycles of 5-fluorouracil (5-FU), cisplatin, and concurrent radiation (either 46 Gy at 2 Gy/d or split course 15 Gy weeks 1 and 3 [68] ). The 259 patients who had at least a partial response were then randomized to surgery vs additional chemoradiation which included 3 cycles of 5-FU, cisplatin, and concurrent radiation (either 20 Gy at 2 Gy/d or split course 15 Gy). There was no significant difference in 2-year survival (34% vs 40%, P = 0.56) or median survival (18 mo vs 19 mo) in patients who underwent surgery vs additional chemoradiation. These data suggest that for patients who initially respond to chemoradiation, they should complete chemoradiation rather than stop and undergo surgery. The German Oesophageal Cancer Study Group compared preoperative chemoradiation followed by surgery vs chemoradiation alone [69]. In this trial, 172 patients < 70 years old with ut3-4n0-1m0 squamous cell cancers of the esophagus were randomized to preoperative therapy (3 cycles of 5-FU, leucovorin, etoposide, and cisplatin, followed by concurrent etoposide, cisplatin, plus 40 Gy) followed by surgery vs chemoradiation alone (the same chemotherapy but the radiation dose was increased to Gy +/- brachytherapy). In patients who underwent surgical resection, 35% had complete pathologic response and 33% had no evidence of lymph node involvement following neoadjuvant therapy [69]. Despite a decrease in 2-year local failure (36% vs 58%, P = 0.003) there was no statistically significant difference in 3-year survival (31% vs 24%) for those who were randomized to preoperative chemoradiation followed by surgery vs chemoradiation alone. The practice at our institution is to closely observe patients with esophageal squamous cell cancer who have a complete clinical response. Patients with adenocarcinoma continue to require surgical resection as these studies only evaluate patients with squamous cell cancer and studies to address adenocarcinoma have not been done. DO PATIENTS WHO HAVE SURGERY BENEFIT FROM ADJUVANT TREATMENT? In an effort to address locally advanced gastric and GE junction cancers adjuvant chemoradiotherapy was evaluated (MacDonald) [70]. This trial (INT 0116) enrolled 556 patients with gastric and GE junction (approximately 20%) adenocarcinoma. Patients were randomized to surgery alone or surgery followed by adjuvant leucovorinmodulated fluorouracil with concurrent radiation (45 Gy) in cycle 2 of 4 total cycles. There was an improvement in median overall survival with adjuvant therapy 36 mo vs 27 mo in the observation group (P = 0.005) [70]. Treatment related toxicity prevented completion of the treatment in 17% of patients [70]. There are no data evaluating the utility of adjuvant therapy in patients with more proximal esophageal tumors. At our institution, we plan neoadjuvant chemoradiation in locally advanced esophageal cancer patients. If following surgery, the pathology upstages the cancer, we plan for adjuvant treatment only in cancers of the GE junction. WHAT IS OUR PRACTICE AND RATIONALE? In summary, although there is good rationale for its use, it is not clear that the combination of surgery and chemoradiation regardless of the sequence, improves the survival results of either treatment alone. The survival benefit is likely to be 5%-10% with multimodality therapy. Currently, the standard of care in treatment of locally advanced tumors at our institution is to place patients on neoadjuvant chemoradiotherapy provided that it is feasible. We take into account the tumor location, size of the radiation field, comorbidities, and performance status in determining what the best multimodality approach is. There are many institutions, especially in Europe, who use neoadjuvant chemotherapy only. It is our practice to use neoadjuvant chemoradiotherapy because of the findings of the CALGB 9781, Walsh study, multiple meta-analysis and more recently the POET study [27,61,62,67]. The Gebski metaanalysis quoted a 13% absolute benefit in 2 year survival with neoadjuvant chemoradiotherapy and a 7% absolute benefit with neoadjuvant chemotherapy [27]. This is almost a doubling of the benefit conferred with perioperative chemotherapy alone. Additionally, the POET study, the only study which compares neoadjuvant chemotherapy to neoadjuvant chemoradiotherapy demonstrates a trend toward increased pathologic complete response at resection and survival with neoadjuvant chemoradiotherapy [67]. This study was performed in patients with GE junction 6742 December 14, 2012 Volume 18 Issue 46

64 Villaflor VM et al. Multidisciplinary approach for esophageal cancer tumors and was closed early due to poor recruitment. Additionally, we feel surgery may not be required in patients with esophageal squamous cell carcinoma provided they have a complete response to neoadjuvant therapy. Given that squamous cell carcinomas often recur locally, observation alone may be acceptable in this small group of patients. For those patients who have adenocarcinoma, we feel resection is still standard of care. WHAT IS BEING EVALUATED CURRENTLY IN ESOPHAGEAL CANCER? Despite improvements noted with multimodality treatment in esophageal cancer, cure rates are consistently dismal [27]. With new interest in targeted agents in cancer demonstrating benefit in malignancies of the head and neck, breast, lung, colon and pancreas have generated evaluation in the esophagus [71-73]. Multiple molecular pathways have been evaluated at the molecular level with potential targets in esophageal cancer including cyclin dependent kinases, nuclear factor κ, matrix metalloproteinases, inhibition of cyclooxygenase-2, c-met (a protooncogene that encodes a protein known as hepatocyte growth factor), epidermal growth factor receptor (EGFR) and vascular endothelial growth factor [72]. Over-expression of EGFR proteins may occur in 30%-70% of both adenocarcinoma and squamous cell carcinoma of the esophagus. Over-expression is associated with increased aggressiveness of the malignancy and poor prognosis [74-76]. Clinical trials have been initiated trying to take advantage of this protein. The Southwest Oncology Group initially targeted this protein by using single agent cetuximab as a second-line therapy with discouraging results [77]. More recent studies have evaluated cetuximab or other monoclonal EGFR antibodies with chemotherapy appear to be more promising [78-80]. Recently, EGFR-2 (Her-2-neu) has also been evaluated in gastric and esophageal cancers over expressing human EGFR-2 HER2 (ToGA trial) with promising results [81]. These targeted agents are currently undergoing evaluation in a multimodality setting with chemoradiotherapy. Safran et al [82] have evaluated 57 patients with esophageal cancer with weekly carboplatin, paclitaxel, cetuximab, and concurrent radiation (50.4 Gy). Complete clinical response was achieved in 70% of patients. Forty-nine of patients went on to surgery with a pathologic complete response rate of 27%. The RTOG 0436 is an ongoing phase Ⅲ trial evaluating weekly carboplatin, paclitaxel, and concurrent radiation with or without cetuximab in locally advanced inoperable patients. Additionally, given the results on the ToGA trial trastuzumab is currently under investigation with cisplatin, paclitaxel, and concurrent radiation for locally-advanced, HER2 overexpressing adenocarcinoma of the esophagus [83]. Survival benefits of neoadjuvant therapy appear small, but is should be noted this is similar to other treatments for other lethal malignancies [84]. The need to treat approximately 8 patients with a toxic regimen to cure one additional patient is not ideal, yet these odds must be discussed with a patient who is felt to be medically fit to withstand an esophagectomy [64]. Additionally, patients will often question about adjuvant therapy. While appropriate, in patients who have GE junction tumors only this is poorly tolerated post surgically, involves a larger radiation field, and radiation doses are lower [70]. Hence, our recommendations in the locally advanced resectable patient remain neoadjuvant chemoradiotherapy followed by esophagectomy. How targeted therapies will affect our approach in locally advanced esophageal carcinoma and is currently under investigation [73,76]. 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66 Villaflor VM et al. Multidisciplinary approach for esophageal cancer 48 Nagpal K, Ahmed K, Vats A, Yakoub D, James D, Ashrafian H, Darzi A, Moorthy K, Athanasiou T. Is minimally invasive surgery beneficial in the management of esophageal cancer? A meta-analysis. Surg Endosc 2010; 24: Sgourakis G, Gockel I, Radtke A, Musholt TJ, Timm S, Rink A, Tsiamis A, Karaliotas C, Lang H. Minimally invasive versus open esophagectomy: meta-analysis of outcomes. Dig Dis Sci 2010; 55: Low DE, Kunz S, Schembre D, Otero H, Malpass T, Hsi A, Song G, Hinke R, Kozarek RA. Esophagectomy--it s not just about mortality anymore: standardized perioperative clinical pathways improve outcomes in patients with esophageal cancer. J Gastrointest Surg 2007; 11: ; discussion Pennathur A, Farkas A, Krasinskas AM, Ferson PF, Gooding WE, Gibson MK, Schuchert MJ, Landreneau RJ, Luketich JD. Esophagectomy for T1 esophageal cancer: outcomes in 100 patients and implications for endoscopic therapy. 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High volume centers for esophagectomy: what is the number needed to achieve low postoperative mortality? Dis Esophagus 2004; 17: Wouters MW, Karim-Kos HE, le Cessie S, Wijnhoven BP, Stassen LP, Steup WH, Tilanus HW, Tollenaar RA. Centralization of esophageal cancer surgery: does it improve clinical outcome? Ann Surg Oncol 2009; 16: Yasunaga H, Matsuyama Y, Ohe K. Effects of hospital and surgeon case-volumes on postoperative complications and length of stay after esophagectomy in Japan. Surg Today 2009; 39: Funk LM, Gawande AA, Semel ME, Lipsitz SR, Berry WR, Zinner MJ, Jha AK. Esophagectomy outcomes at low-volume hospitals: the association between systems characteristics and mortality. Ann Surg 2011; 253: Anderson O, Ni Z, Møller H, Coupland VH, Davies EA, Allum WH, Hanna GB. Hospital volume and survival in oesophagectomy and gastrectomy for cancer. Eur J Cancer 2011; 47: Tepper J, Krasna MJ, Niedzwiecki D, Hollis D, Reed CE, Goldberg R, Kiel K, Willett C, Sugarbaker D, Mayer R. 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67 Villaflor VM et al. Multidisciplinary approach for esophageal cancer cisplatin-5-fluorouracil alone in first-line metastatic squamous cell carcinoma of the esophagus: a randomized phase II study of the Arbeitsgemeinschaft Internistische Onkologie. Ann Oncol 2009; 20: Pinto C, Di Fabio F, Barone C, Siena S, Falcone A, Cascinu S, Rojas Llimpe FL, Stella G, Schinzari G, Artale S, Mutri V, Giaquinta S, Giannetta L, Bardelli A, Martoni AA. Phase II study of cetuximab in combination with cisplatin and docetaxel in patients with untreated advanced gastric or gastro-oesophageal junction adenocarcinoma (DOCETUX study). Br J Cancer 2009; 101: Pinto C, Di Fabio F, Siena S, Cascinu S, Rojas Llimpe FL, Ceccarelli C, Mutri V, Giannetta L, Giaquinta S, Funaioli C, Berardi R, Longobardi C, Piana E, Martoni AA. Phase II study of cetuximab in combination with FOLFIRI in patients with untreated advanced gastric or gastroesophageal junction adenocarcinoma (FOLCETUX study). Ann Oncol 2007; 18: Bang YJ, Van Cutsem E, Feyereislova A, Chung HC, Shen L, Sawaki A, Lordick F, Ohtsu A, Omuro Y, Satoh T, Aprile G, Kulikov E, Hill J, Lehle M, Rüschoff J, Kang YK. Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial. Lancet 2010; 376: Safran H, Suntharalingam M, Dipetrillo T, Ng T, Doyle LA, Krasna M, Plette A, Evans D, Wanebo H, Akerman P, Spector J, Kennedy N, Kennedy T. Cetuximab with concurrent chemoradiation for esophagogastric cancer: assessment of toxicity. Int J Radiat Oncol Biol Phys 2008; 70: Safran H, Dipetrillo T, Akerman P, Ng T, Evans D, Steinhoff M, Benton D, Purviance J, Goldstein L, Tantravahi U, Kennedy T. Phase I/II study of trastuzumab, paclitaxel, cisplatin and radiation for locally advanced, HER2 overexpressing, esophageal adenocarcinoma. Int J Radiat Oncol Biol Phys 2007; 67: Pignon JP, Tribodet H, Scagliotti GV, Douillard JY, Shepherd FA, Stephens RJ, Dunant A, Torri V, Rosell R, Seymour L, Spiro SG, Rolland E, Fossati R, Aubert D, Ding K, Waller D, Le Chevalier T. Lung adjuvant cisplatin evaluation: a pooled analysis by the LACE Collaborative Group. J Clin Oncol 2008; 26: Le Prise E, Etienne PL, Meunier B, Maddern G, Ben Hassel M, Gedouin D, Boutin D, Campion JP, Launois B. A randomized study of chemotherapy, radiation therapy, and surgery versus surgery for localized squamous cell carcinoma of the esophagus. Cancer 1994; 73: Apinop C, Puttisak P, Preecha N. A prospective study of combined therapy in esophageal cancer. Hepatogastroenterology 1994; 41: Lee JL, Park SI, Kim SB, Jung HY, Lee GH, Kim JH, Song HY, Cho KJ, Kim WK, Lee JS, Kim SH, Min YI. A single institutional phase III trial of preoperative chemotherapy with hyperfractionation radiotherapy plus surgery versus surgery alone for resectable esophageal squamous cell carcinoma. Ann Oncol 2004; 15: Urba SG, Orringer MB, Turrisi A, Iannettoni M, Forastiere A, Strawderman M. Randomized trial of preoperative chemoradiation versus surgery alone in patients with locoregional esophageal carcinoma. J Clin Oncol 2001; 19: Burmeister BH, Smithers BM, Gebski V, Fitzgerald L, Simes RJ, Devitt P, Ackland S, Gotley DC, Joseph D, Millar J, North J, Walpole ET, Denham JW. Surgery alone versus chemoradiotherapy followed by surgery for resectable cancer of the oesophagus: a randomised controlled phase III trial. Lancet Oncol 2005; 6: Mariette C, Seitz JF, Maillard E, Mornex F, Thomas PA, Raoul J, Boige V, Pezet D, Genet C, Bedenne L. Surgery alone versus chemoradiotherapy followed by surgery for localized esophageal cancer: analysis of a randomized controlled phase III trial FFCD ProcASCO 2010; 28: 15s S- Editor Gou SX L- Editor A E- Editor Xiong L 6746 December 14, 2012 Volume 18 Issue 46

68 Online Submissions: doi: /wjg.v18.i World J Gastroenterol 2012 December 14; 18(46): ISSN (print) ISSN (online) 2012 Baishideng. All rights reserved. TOPIC HIGHLIGHT Marco Giuseppe Patti, MD, Professor, Series Editor Laparoscopic rectal cancer surgery: Where do we stand? Mukta K Krane, Alessandro Fichera Mukta K Krane, Alessandro Fichera, Department of Surgery, University of Chicago Pritzker School of Medicine, 5841 South Maryland Ave, MC 5095, Chicago, IL 60637, United States Author contributions: Krane MK and Fichera A equally contributed to this paper. Correspondence to: Mukta K Krane, MD, Assistant Professor, Department of Surgery, University of Chicago Pritzker School of Medicine, 5841 South Maryland Ave, MC 5095, Chicago, IL 60637, United States. [email protected] Telephone: Fax: Received: April 18, 2012 Revised: October 10, 2012 Accepted: October 16, 2012 Published online: December 14, 2012 Abstract Large comparative studies and multiple prospective randomized control trials (RCTs) have reported equivalence in short and long-term outcomes between the open and laparoscopic approaches for the surgical treatment of colon cancer which has heralded widespread acceptance for laparoscopic resection of colon cancer. In contrast, laparoscopic total mesorectal excision (TME) for the treatment of rectal cancer has been welcomed with significantly less enthusiasm. While it is likely that patients with rectal cancer will experience the same benefits of early recovery and decreased postoperative pain from the laparoscopic approach, whether the same oncologic clearance, specifically an adequate TME can be obtained is of concern. The aim of the current study is to review the current level of evidence in the literature on laparoscopic rectal cancer surgery with regard to short-term and long-term oncologic outcomes. The data from 8 RCTs, 3 metaanalyses, and 2 Cochrane Database of Systematic Reviews was reviewed. Current data suggests that laparoscopic rectal cancer resection may benefit patients with reduced blood loss, earlier return of bowel function, and shorter hospital length of stay. Concerns that laparoscopic rectal cancer surgery compromises shortterm oncologic outcomes including number of lymph nodes retrieved and circumferential resection margin and jeopardizes long-term oncologic outcomes has not conclusively been refuted by the available literature. Laparoscopic rectal cancer resection is feasible but whether or not it compromises short-term or long-term results still needs to be further studied Baishideng. All rights reserved. Key words: Rectal cancer; Laparoscopy; Total mesorectal excision; Anterior resection; Abdominoperineal resection Peer reviewers: Dietmar Öfner, MD, MAS, MSc, Professor, Department of Surgery, Paracelsus Private Medical University Salzburg, Müllner Hauptstrasse 48, Salzburg 5020, Austria; Kjetil Soreide, MD, PhD, Associate Professor, Department of Surgery, Stavanger University Hospital, Armauer Hansensvei 20, POB 8100, N-4068 Stavanger, Norway, Philip H Gordon, Professor, Department of Surgery, McGill University, 3755 Cote Ste. Catherine Road, Suite G-314, Montreal, Quebec H3T 1E2, Canada; Vamsi R Velchuru, MRCS, FRCSEd, RCS (Gen Surg), James Paget University Hospital, Great Yarmouth, 6 Pickwick Drive, Off Market Lane, Blundeston NR32 5BX, United Kingdom Krane MK, Fichera A. Laparoscopic rectal cancer surgery: Where do we stand? World J Gastroenterol 2012; 18(46): Available from: URL: v18/i46/6747.htm DOI: i INTRODUCTION Laparoscopic colon resection was introduced in 1991 [1,2]. Concern for port site metastasis and inadequate oncologic clearance initially hampered its adoption in the treatment of colon and rectal malignancy [3-6]. However, recently large comparative studies and multiple prospective randomized control trials (RCTs) have reported equivalence in resection margin, lymph node collection, tumor recurrence, postoperative complications, and long-term outcomes between open and laparoscopic 6747 December 14, 2012 Volume 18 Issue 46

69 Krane MK et al. Laparoscopic surgery for rectal cancer resection for colon cancer [7-12]. In addition, these studies demonstrated earlier recovery of bowel function, less postoperative pain, and decreased hospital stay with the laparoscopic approach which has heralded widespread acceptance for laparoscopic resection of colon cancer [8,9,13-16]. In contrast, laparoscopic total mesorectal excision (TME) for the treatment of rectal cancer has been welcomed with significantly less enthusiasm. While it is likely that patients with rectal cancer will experience the same benefits of early recovery and decreased postoperative pain from the laparoscopic approach, whether the same oncologic clearance, specifically an adequate TME can be obtained is of concern [17-23]. Involvement of the circumferential resection margin (CRM) after TME is a prognostic factor for local recurrence [24-28]. Marijnen et al [29] found that in the Dutch Rectal Cancer Trial, 13.1% of patients with a positive CRM developed a local recurrence within 2 years of followup, whereas patients with a margin > 2 mm had a local recurrence rate of 3.3% at 2 years (P < ). Postoperative radiation did not lead to a reduction in the local recurrence rate (17.3% vs 15.7% local recurrence in patients with CRM < 1 mm with and without adjuvant radiotherapy respectively, P = 0.98) [29]. In addition, preoperative radiotherapy had no significant effect on the prevention of local recurrence in patients with positive CRM (9.3% in the irradiated group vs 16.4% in the surgery alone group, P = 0.08) highlighting the importance of adequate surgery. In conventional open resection of rectal cancer, considerable variation between surgeons in oncologic outcomes has been demonstrated [30]. Differences in local recurrence and disease-free survival may be amplified by the technical challenges of laparoscopic proctectomy. While, the laparoscopic approach provides a magnified view compared to open surgery, TME and autonomic nerve preservation which are prerequisites for satisfactory oncologic and functional results require significant laparoscopic expertise [31]. A number of studies have reported on the safety and feasibility of laparoscopic low anterior resection (LAR) and abdominoperineal resection (APR) with TME but there is no level one evidence supporting laparoscopic TME in terms of oncologic outcomes [19,20,32-36]. The aim of this study is to provide a systematic review of the short-term and longterm oncologic outcomes of laparoscopic rectal cancer resection. DATA SOURCE Peer-reviewed papers published on laparoscopic rectal cancer resection were found by searching the following terms in the Ovid Medline, PubMed, and Cochrane Database of Systematic Reviews from 1993 to 2010: laparoscopy, laparoscopic surgery, and rectal cancer. Review articles found using the search terms colon cancer or rectal cancer and laparoscopy were also reviewed to find pertinent articles. All relevant articles were assessed and inclusion and exclusion criteria applied. Study designs included prospective RCTs, metaanalyses, and Cochrane Database of Systematic Reviews. Studies were included if short-term outcomes, morbidity and mortality, or oncologic data specifically, recurrence rates, number of lymph nodes retrieved, margin status, and overall survival for patients undergoing curative laparoscopic rectal cancer resection were reported. When more than one trial containing overlapping patient inclusion periods and data was reported from the same institution, the most recent publication was included. Studies were excluded if they (1) reported both colonic and rectal outcomes, but did not analyze rectal cancer outcomes individually; (2) were non-randomized comparative trials, descriptive trials, or case reports; (3) were not published in the English language; and (4) reported on patients undergoing palliative treatment (non-curative surgical intent). The majority of data on laparoscopic resection for rectal cancer come from non-randomized comparative and descriptive studies. The literature review yielded a total of 79 studies published in the English language from 1993 to Sixty-five studies were excluded because they were non-randomized comparative trials or descriptive trials. One meta-analysis was excluded because individual studies were not analyzed according to the site of disease or the type of resection. The remaining 2 Cochrane reviews, 3 meta-analyses, and 8 RCTs comparing laparoscopic vs open TME for rectal cancer form the basis of this review. When assessing the data on laparoscopic resection of rectal cancer it is important to remember that results may vary greatly based on level of the tumor, APR vs LAR, use of neoadjuvant chemoradiation, and completeness of TME. OUTCOMES OF INTEREST Intraoperative outcomes include: duration of operation, blood loss, length of incision, and conversion rate. Short-terms parameters of interest include: early postoperative complications (hemorrhage, anastomotic leak, wound complications, chest infection, prolonged ileus, incidence of pulmonary embolism or deep vein thrombosis, and urinary infection/retention), and mortality. Oncologic outcomes reviewed include: number of lymph nodes retrieved, margin status, completeness of TME, local recurrence, and overall survival. Intraoperative results The proven benefits of laparoscopy noted in colon cancer surgery including decreased intraoperative blood loss, smaller length of incision, less postoperative pain, faster recovery of intestinal function, and shorter length of hospital stay likely also apply to rectal cancer surgery [37]. In RCTs (Table 1) the mean operative time for open surgical resection of rectal cancer ranged from 106 to 284 min compared to 120 to 245 min for laparoscopic resection (Table 2). As expected, duration of operation was significantly longer in the laparoscopic group com December 14, 2012 Volume 18 Issue 46

70 Krane MK et al. Laparoscopic surgery for rectal cancer Table 1 Patient characteristics from randomized control trials Ref. Patients M/F BMI Age (yr) % Pre-op ChemoRT Total Open Lap Open Lap Open Lap Open Lap Open Lap Kang et al [40] /60 110/ (3.2) 24.1 (3.2) 59.1 (9.9) 57.8 (11.1) Ng et al [45] /29 37/39 NA NA 65.7 (12) 66.5 (11.9) NA NA Lujan et al [31] /39 62/39 NA NA 66.0 (9.9) 67.8 (12.9) Ng et al [39] /18 31/20 NA NA 63.5 (12.6) 63.7 (11.8) 0 0 Guillou et al [7] /Jayne et al [12] NA NA 26 (4) 25 (4) 69 (12) 69 (11) NA NA Braga et al [38] /21 55/28 NA NA 65.3 (10.3) 62.8 (12.6) Zhou et al [35] /46 46/36 NA NA 45 (30-81) 44 (26-85) NA NA Araujo et al [34] /5 9/ ( ) 23.5 ( ) 56.4 (24-78) 59.1 (31-75) 15 (100%) 13 (100%) BMI: Body mass index; ChemoRT: Chemoradiation. Table 2 Intraoperative characteristics of patients from randomized control trials Ref. Number of patients (%) Conv % Op time (min) Blood loss (ml) Length of incision (cm) Total Open Lap Open Lap Open Lap Open Lap Total LAR APR Total LAR APR Kang et al [40] (75.9) Ng eet al [45] (100) Lujan et al [31] (78.6) Ng et al [39] (0) Guillou et al [7] / Jayne et al [12] (69.9) Braga et al [38] (87.1) Zhou et al [35] (100) Araujo et al [34] (0) 24 (14.1) (88.8) (100) (76.2) 51 0 (0) (72.6) (92) (100) 13 0 (0) 19 (11.2) 0 (0) 24 (23.8) 51 (100) 63 (27.4) 7 (84) 0 (0) 13 (100) (62.9) (70.3) (59.4) (43.4) (75.4) a ( ) ( ) a ( ) 5.0 ( ) NS 0 (0) (59.3) a (0-2542) (45.1) a (± 174.3) (46.2) a (0-4720) (0-3000) NA 22 (21.4) (± 113.3) a NA NA (0-3000) 48 (100) 34 (30.1) ( ) 135 ( ) NA NA 22 (18-29) 10 (6-17) 5.8 (± 0.8) a 11 (12.9) (72) (72) a ( ) ( ) a (± 3.1) 0 (0) 15 (100) NA 106 (80-230) ( ) a (50-200) 20 NA NA (5-120) a a NA NA NA NA Conv: Conversion rate; LAR: Low anterior resection; APR: Abdominoperineal resection; NA: Not available. a P < 0.05 vs Open. pared to the open group in 6 of the 8 RCTs [7,22,31,38-40]. Similar results were reported in RCTs of open vs laparoscopic resection for colon cancer. Zhou et al [35] reported both shorter open and laparoscopic operative times compared to other trials with no significant difference between the two operative approaches (120 min vs 106 min for laparoscopic vs open resection respectively, P = 0.051). However, no details were provided on tumor stage, conversion rate, or whether the analysis was performed on an intent-to-treat basis. Araujo et al [34] was the only RCT to demonstrate significantly shorter operative times with laparoscopic compared to open resection (228 min vs 284 min respectively, P = 0.04). However, they attributed these results to fact that the surgical team performing laparoscopic APR was the same whereas open APR was often performed by different surgical teams. In addition, extraction of the specimen from the perineum likely decreased operative time because there was not an abdominal incision to close. Two meta-analyses included operative time as an outcome of interest. Aziz et al [41] included 22 studies comparing laparoscopic vs open rectal cancer resection in 2071 patients and found that operative time was significantly increased with the laparoscopic group as compared to the open group with a weighted mean difference (WMD) of (95% CI, ). Gao et al [42] performed a meta-analysis of short-term outcomes after laparoscopic resection for rectal cancer based on 11 studies and included 643 patients which reported no difference in operating time between open and laparoscopic approaches with a WMD of 1.59 ( ). Intraoperative blood loss was significantly less for the laparoscopic group compared to the open group in 4 of 6 RCTs and ranged from 20 ml to ml and from 92 ml to ml in the laparoscopic and open groups respectively (Table 2) [31,35,38,40]. Araujo et al [34] did not specifically report on the amount of intraoperative blood loss but there was no statistically significant difference in the need for blood transfusions between the two groups which was attributed to the fact that in an APR the majority of blood loss occurs during the perineal portion of the case which is the same regardless of surgical access December 14, 2012 Volume 18 Issue 46

71 Krane MK et al. Laparoscopic surgery for rectal cancer Table 3 Short-term oncologic outcomes of patients from randomized control trials Ref. LN Positive margin (CRM/distal) (%) Open Lap Open Lap Kang et al [40] 18 (13-24) 17 (12-22) 7 (4.1)/NA 5 (2.9)/NA Ng eet al [45] 12 (7) 11.5 (7.9) 1 (1.3)/NA 2 (2.6)/NA Lujan et al [31] (5.10) (6.26) a 3 (2.9)/0 4 (4.0)/0 Ng et al [39] 13.0 (7) 12.4 (6.7) 2 (4.2)/NA 3 (5.9)/NA Guillou et al [7] / NA NA (14)/NA (16)/NA Jayne et al [12] Braga et al [38] 13.6 (6.9) 12.7 (7.3) 2 (2.4)/0 1 (1.2)/0 Zhou et al [35] NA NA NA NA Araujo et al [34] a NA NA LN: Lymph nodes; CRM: Circumferential resection margin; NA: Not available. a P < 0.05 vs Open. A recent Cochrane review by Breukink et al [43] evaluating the safety and efficacy of elective laparoscopic TME for the resection of rectal cancer found that in the majority of studies blood loss was reduced with the laparoscopic approach although this did not translate to fewer blood transfusions. Length of incision was measured in 3 of 8 RCTs and ranged from an average of 5 cm to 10 cm with the laparoscopic approach compared to an average of 19.1 cm to 22 cm with the open approach (Table 2) [7,38,40]. Seven of the 8 trials reported a conversion rate which ranged from 0%-34% (Table 2) [7,12,22,31,34,38-40]. Conversion to the open approach was commonly defined as length of incision greater than the size needed for tumor extraction or premature abdominal incision to allow improved mobilization. In the majority of studies conversion to open surgery was required because of local tumor invasion or difficult dissection in a narrow pelvis although bulky tumor, dilated small bowel, dense adhesions, bleeding, rectal perforation, difficulty mobilizing the splenic flexure, failure to identify or injury to the ureter, ischemia of the descending colon, and anastomotic failure were also cited. Breukink et al [44] reported that 36 of 48 studies assessed conversion and showed a highly variable rate ranging from 0% to 33%. However, they report that the lack of consensus in the definition made results difficult to interpret. In addition, surgeon experience and patient selection criteria were often not mentioned. Two trials reported particularly high rates of conversion. Ng et al [45] had a conversion rate of 30.3% but they did not routinely perform preoperative staging with computed tomography scans and therefore frequently converted after diagnostic laparoscopy. Twelve of the 23 patients randomized to laparoscopic surgery were converted to open due to local tumor invasion, bulky tumor, or dilated small bowel which may have been recognized by preoperative imaging. In the CLASICC trial the conversion rate for laparoscopic resection of rectal cancer was reported at 34% and attributed to excessive tumor fixation and uncertainty of tumor clearance [7]. Surgeon learning curve may account for this high rate of conversion as evidenced by the fact that the overall rate of conversion dropped by year of study from 38% in year one to 16% in year six. However, consistent with several non-randomized reports, in the CLASICC trial patients converted to open resection had a higher operative mortality compared to patients in the laparoscopic or open groups (9% vs 1% vs 5% respectively) [7]. Conversion was also associated with worse oncologic outcomes in nonrandomized comparative and descriptive studies [46]. Short-term oncologic outcomes While the number of lymph nodes retrieved can vary based on age, gender, tumor site, use of pre-operative radiation, and tumor grade, the extent and quality of surgical resection can also have an impact on the number of nodes collected and is therefore often considered a surrogate marker of the oncologic completeness of the resection [47-53]. The American Joint Committee on Cancer recommends that at least 12 lymph nodes be examined in patients with rectal cancer to confirm the absence of nodal involvement by the tumor [54]. In addition, a number of studies have reported that the number of lymph nodes examined may be associated with patient outcome [55,56]. Six of the 8 RCTs reported the mean number of lymph nodes retrieved with a range of 5.5 to 17 nodes in the laparoscopic group compared to 11.6 to 18 nodes in the open group (Table 3) [22,31,34,38-40]. In 4 of the 6 trials the number of lymph nodes isolated was not significantly different based on surgical approach. Araujo et al [34] reported a significantly lower yield of lymph nodes with laparoscopic rectal resection compared to open resection (5.5 vs 11.9 respectively, P = 0.04). However, the number of lymph nodes obtained in the study by Lujan et al [31] was higher in the laparoscopic group (13.63 vs in the laparoscopic vs open approach respectively, P = 0.026). They suggested that laparoscopy offered better dissection and accuracy due to better visualization and exposure of structures with less manipulation of the mesorectum especially in a narrow pelvis. Four of the 8 RCTs reported the use of pre-operative chemoradiation. In these trials, the mean number of lymph nodes retrieved ranged from 5.5 to 17 nodes in the laparoscopic group and from 11.6 to 18 nodes in the open group [31,34,38,40]. Anderson et al [57] found that in the 17 trials that reported the number of lymph nodes retrieved, the mean number of nodes was 10 for the laparoscopic group and 12 for the open group (P = 0.001) with the majority of trials reporting a median of 11 or fewer nodes obtained. In 9 of these 17 trials, both groups were treated with preoperative radiation therapy and reported a mean of 10 lymph nodes harvested in the laparoscopic group and 11 in the open group. One of the greatest concerns of laparoscopic TME is that obtaining a complete oncologic resection will be more difficult. Involvement of the circumferential or distal margin is one of the most important prognostic factors in rectal resection with TME and can lead to an increase in local recurrence and a reduction in survival. Radial margins of less than 2 mm are associated with 6750 December 14, 2012 Volume 18 Issue 46

72 Krane MK et al. Laparoscopic surgery for rectal cancer Table 4 Short-term outcomes of patients in randomized control trial Ref. Length of stay (d) Anastomotic leak (%) Wound infection (%) Ileus (%) Pain/PCA use (mg) or (number of shots) Mortality Open Lap Open Lap Open Lap Open Lap Open Lap Open Lap 9 26 Kang et al [40] 9 (8-12) 8 (7-12) 0 2 (1.2) 11 (6.5) 2 (1.2) a 22 (12.9) 17 (10) ( ) ( ) 0 0 Ng eet al [45] 10.0 (3-39) 8.4 (2-32) a 4 (5.2) 1 (1.3) 9 (11.7) 5 (6.6) 2 (2.6) 1 (1.3) 8.3 (0-49) 4.9 (0-23) a 3 (3.9) 2 (2.6) Lujan et al [31] 9.9 (6.8) 8.2 (7.3) 10 (12) 5 (6) 2 (1.9) 0 (0) 8 (7.8) 6 (5.9) NA NA 3 (2.9) 2 (1.9) Ng et al [39] 11.5 (5-38) 10.8 (5-27) NA NA 4 (8.3) 0 (0) 2 (4.2) 1 (2.0) 11.4 (0-49) 6.0 (0-47) a 1 (2.8) 1 (2.5) Guillou et al [7] / 13 (9-18) 11 (9-15) (7) (10) 15 (12) 33 (13) NA NA NA NA NA NA Jayne et al [12] Braga et al [38] 13.6 (6-80) 10 (6-27) a 9 (10.6) 8 (9.6) 13 (15.3) 6 (7.2) 2 (2.3) 2 (2.4) NA NA 1 (1.2) 1 (1.2) Zhou et al [35] 13.3 (3.4) 8.1 (3.1) a 3 (3.4) 1 (1.2) NA NA NA NA NA NA 0 (0) 0 (0) Araujo et al [34] < NA NA NA NA NA NA NA NA PCA: Patient controlled analgesia; NA: Not available. a P < 0.05 vs Open. a local recurrence rate of 16% compared to a significantly reduced local recurrence rate of 6% with margins greater than 2 mm [27]. Six of the 8 RCTs reported the involvement of the CRM and no difference was found by surgical approach (Table 3) [7,31,38-40,45]. In the majority of trials the rate of CRM involvement was less than 5%. Patients with positive radial margins often had tumor invading the pelvic side wall or adjacent structure and were frequently converted from a laparoscopic to an open procedure [39]. In the CLASICC study, the only multicenter trial, a positive CRM was identified in 14 of 97 (14%) patients with open surgery and in 30 of 193 (16%) patients with laparoscopic rectal resection (P = 0.8) [7]. Of patients undergoing anterior resection, the CRM was positive in 16 of 129 (12%) individuals in the laparoscopic group and in 4 of 64 (6%) individuals in the open group (P = 0.19). While there is a non-significant higher positivity of the CRM in the laparoscopic anterior resection group, this is once again likely due to the fact that the learning curve was not completed before the start of this study. Two RCTs reported on distal margin status and the incidence of distal margin positivity was not significantly different between the two surgical approaches and in fact was 0% [31,38]. All 3 meta-analyses and the Cochrane review by Breukink et al [44] found no difference in positive margins based on surgical access. Postoperative course: Less postoperative pain, faster recovery of intestinal function, and shorter length of stay are important benefits of laparoscopic colorectal surgery. Only 3 of 8 RCTs compared the exact amount of post-operative pain medication and 2 of these studies reported a significant reduction in analgesic use in the laparoscopic group (Table 4) [39,40,45]. Zhou et al [35] did not quantify the exact usage of pain medication, but found no significant difference in the number of days parental analgesics were necessary (4.1 vs 3.9 in the open and laparoscopic groups respectively, P = 0.225). Resumption of bowel function was usually reported on post-operative days 3 to 5 and ability to tolerate a solid food diet was reported on post-operative days 3 to 6 [7,31,35,39,40,45]. In the majority of RCTs earlier bowel movements and diet advancement was reported with the laparoscopic approach. The return of bowel function and reduction in wound pain was thought to contribute to earlier discharge after laparoscopic surgery. While in a majority of trials, the length of stay was not significantly different between surgical approaches, there was a trend toward decreased length of stay with laparoscopic rectal surgery. Breukink et al [58] found that laparoscopic TME resulted in earlier return of normal diet, less pain, less narcotic use and a shorter hospital stay. Complications: Rectal cancer surgery is associated with a high rate of morbidity and mortality. Post-operative mortality in RCTs ranged from 1%-4% and demonstrated no statistically significant difference based on surgical approach (Table 4). The rate of post-operative complications ranged from 6% to 69% and with the exception of Zhou et al [35] did not differ significantly between laparoscopic and open groups. Wound infection and urinary tract infection accounted for the majority of perioperative complications in both groups. There was a higher incidence of wound infection with the open approach however this did not reach statistical significance. Breukink et al [58] found no difference in morbidity between the laparoscopic and open groups although there was a trend toward lower morbidity with laparoscopic TME. Aziz et al [41] found no difference in perioperative morbidity between the 2 groups while Gao et al [42] found that the overall morbidity rate of the laparoscopic group was significantly lower than that of the open group. Anastomotic leak is the most serious complication after sphincter sparing rectal cancer resection especially with neoadjuvant chemoradiation. In addition, development of an anastomotic leak is reported to be associated with decreased long-term survival and higher rates of local recurrence after curative resection for colorectal cancer [59-63]. Operative expertise and selective diversion in high risk patients has resulted in a anastomotic leak rate of 1%-17% in most published series studying laparoscopic resection for rectal cancer [46,64,65]. Consistent with reports from non-randomized comparative trials, RCTs demonstrated no significant difference in the incidence 6751 December 14, 2012 Volume 18 Issue 46

73 Krane MK et al. Laparoscopic surgery for rectal cancer Table 5 Long-term oncologic outcomes of patients in randomized control trials Ref. Mean F/U (mo) LR (%) DFS (%) OS (%) Open Lap Open Lap Open Lap Open Lap Kang et al [40] NA NA NA NA NA NA NA NA Ng eet al [45] ( ) ( ) (5.1) 82.9 (4.9) 55.1 (6.5) 63.9 (6.6) Lujan et al [31] 34.1 (20) 32.8 (18.9) Ng et al [39] 90.1 ( ) 87.2 ( ) (8.1) 78.1 (6.9) 76.5 (7.3) 75.2 (7.2) Guillou et al [7] /Jayne et al [12] 36.8 ( ) 36.8 ( ) / / / /65.2 Braga et al [38] NA NA NA NA Zhou et al [35] NA NA NA NA NA NA Araujo et al [34] NA NA NA NA F/U: Follow-up; LR: Local recurrence; DFS: Disease-free survival; OS: Overall survival; NA: Not available. of anastomotic leak between the laparoscopic and open technique for the resection of rectal cancer (Table 4). While the incidence of perioperative morbidity was not different based on surgical access, fewer patients had long-term complications with laparoscopic rectal cancer resection compared to the open approach. Adhesion related bowel obstruction was the most common longterm morbidity. With a median follow-up of greater than 9 years, Ng et al [45] found that adhesion-related obstruction requiring hospitalization (18.9% vs 2.7%) and reoperation (6.8% vs 0%) was higher in the open group. They report a cumulative probability of adhesion-related bowel obstruction at 10 years of 20.5% in the open group and 3.9% in the laparoscopic group (P = 0.001) [45]. Kuhry et al [66] performed a systematic review including 12 trials (3346 patients) to evaluate the long-term outcomes of laparoscopically assisted vs open surgery for resectable colorectal cancer. Data on long-term complications was not separated by site of disease but the overall occurrence of incisional hernia (7.9% vs 10.9%, P = 0.32) and reoperation for adhesions (1.1% vs 2.5%, P = 0.30) was not statistically difference between laparoscopic and open resection. Long-term studies need to be done to determine if laparoscopy decreases the incidence of intra-abdominal adhesion formation by reduced surgical trauma, less tissue handling, and smaller incisions. Long-term oncologic outcomes A number of the clinical trials were performed to determine the safety and feasibility of the laparoscopic approach for rectal adenocarcinoma and therefore the data we have for long-term outcomes is limited (Table 5). Braga et al [38] found no difference in local recurrence (4.0% in the laparoscopic group vs 5.2% in the open group, P = 0.97), overall five-year survival, or diseasefree five-year survival based on surgical approach. With a median follow-up of 87.2 mo in the laparoscopic group and 90.1 mo in the open group, Ng et al [39] demonstrated that after curative resection, the probability of five-year survival was 75.2% vs 76.5% for laparoscopic vs open APR respectively (P = 0.20). In addition, stage-by-stage comparison for the two groups showed no statistical difference. There were no port site recurrences and overall recurrence rates were not significantly different between the two groups (laparoscopic 20% vs open 25%, P = 0.60). Despite the higher rate of circumferential margin positivity in patients undergoing laparoscopic anterior resection in the CLASICC trial, there was no difference in local recurrence, three- year overall or three-year disease free survival between the two approaches (open OS 66.7% and laparoscopic OS 74.6%, P = 0.17; open DFS 70.4% and laparoscopic DFS 70.9%, P = 0.72; open LR 7.0% and laparoscopic LR 7.98%, P = 0.70) [12]. In addition, there was no significant difference in the rates of local recurrence, three-year overall survival, or three-year disease-free survival in patients undergoing laparoscopic vs open APR [12]. However, the sample size is small and therefore larger studies are needed for conclusive results. Ng et al [45] published results of a randomized trial of laparoscopic vs open anterior resection for upper rectal cancer with a median follow-up of 9 years. No difference in local recurrence, overall survival, or disease-free survival was reported. Although these studies suggest comparative oncologic outcomes between laparoscopic and open rectal cancer resection, they include small sample sizes and are almost all are single institution studies, highlighting the need for large, multi-center RCTs to provide confirmatory data. In a meta-analysis by Anderson et al [57] 18 of 24 studies reported recurrence rates. With a mean follow-up of 35 mo for both groups, overall local recurrence was not statistically different between the 2 groups (laparoscopic 7% vs open 8%, P = NS). Eleven studies provided sufficient data to compare overall survival. Overall survival was 72% for patients undergoing laparoscopic rectal cancer resection and 65% for open resection at an average of 4.4 years (P = 0.5). Subset analysis by Kuhry et al [66] demonstrated no significant difference between laparoscopic and open rectal cancer resection in terms of local recurrence (laparoscopic 7.2% vs open 7.8%, P = 0.46), development of distant metastases (laparoscopic 13.5% vs open 9.1%, P = 0.60), or cancer-related mortality (laparoscopic 9% vs open 10%, P = 0.16). While, this data is encouraging, it is no conclusive. CONCLUSION The primary goal of this study was to outline and review 6752 December 14, 2012 Volume 18 Issue 46

74 Krane MK et al. Laparoscopic surgery for rectal cancer the short-term and long-term oncologic outcomes and complications of laparoscopic rectal cancer resection compared to the gold standard of conventional open resection currently available in the literature. Due to the heterogeneity in tumor stage, surgeon experience, and surgical technique, descriptive and non-randomized trials were not included in this review. However, because of the relatively few RCTs, information on the long-term outcomes is sparse and our conclusions are thus based on a small number of patients. A second limitation is that in a number of these trials data accrual started before the effectiveness of neoadjuvant therapy had been proven and thus the majority of patients did not receive pre-operative chemoradiation which is the current standard of care. Given these limitations, we found no difference in adequacy of oncologic resection, perioperative morbidity, recurrence rates, overall survival, or diseasefree survival between open and laparoscopic rectal cancer resection. In conclusion, RCTs have demonstrated that laparoscopy does not adversely affect cancer related survival in patients with adenocarcinoma of the colon. Concerns about the technical difficulty of TME may have contributed to the exclusion of rectal cancer patients from most of these large multicenter RCTs resulting in little data on oncologic outcomes with laparoscopic rectal cancer resection. Laparoscopic rectal dissection is technically more demanding than open and constraints of a narrow pelvis may result in difficulty assessing and obtaining adequate surgical margins. However, there are several proposed benefits of laparoscopic rectal resection. A clear and magnified view of the pelvis provided by the improved optics of laparoscopy may aid sharp dissection for TME and assist in identification of vital pelvic structures including the ureters and autonomic nerves. In addition, pneumoperitoneum may separate the parietal and visceral fascia of the mesorectum facilitating dissection in this plane. Laparoscopic rectal cancer resection has a steep learning curve but increased experience with both open and laparoscopic TME will lead to shorter operating times and decreased morbidity [67]. Current data suggests that laparoscopic rectal cancer resection may benefit patients because of reduced blood loss, earlier return of bowel function, and shorter hospital length of stay [68,69]. Concerns that laparoscopic rectal cancer surgery may compromise short-term oncologic outcomes including number of lymph nodes harvested and CRM positivity do not appear to be supported by the available literature. 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Radiotherapy does not compensate for positive resection margins in rectal cancer patients: report of a multicenter randomized trial. Int J Radiat Oncol Biol Phys 2003; 55: Akbari RP, Read TE. Laparoscopic rectal surgery: rectal cancer, pelvic pouch surgery, and rectal prolapse. Surg Clin North Am 2006; 86: Lujan J, Valero G, Hernandez Q, Sanchez A, Frutos MD, Parrilla P. Randomized clinical trial comparing laparoscopic and open surgery in patients with rectal cancer. Br J Surg 2009; 96: Staudacher C, Vignali A, Saverio DP, Elena O, Andrea T. Laparoscopic vs. open total mesorectal excision in unselected patients with rectal cancer: impact on early outcome. Dis Colon Rectum 2007; 50: Law WL, Chu KW, Tung HM. Early outcomes of 100 patients with laparoscopic resection for rectal neoplasm. Surg Endosc 2004; 18: Araujo SE, da Silva esousa AH, de Campos FG, Habr- Gama A, Dumarco RB, Caravatto PP, Nahas SC, da Silva J, Kiss DR, Gama-Rodrigues JJ. Conventional approach x laparoscopic abdominoperineal resection for rectal cancer treatment after neoadjuvant chemoradiation: results of a prospective randomized trial. Rev Hosp Clin Fac Med Sao Paulo 2003; 58: Zhou ZG, Hu M, Li Y, Lei WZ, Yu YY, Cheng Z, Li L, Shu Y, Wang TC. Laparoscopic versus open total mesorectal excision with anal sphincter preservation for low rectal cancer. Surg Endosc 2004; 18: Baik SH, Gincherman M, Mutch MG, Birnbaum EH, Fleshman JW. Laparoscopic vs open resection for patients with rectal cancer: comparison of perioperative outcomes and long-term survival. Dis Colon Rectum 2011; 54: McKay GD, Morgan MJ, Wong SK, Gatenby AH, Fulham SB, Ahmed KW, Toh JW, Hanna M, Hitos K. Improved short-term outcomes of laparoscopic versus open resection for colon and rectal cancer in an area health service: a multicenter study. Dis Colon Rectum 2012; 55: Braga M, Frasson M, Vignali A, Zuliani W, Capretti G, Di Carlo V. Laparoscopic resection in rectal cancer patients: outcome and cost-benefit analysis. Dis Colon Rectum 2007; 50: Ng SS, Leung KL, Lee JF, Yiu RY, Li JC, Teoh AY, Leung WW. Laparoscopic-assisted versus open abdominoperineal resection for low rectal cancer: a prospective randomized trial. Ann Surg Oncol 2008; 15: Kang SB, Park JW, Jeong SY, Nam BH, Choi HS, Kim DW, Lim SB, Lee TG, Kim DY, Kim JS, Chang HJ, Lee HS, Kim SY, Jung KH, Hong YS, Kim JH, Sohn DK, Kim DH, Oh JH. Open versus laparoscopic surgery for mid or low rectal cancer after neoadjuvant chemoradiotherapy (COREAN trial): short-term outcomes of an open-label randomised controlled trial. Lancet Oncol 2010; 11: Aziz O, Constantinides V, Tekkis PP, Athanasiou T, Purkayastha S, Paraskeva P, Darzi AW, Heriot AG. Laparoscopic versus open surgery for rectal cancer: a meta-analysis. Ann Surg Oncol 2006; 13: Gao F, Cao YF, Chen LS. Meta-analysis of short-term outcomes after laparoscopic resection for rectal cancer. Int J Colorectal Dis 2006; 21: Breukink SO, van der Zaag-Loonen HJ, Bouma EM, Pierie JP, Hoff C, Wiggers T, Meijerink WJ. Prospective evaluation of quality of life and sexual functioning after laparoscopic total mesorectal excision. Dis Colon Rectum 2007; 50: Breukink S, Pierie J, Wiggers T. Laparoscopic versus open total mesorectal excision for rectal cancer. Cochrane Database Syst Rev 2006; (4): CD Ng SS, Leung KL, Lee JF, Yiu RY, Li JC, Hon SS. Long-term morbidity and oncologic outcomes of laparoscopic-assisted anterior resection for upper rectal cancer: ten-year results of a prospective, randomized trial. Dis Colon Rectum 2009; 52: Poon JT, Law WL. Laparoscopic resection for rectal cancer: a review. Ann Surg Oncol 2009; 16: Vaccaro CA, Im V, Rossi GL, Quintana GO, Benati ML, Perez de Arenaza D, Bonadeo FA. Lymph node ratio as prognosis factor for colon cancer treated by colorectal surgeons. Dis Colon Rectum 2009; 52: Riediger H, Keck T, Wellner U, zur Hausen A, Adam U, Hopt UT, Makowiec F. The lymph node ratio is the strongest prognostic factor after resection of pancreatic cancer. J Gastrointest Surg 2009; 13: Kim YW, Kim NK, Min BS, Lee KY, Sohn SK, Cho CH, Kim H, Keum KC, Ahn JB. The prognostic impact of the number of lymph nodes retrieved after neoadjuvant chemoradio December 14, 2012 Volume 18 Issue 46

76 Krane MK et al. Laparoscopic surgery for rectal cancer therapy with mesorectal excision for rectal cancer. J Surg Oncol 2009; 100: Newland RC, Dent OF, Lyttle MN, Chapuis PH, Bokey EL. Pathologic determinants of survival associated with colorectal cancer with lymph node metastases. A multivariate analysis of 579 patients. Cancer 1994; 73: Sarli L, Bader G, Iusco D, Salvemini C, Mauro DD, Mazzeo A, Regina G, Roncoroni L. Number of lymph nodes examined and prognosis of TNM stage II colorectal cancer. Eur J Cancer 2005; 41: Wong SL, Ji H, Hollenbeck BK, Morris AM, Baser O, Birkmeyer JD. Hospital lymph node examination rates and survival after resection for colon cancer. JAMA 2007; 298: Sjo OH, Merok MA, Svindland A, Nesbakken A. Prognostic impact of lymph node harvest and lymph node ratio in patients with colon cancer. Dis Colon Rectum 2012; 55: Compton CC, Greene FL. The staging of colorectal cancer: 2004 and beyond. CA Cancer J Clin 2004; 54: Tepper JE, O Connell MJ, Niedzwiecki D, Hollis D, Compton C, Benson AB, Cummings B, Gunderson L, Macdonald JS, Mayer RJ. Impact of number of nodes retrieved on outcome in patients with rectal cancer. J Clin Oncol 2001; 19: Le Voyer TE, Sigurdson ER, Hanlon AL, Mayer RJ, Macdonald JS, Catalano PJ, Haller DG. Colon cancer survival is associated with increasing number of lymph nodes analyzed: a secondary survey of intergroup trial INT J Clin Oncol 2003; 21: Anderson C, Uman G, Pigazzi A. Oncologic outcomes of laparoscopic surgery for rectal cancer: a systematic review and meta-analysis of the literature. Eur J Surg Oncol 2008; 34: Breukink SO, Pierie JP, Hoff C, Wiggers T, Meijerink WJ. Technique for laparoscopic autonomic nerve preserving total mesorectal excision. Int J Colorectal Dis 2006; 21: Walker KG, Bell SW, Rickard MJ, Mehanna D, Dent OF, Chapuis PH, Bokey EL. Anastomotic leakage is predictive of diminished survival after potentially curative resection for colorectal cancer. Ann Surg 2004; 240: McArdle CS, McMillan DC, Hole DJ. Impact of anastomotic leakage on long-term survival of patients undergoing curative resection for colorectal cancer. Br J Surg 2005; 92: Law WL, Choi HK, Lee YM, Ho JW, Seto CL. Anastomotic leakage is associated with poor long-term outcome in patients after curative colorectal resection for malignancy. J Gastrointest Surg 2007; 11: Lin JK, Yueh TC, Chang SC, Lin CC, Lan YT, Wang HS, Yang SH, Jiang JK, Chen WS, Lin TC. The influence of fecal diversion and anastomotic leakage on survival after resection of rectal cancer. J Gastrointest Surg 2011; 15: Mirnezami A, Mirnezami R, Chandrakumaran K, Sasapu K, Sagar P, Finan P. Increased local recurrence and reduced survival from colorectal cancer following anastomotic leak: systematic review and meta-analysis. Ann Surg 2011; 253: Tan WS, Tang CL, Shi L, Eu KW. Meta-analysis of defunctioning stomas in low anterior resection for rectal cancer. Br J Surg 2009; 96: Shiomi A, Ito M, Saito N, Ohue M, Hirai T, Kubo Y, Moriya Y. Diverting stoma in rectal cancer surgery. A retrospective study of 329 patients from Japanese cancer centers. Int J Colorectal Dis 2011; 26: Kuhry E, Schwenk W, Gaupset R, Romild U, Bonjer J. Longterm outcome of laparoscopic surgery for colorectal cancer: a cochrane systematic review of randomised controlled trials. Cancer Treat Rev 2008; 34: Kayano H, Okuda J, Tanaka K, Kondo K, Tanigawa N. Evaluation of the learning curve in laparoscopic low anterior resection for rectal cancer. Surg Endosc 2011; 25: Staudacher C, Di Palo S, Tamburini A, Vignali A, Orsenigo E. Total mesorectal excision (TME) with laparoscopic approach: 226 consecutive cases. Surg Oncol 2007; 16 Suppl 1: S113-S Pugliese R, Di Lernia S, Sansonna F, Maggioni D, Ferrari GC, Magistro C, Costanzi A, De Carli S, Artale S, Pugliese F. Laparoscopic resection for rectal adenocarcinoma. Eur J Surg Oncol 2009; 35: Buunen M, Bonjer HJ, Hop WC, Haglind E, Kurlberg G, Rosenberg J, Lacy AM, Cuesta MA, D Hoore A, Fürst A, Lange JF, Jess P, Bulut O, Poornoroozy P, Jensen KJ, Christensen MM, Lundhus E, Ovesen H, Birch D, Iesalnieks I, Jäger C, Kreis M, van riet Y, van der Harst E, Gerhards MF, Bemelman WA, Hansson BM, Neijenhuis PA, Prins HA, Balague C, Targarona E, Luján Mompeán JA, Franco Osorio JD, Garcia Molina FJ, Skullman S, Läckberg Z, Kressner U, Matthiessen P, Kim SH, Poza AA. COLOR II. A randomized clinical trial comparing laparoscopic and open surgery for rectal cancer. Dan Med Bull 2009; 56: S- Editor Lv S L- Editor A E- Editor Xiong L 6755 December 14, 2012 Volume 18 Issue 46

77 Online Submissions: doi: /wjg.v18.i World J Gastroenterol 2012 December 14; 18(46): ISSN (print) ISSN (online) 2012 Baishideng. All rights reserved. Marco Giuseppe Patti, MD, Professor, Series Editor Minimally invasive approaches for the treatment of inflammatory bowel disease TOPIC HIGHLIGHT Marco Zoccali, Alessandro Fichera Marco Zoccali, Department of Surgery, University of Chicago Pritzker School of Medicine, Chicago, IL 60637, United States Alessandro Fichera, Department of Surgery, University of Washington Medical Center, Seattle, WA 98195, United States Author contributions: Zoccali M and Fichera A equally contributed to conception and design of the paper; Zoccali M was responsible for searching the literature, interpreting data and drafting the article; Fichera A was responsible for revising the article critically for important intellectual content and gave final approval of the version to be submitted and any revised version to be published. Correspondence to: Alessandro Fichera, MD, FACS, FAS- CRS, Professor, Department of Surgery, University of Washington Medical Center, 1959 NE Pacific Street, Box , Room BB-414, Seattle, WA 98195, United States. [email protected] Telephone: Fax: Received: April 18, 2012 Revised: July 13, 2012 Accepted: August 4, 2012 Published online: December 14, 2012 Abstract Despite significant improvements in medical management of inflammatory bowel disease, many of these patients still require surgery at some point in the course of their disease. Their young age and poor general conditions, worsened by the aggressive medical treatments, make minimally invasive approaches particularly enticing to this patient population. However, the typical inflammatory changes that characterize these diseases have hindered wide diffusion of laparoscopy in this setting, currently mostly pursued in high-volume referral centers, despite accumulating evidences in the literature supporting the benefits of minimally invasive surgery. The largest body of evidence currently available for terminal ileal Crohn s disease shows improved short term outcomes after laparoscopic surgery, with prolonged operative times. For Crohn s colitis, high quality evidence supporting laparoscopic surgery is lacking. Encouraging preliminary results have been obtained with the adoption of laparoscopic restorative total proctocolectomy for the treatment of ulcerative colitis. A consensus about patients selection and the need for staging has not been reached yet. Despite the lack of conclusive evidence, a wave of enthusiasm is pushing towards less invasive strategies, to further minimize surgical trauma, with single incision laparoscopic surgery being the most realistic future development Baishideng. All rights reserved. Key words: Laparoscopic surgery; Inflammatory bowel disease; Ulcerative colitis; Crohn s disease Peer reviewers: Julio Mayol, MD, PhD, Department of Digestive Surgery, Hospital Clinico San Carlos, Madrid, Spain; Dr. Abdul-Wahed Meshikhes, MD, FRCS, Chairman, Consultant Surgeon, Department of Surgery, King Fahad Specialist Hospital, Amir Bin Thabit St, Dammam 31444, Eastern Province, Saudi Arabia; Arjuna De Silva, Professor in Medicine, Department of Medicine, Faculty of Medicine, University of Kelaniya, PO BOX6, Tallagolla Road, Ragama, Sri Lanka Zoccali M, Fichera A. Minimally invasive approaches for the treatment of inflammatory bowel disease. World J Gastroenterol 2012; 18(46): Available from: URL: wjgnet.com/ /full/v18/i46/6756.htm DOI: dx.doi.org/ /wjg.v18.i INTRODUCTION The past 20 years have seen dramatic improvements in the treatment of inflammatory bowel disease (IBD) [1]. Medical therapy, especially with the advent of biologics, has significantly increased efficacy of disease control, even if the actual reduction of the need for surgery is still debated, and concerns have been raised about po December 14, 2012 Volume 18 Issue 46

78 Zoccali M et al. Minimally invasive approach for IBD tential negative impact on postoperative outcomes [2,3]. In this setting, the introduction and implementation of minimally invasive surgical techniques has substantially improved outcomes and quality of life in this particularly frail patient population [4,5]. After the first description of laparoscopic colectomy about 20 years ago, laparoscopic surgery slowly has gained wide acceptance for the treatment of colorectal diseases, showing several advantages in short-term outcomes over open surgery in randomized trials and meta-analysis, with comparable safety and long-term results [6-9]. However, the diffusion of laparoscopy for IBD is proceeding particularly cautiously, given the magnitude of the procedures required for the most complex cases and the difficulty in handling severely inflamed tissues, as proven by the high conversion rates observed even in the hands of surgeons with documented experience in IBD and laparoscopic surgery [10,11]. Cronh s disease (CD) and ulcerative colitis (UC) represent real surgical challenges, due to thickened mesentery, strictures, abscesses, inflammatory masses, and enteric fistulae in CD, and intense inflammation leading to colonic distension and high risk of bleeding and accidental perforation in UC [12,13]. The quest for further reduction of surgical trauma is ongoing, and if the natural orifice transluminal endoscopic surgery has unsolved issues related to the violation of uninvolved hollow viscera, costs and specific training, single incision laparoscopic surgery (SILS) seems to be a reasonable approach capable of minimizing the overall trauma and extent of incisions, with benefits in short term outcomes and cosmesis [14-17]. The aim of this article is to provide a comprehensive review of the state of the art in minimally invasive approaches to IBD, highlighting the current standard of care, with a glance at the most promising future directions. CD Approximately 70% of patients with a diagnosis of CD will eventually require a surgical treatment, due to failure of medical therapy, septic complications, recurrent intestinal obstruction, and malnutrition [18]. The treatment of CD has traditionally represented a challenge even in open surgery, with just two prospective randomized trials comparing laparoscopic vs standard approach published to date [19,20], and with the long-term results of these studies only recently available [21,22]. The panintestinal involvement and inflammatory complications, along with the additional risk for postoperative complication, increased by the aggressive medical management, make CD patients particularly poor laparoscopic candidates [23]. Concerns have been raised about missing occult segments of disease and critical strictures due to the lack of tactile sensation, technical difficulty due to inflamed bowel mesentery and the presence of adhesions, fistulas, and abscesses [24]. In order to overcome this issues, some authors have advocated the use of laparoscopic-assisted or hand-assisted laparoscopic surgery procedures, with the rationale that an incision is needed for specimen extraction, and the handling of inflamed Crohn s tissue is easier and safer when an assisted method is used, while maintaining the advantages of a minimally invasive approach [25]. The intrinsic difficulty of this surgery is further confirmed by a study by Hamel at al [26], that showed no differences in morbidity or conversion between the earlier and the latter time periods of the experience, thus negating the effects of the learning curve. Alves et al [27] looked at the risk factors of conversion in a prospective study on 69 patients undergoing primary laparoscopic ileocecal resection, observing a conversion rate of 30%, with recurrent CD, intra-abdominal abscess and fistula independent risk factors on multivariate analysis. Even if minimally invasive surgery for CD is technically complex, requiring specific training and longer operating time [28], data in the literature confirm the safety and efficacy of this approach in terms of postoperative pain, cosmesis, return to normal activity, and, more importantly, surgical recurrence rates [29]. Despite this evidence, in a recent study by Lesperance et al [30] on patients admitted for CD that required surgical treatment from the Nationwide Inpatient Sample, only 2826 cases (6%) underwent a laparoscopic resection, demonstrating that the vast majority of CD patients are still undergoing open conventional surgery, with a minimal invasive approach mostly reserved for patients who are younger (< 35 years old), female, admitted to a teaching hospital, with ileocecal, uncomplicated disease. The increased adoption of the laparoscopic approach for the treatment of CD in teaching hospitals confirms the peculiar technical complexity of minimally invasive procedures in this setting, requiring more skilled colorectal surgeons, as can be found in referral centers where specific laparoscopic training programs are implemented. Terminal ileal CD The small samples size and selection bias explain the conflicting results in the initial published series of ileocolonic CD treated by laparoscopic surgery [28,31-34]. In our series of selected consecutive patients with elective, complex and even recurrent terminal ileal CD, laparoscopic patients had faster postoperative recovery - partially related to less postoperative pain and consequent decreased need for intravenous narcotics - and similar operating times compared to the open cohort, without increased complication and recurrence rates, with potential overall cost savings [35]. In regards to the issue of costs associated with laparoscopy, Young-Fadok et al [36], in a case match study comparing 33 cases of laparoscopic ileocolic resections with 33 open, showed significantly lower direct and indirect costs in the laparoscopic group. The strongest evidence available comes from the only two prospective randomized trials present in the literature, both conducted on small samples of highly selected patients. Although such populations might be far from the reality of a tertiary referral center, it is the only way to randomize CD patients given the panintestinal, relenting nature and often unpredictable presentation of the disease. In the trial by Maartense et al [19], patients with 6757 December 14, 2012 Volume 18 Issue 46

79 Zoccali M et al. Minimally invasive approach for IBD a fixed palpable inflammatory mass, prior median laparotomy, earlier bowel resection, or pregnancy were excluded. In this study, the laparoscopic approach showed longer median operating time, shorter hospital stay, lower 30-d post-operative morbidity, but no differences in quality of life, the primary endpoint of this study. After a median follow-up of 6.7 year, there were no differences in recurrence rate and need for reoperation between open and laparoscopic group, with a 58% relapse free rate and no patients in the laparoscopic group requiring a reoperation for incisional hernia or adhesive small bowel obstruction [21]. Even if a minimal invasive approach did not impact the overall quality of life, body image and cosmesis scores were significantly higher after laparoscopy [21]. These data differ from the previous observation by Thaler and colleagues, that found long-term quality of life significantly reduced in patients with CD compared to general healthy population, irrespective of the surgical approach, with recurrence identified as the only significant predictor of poor quality of life [37]. In the other randomized trial, Milsom et al [20] included only patients with isolated Crohn s disease of the terminal ileum with or without cecal involvement. The results of this study demonstrated that laparoscopy offers faster recovery of pulmonary function, fewer minor complications, and a trend towards shorter length of stay compared with conventional surgery, even if no differences in the amount of morphine equivalents, return of bowel function and length of stay were found. After a mean followup of 10.5 years there were no significant differences between groups with regard to use of medications to treat CD and recurrence rates, both clinical and surgical. Furthermore, two laparoscopic patients underwent lysis of adhesions while none did in the open group, with an incidence of incisional hernia repair of 4% in the laparoscopic group vs 14% in the open (both differences were not statistically significant) [22]. Recently, Dasari et al [38] conducted a meta-analysis of the aforementioned trials, and found that laparoscopic patients had a trend towards less wound infection and shorter hospital stay, with comparable incidence of other postoperative complications, duration of postoperative ileus, incidence of anastomotic leak and intraabdominal abscess, 30-d reoperation rate, and actuarial disease recurrence rates. To date, three meta-analysis comparing laparoscopic and open surgery for ileocolonic CD have been conducted, all demonstrating that laparoscopic surgery is associated with prolonged operative time, shorter duration of postoperative ileus, shorter hospital stay and lower incidence of early postoperative complications [39-41]. Other significant findings from these studies also include similar intraoperative blood loss and complications [41], with a trend toward lower overall costs with laparoscopic surgery [39], and no differences in the rate of disease recurrence [40]. With regard to the long-term outcomes, the study from Washington University, comparing 63 CD patients treated laparoscopically with 50 open ileocolic resections, found that the two groups had a recurrence rate of 9.5% and 24%, respectively (difference not statistically significant), with the laparoscopic group having shorter mean followup, thus confirming the non-inferiority of the laparoscopic approach. Interestingly, 50% of the recurrences in the laparoscopic group and 33% in the open group were able to be retreated laparoscopically [29]. Laparoscopy in complicated/recurrent CD In complicated CD laparoscopy is even more challenging. Seymour and Kavic analyzed their series of 17 patients managed with laparoscopic approach for complicated CD (defined as for the presence of fistulas, multiple or long-segment disease, abscesses and previous operations). In this study, conversion to open procedures was not always required, but operative time and postoperative hospital stay were longer compared to laparoscopic ileocecal resections for uncomplicated disease, with major complications occurring in 18% of patients [42]. In the literature, surgical recurrence rates are reported as high as 70% to 90%, and multiple procedures are required in more than 30% [12]. In a recent study from France, of 62 reoperations for CD recurrence in 57 patients, 29 were performed laparoscopically. While no differences between the two groups were observed in terms of use of a temporary stoma, mean operating time, postoperative mortality (nil in both groups), overall morbidity rate, severe complications, median hospital stay, and conversion rates, a higher number of intraoperative intestinal injuries was reported in the laparoscopic group (5 vs 0) (P = 0.01). The occurrence of fistulizing disease was a risk factor for conversion, and conversion did not seem to affect complication rate [43]. A study from Japan looked at 16 laparoscopic procedures for CD recurrence at the anastomotic site out of 61 attempted laparoscopically by experienced surgeons in 52 patients. The result of this study showed that while the operating time was significantly longer in the recurrent group, there were no differences in the rates of postoperative complications and hospital stay, with the repeated laparoscopic operations performed using the same small incision as that of the primary operation. The advantage of a minimally invasive primary approach are supported by the fact that the operating time was shorter and blood loss was less in patients who underwent the primary procedure laparoscopically [44]. Finally, in the experience by Goyer et al [45] on 54 complex CD (defined as recurrent or complicated by abscess and/or fistula) compared with 70 patients with uncomplicated CD, the complex group had increased operative time, conversion rates and use of temporary stoma. Conversely, no differences were noted in overall postoperative morbidity, including major surgical postoperative complications and hospital stay, leading to the conclusion that complex CD should not be considered an absolute contraindication to a laparoscopic approach in experienced hands. Crohn s colitis In contrast with the data available on minimally invasive 6758 December 14, 2012 Volume 18 Issue 46

80 Zoccali M et al. Minimally invasive approach for IBD surgery for terminal ileal CD, very few series have been published on CD of the colon. The feasibility and safety of a laparoscopic approach to subtotal colectomy for CD was addressed by Hamel et al [46], who observed a higher rate of intraoperative complications compared to ileocolic resection, while hospital stay and postoperative complication rate did not differ between the two groups. Contrasting results come from a recent case match study by the Cleveland Clinic group on 27 laparoscopic and 27 open cases, with a conversion rate of 26%. In this series, laparoscopic colectomies took longer with similar blood loss and postoperative complications, along with a trend towards shorter time to first bowel movement and length of stay, which became statistically significant in favor of laparoscopy when overall length of stay included 30-d readmissions [47]. In our own personal experience on 125 patients who underwent colectomy for CD, 44% by a laparoscopic approach, the conversion rate was 10.9%, median operative time, blood loss, return of bowel function and length of post-op stay were reduced in the laparoscopic group, while postoperative complications and disease recurrence rates were similar, suggesting that a laparoscopic approach for CD of the colon is safe and feasible in the hands of experienced surgeons [48]. Laparoscopy has a role also in creating diverting stomas for severe perianal CD, reducing the number of incisions to few trocars and the ostomy site. In a study by Liu et al [49] on 80 patients who underwent laparoscopic stoma creation over a 10-year period (ileostomy 30, colostomy 49, conversion 1), the overall morbidity rate was 11% with five major complications requiring reoperation, and no further stoma complications recorded within a 1-year follow-up. UC Despite significant advances in the medical treatment of UC, surgery remains definitive cure for these patients after failure of medical management or diagnosis of neoplastic degeneration [50,51]. A restorative procedure with the creation of an ileal pouch anal anastomosis (IPAA) is universally considered the standard of care. The earliest reports of a laparoscopic approach to ulcerative colitis was published in 1992 by Peters et al [52], who described the technique of laparoscopic proctocolectomy for two UC patients. The same year, Wexner and colleagues reported the first case-controlled series on the outcome of laparoscopic-assisted proctocolectomy with IPAA, showing a longer operative time compared to open procedure, and comparable postoperative ileus and hospital stay, with no shot-term benefits in favor of laparoscopy [53]. Since then, numerous series have been reported both in the adult and pediatric patient populations [54-56], but only from single institutions with short follow-ups [5,57]. Universally, these initial studies showed that laparoscopy took longer, with the exception of the series published by Araki et al [58]. In these studies only the colonic mobilization was performed laparoscopically, with vessel transection and rectal mobilization carried out through a mini laparotomy [53,58-61], with the exception of the series reported by Marcello et al [55], where a totally laparoscopic techniques was adopted, reserving a mini laparotomy only for specimen extraction. Subsequently, in a study from the Netherlands, 60 patients were randomized for hand assisted or laparoscopic restorative proctocolectomy with IPAA. The results from this study failed to show statistically significant differences in terms of morbidity, postoperative stay, quality of life at 3 mo after surgery, and overall costs, but the operative time for laparoscopy was significantly longer [62]. In a subsequent study, Polle et al [63] observed that female patients reported higher body image and cosmesis scores compared to open group, while there were no differences in functional outcome, morbidity, and overall quality of live. Similarly, Dunker et al [59] compared 16 patients who underwent restorative surgery with laparoscopic technique with 19 open patients. The authors found that laparoscopic patients showed significantly higher satisfaction with the cosmetic results and better body image, but once again functional outcome and quality of life were similar between groups. It seems evident, as it may have been expected, that laparoscopic IPAA offers significant advantages over the open conventional procedure in terms of body image and cosmesis, important factors in the acceptance of surgery in this young patient population, while conflicting results have been reported in terms of postoperative recovery. Faster return of bowel function after laparoscopy and decreased use of narcotics have been reported by some authors, not always translating into shorter hospital stay [57,62]. On the other hand, concerns have been raised regarding the duration of surgery often noted to be longer than open surgery even by very experienced laparoscopic surgeons, often resulting in higher costs. In regards to long-term pouch function, quality of life and complications, very few studies are available with adequate follow-up [5,57,62,63,59]. These observations were confirmed in a Cochrane review on 607 patients from 12 studies, only one randomized, which did not found any significant differences in complications, readmission, reoperation rates and mortality. However, once again, it showed that laparoscopic IPAA is associated with a significantly longer operating time, along with the inability to confirm conclusively the presumed shortterm benefits of laparoscopy, with length of follow-up too short for evaluating long-term outcomes [64]. Similar results were obtained in a subsequent meta-analysis on 16 studies, only one randomized, by Wu et al [13]. Postoperative fasting time and hospital stay were shorter for laparoscopy, and overall complication rates were higher after open surgery. Once again, laparoscopy took significantly longer and no advantages were demonstrated in terms of recovery of bowel function, postoperative septic complications, anastomotic leakage, postoperative bowel obstruction, blood loss, and mortality [13]. In our personal experience with 73 laparoscopic IPAA with a mean follow-up of 24 mo, the minimally invasive ap December 14, 2012 Volume 18 Issue 46

81 Zoccali M et al. Minimally invasive approach for IBD proach offered a statistically significant earlier return of flatus and resumption of diet, less intraoperative blood loss, and lower incidence of incisional hernias compared to 106 open IPAA, with no differences in overall complication rate, pouch function and quality of life [65]. The controversy about the safety of a single-stage procedure has not been resolved yet. Since long-term functional outcomes after IPAA are threatened by the occurrence of pouch-related septic complications, every effort should be made to reduce such complications and to identify patients at risk for pouch-related sepsis [66]. In a study by Marcello et al [67] on 59 patients who underwent laparoscopic proctocolectomy for UC, where only 9 patients received a diverting stoma at the primary procedure, 9 patients, all on high dose immunosuppressors or elevated body mass index, required a secondary ileostomy for postoperative complications. Better results were reported by Ky et al [68], with only one out 32 patients with an anastomotic leak requiring secondary diversion after one-stage laparoscopic restorative proctocolectomy. It is hard to analyze these data since these results can be influenced by patients selection; pelvic sepsis is reported to occur in up to 23% of patients after IPAA for UC, especially after the introduction of biologic therapy for IBD, in most cases secondary to an anastomotic leak [66,69-72]. In a recent study on 118 UC patients treated with a minimally invasive approach, we compared a 3-stage approach (laparoscopic abdominal colectomy followed by pouch surgery with a diverting loop ileostomy, 50 patients) with a 2-stage approach (laparoscopy colectomy with IPAA and diverting stoma at the initial operation, 68 patients). We observed a significant higher rate of septic complications in the 2-stage group (38.2% vs 21%, P < 0.05), despite 3-stage patients had been receiving a more aggressive medical therapy in the immediate preoperative period [73]. The role of laparoscopy for the treatment of ulcerative colitis in the emergency setting has been investigated by two studies. In the study by Bella and Seymour, 18 patients underwent laparoscopic-assisted restorative proctocolectomy for fulminant colitis, reporting a postoperative complication rate of 33%, with a length of stay of 5.0 d, which was shorter compared to the 8.8 d reported for the 6 open cases analyzed in the study [74]. The other study, by Marcello et al [67], reviewed the data from 19 laparoscopic and 29 conventional total colectomies with end ileostomy and mucous fistula buried within subcutaneous tissue for acute, not fulminant, UC, demonstrating longer operative time (210 vs 120 min) but lower complication rates (16% vs 24%), earlier return of bowel function (1 vs 2 d) and shorter length of stay (4 vs 6 d) for the laparoscopic group. SILS IN IBD During the last few years an increasing number of reports and case series on SILS colorectal resections for both benign and malignant diseases have been reported. Few studies have been published comparing SILS to standard laparoscopy, showing potential for improved short-term outcomes [75-78]. Besides the obvious cosmetic advantage resulting from a reduced number and size of scars - particularly important in a young IBD patient population - limiting the incisions seems to result in less postoperative pain, less use of narcotic pain medications, with consequent faster recovery and earlier discharge, along with a lower incidence of wound-related complications [17,76,78-80]. These data are still preliminary, with only few cases of SILS for UC published to date [4,16,17,75-78,81-92]. We believe that particularly for total abdominal colectomy (TAC) the SILS approach is a very attractive option in this patient population, representing a true scarless procedure, with the only access to the abdominal cavity at the site of the future stoma. Our preliminary results with the adoption of a well-standardized SILS approach to TAC confirm the potential of this technique in improving the postoperative recovery in selected patients, without significant increases in operative time and costs [93]. CONCLUSION During the past three decades the evidence has been accumulating in favor of a minimally invasive approach to IBD. Crohn s disease is probably one of the most challenging diseases to treat laparoscopically for the colorectal surgeons, especially when the disease is located in the colon and involves multiple segments, thus explaining the fact that in the United States the majority of CD patients are still approached with open surgery. Laparoscopic IPAA for UC has been shown to be feasible, but to date the evidence present in the literature is still not conclusive. Current data suggest a shorter length of stay, shorter ileus, faster recovery and less postoperative pain, along with better cosmesis with minimally invasive surgery. On the other hand, significantly longer operative times with laparoscopy are universally reported. Our goal and responsibility is to explore new avenues for a true minimally invasive approach to IBD and to train the next generation of surgeons to facilitate wide spread acceptance of laparoscopy. REFERENCES 1 Baumgart DC, Sandborn WJ. Inflammatory bowel disease: clinical aspects and established and evolving therapies. Lancet 2007; 369: Cohen RD, Tsang JF, Hanauer SB. Infliximab in Crohn s disease: first anniversary clinical experience. Am J Gastroenterol 2000; 95: Kunitake H, Hodin R, Shellito PC, Sands BE, Korzenik J, Bordeianou L. Perioperative treatment with infliximab in patients with Crohn s disease and ulcerative colitis is not associated with an increased rate of postoperative complications. J Gastrointest Surg 2008; 12: ; discussion Reissman P, Salky BA, Pfeifer J, Edye M, Jagelman DG, Wexner SD. Laparoscopic surgery in the management of inflammatory bowel disease. 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83 Zoccali M et al. Minimally invasive approach for IBD of laparoscopic and open ileocecal resection for Crohn s disease: a metaanalysis. Surg Endosc 2006; 20: Seymour NE, Kavic SM. Laparoscopic management of complex Crohn s disease. JSLS 2003; 7: Brouquet A, Bretagnol F, Soprani A, Valleur P, Bouhnik Y, Panis Y. A laparoscopic approach to iterative ileocolonic resection for the recurrence of Crohn s disease. Surg Endosc 2010; 24: Hasegawa H, Watanabe M, Nishibori H, Okabayashi K, Hibi T, Kitajima M. Laparoscopic surgery for recurrent Crohn s disease. Br J Surg 2003; 90: Goyer P, Alves A, Bretagnol F, Bouhnik Y, Valleur P, Panis Y. Impact of complex Crohn s disease on the outcome of laparoscopic ileocecal resection: a comparative clinical study in 124 patients. Dis Colon Rectum 2009; 52: Hamel CT, Hildebrandt U, Weiss EG, Feifelz G, Wexner SD. Laparoscopic surgery for inflammatory bowel disease. 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Dis Colon Rectum 2002; 45: ; discussion Mor IJ, Vogel JD, da Luz Moreira A, Shen B, Hammel J, Remzi FH. Infliximab in ulcerative colitis is associated with an increased risk of postoperative complications after restorative proctocolectomy. Dis Colon Rectum 2008; 51: ; discussion Selvasekar CR, Cima RR, Larson DW, Dozois EJ, Harrington JR, Harmsen WS, Loftus EV, Sandborn WJ, Wolff BG, Pemberton JH. Effect of infliximab on short-term complications in patients undergoing operation for chronic ulcerative colitis. J Am Coll Surg 2007; 204: ; discussion Lim WC, Hanauer SB. Emerging biologic therapies in inflammatory bowel disease. Rev Gastroenterol Disord 2004; 4: Heuschen UA, Hinz U, Allemeyer EH, Autschbach F, Stern J, Lucas M, Herfarth C, Heuschen G. Risk factors for ileoanal J pouch-related septic complications in ulcerative colitis and familial adenomatous polyposis. Ann Surg 2002; 235: Pandey S, Luther G, Umanskiy K, Malhotra G, Rubin MA, Hurst RD, Fichera A. Minimally invasive pouch surgery for ulcerative colitis: is there a benefit in staging? Dis Colon Rectum 2011; 54: Bell RL, Seymour NE. Laparoscopic treatment of fulminant ulcerative colitis. Surg Endosc 2002; 16: Adair J, Gromski MA, Lim RB, Nagle D. Single-incision laparoscopic right colectomy: experience with 17 consecutive cases and comparison with multiport laparoscopic right colectomy. Dis Colon Rectum 2010; 53: Champagne BJ, Lee EC, Leblanc F, Stein SL, Delaney CP. Single-incision vs straight laparoscopic segmental colectomy: 6762 December 14, 2012 Volume 18 Issue 46

84 Zoccali M et al. Minimally invasive approach for IBD a case-controlled study. Dis Colon Rectum 2011; 54: Gandhi DP, Ragupathi M, Patel CB, Ramos-Valadez DI, Pickron TB, Haas EM. Single-incision versus hand-assisted laparoscopic colectomy: a case-matched series. J Gastrointest Surg 2010; 14: Ross H, Steele S, Whiteford M, Lee S, Albert M, Mutch M, Rivadeneira D, Marcello P. Early multi-institution experience with single-incision laparoscopic colectomy. Dis Colon Rectum 2011; 54: Dunker MS, Stiggelbout AM, van Hogezand RA, Ringers J, Griffioen G, Bemelman WA. Cosmesis and body image after laparoscopic-assisted and open ileocolic resection for Crohn s disease. Surg Endosc 1998; 12: Munro MG. Laparoscopic access: complications, technologies, and techniques. Curr Opin Obstet Gynecol 2002; 14: Remzi FH, Kirat HT, Kaouk JH, Geisler DP. Single-port laparoscopy in colorectal surgery. Colorectal Dis 2008; 10: Geisler DP, Condon ET, Remzi FH. Single incision laparoscopic total proctocolectomy with ileopouch anal anastomosis. Colorectal Dis 2010; 12: Remzi FH, Kirat HT, Geisler DP. Laparoscopic single-port colectomy for sigmoid cancer. Tech Coloproctol 2010; 14: Bucher P, Pugin F, Morel P. Single-port access laparoscopic radical left colectomy in humans. Dis Colon Rectum 2009; 52: Cahill RA, Lindsey I, Jones O, Guy R, Mortensen N, Cunningham C. Single-port laparoscopic total colectomy for medically uncontrolled colitis. Dis Colon Rectum 2010; 53: Boni L, Dionigi G, Cassinotti E, Di Giuseppe M, Diurni M, Rausei S, Cantore F, Dionigi R. Single incision laparoscopic right colectomy. Surg Endosc 2010; 24: Brunner W, Schirnhofer J, Waldstein-Wartenberg N, Frass R, Weiss H. Single incision laparoscopic sigmoid colon resections without visible scar: a novel technique. Colorectal Dis 2010; 12: Leblanc F, Makhija R, Champagne BJ, Delaney CP. Single incision laparoscopic total colectomy and proctocolectomy for benign disease: initial experience. Colorectal Dis 2011; 13: Pietrasanta D, Romano N, Prosperi V, Lorenzetti L, Basili G, Goletti O. Single-incision laparoscopic right colectomy for cancer: a single-centre preliminary experience. Updates Surg 2010; 62: Leblanc F, Champagne BJ, Augestad KM, Stein SL, Marderstein E, Reynolds HL, Delaney CP. Single incision laparoscopic colectomy: technical aspects, feasibility, and expected benefits. Diagn Ther Endosc 2010; 2010: Law WL, Fan JK, Poon JT. Single-incision laparoscopic colectomy: early experience. Dis Colon Rectum 2010; 53: Uematsu D, Akiyama G, Matsuura M, Hotta K. Single-access laparoscopic colectomy with a novel multiport device in sigmoid colectomy for colon cancer. Dis Colon Rectum 2010; 53: Fichera A, Zoccali M, Gullo R. Single incision ( scarless ) laparoscopic total abdominal colectomy with end ileostomy for ulcerative colitis. J Gastrointest Surg 2011; 15: S- Editor Shi ZF L- Editor A E- Editor Li JY 6763 December 14, 2012 Volume 18 Issue 46

85 Online Submissions: doi: /wjg.v18.i World J Gastroenterol 2012 December 14; 18(46): ISSN (print) ISSN (online) 2012 Baishideng. All rights reserved. Marco Giuseppe Patti, MD, Professor, Series Editor TOPIC HIGHLIGHT Impact of minimally invasive surgery on the treatment of benign esophageal disorders Brian Bello, Fernando A Herbella, Marco E Allaix, Marco G Patti Brian Bello, Fernando A Herbella, Marco E Allaix, Marco G Patti, Department of Surgery, University of Chicago Pritzker School of Medicine, Chicago, IL 60637, United States Author contributions: Bello B, Herbella FA, Allaix ME and Patti MG contributed equally to the conception and design of this work; Bello B, Herbella FA, Allaix ME and Patti MG drafted and revised the article; all authors approved the final version to be published. Correspondence to: Marco G Patti, MD, Department of Surgery, University of Chicago Pritzker School of Medicine, 5841 South Maryland Ave, MC 5031, Room G-201, Chicago, IL 60637, United States. [email protected] Telephone: Fax: Received: April 18, 2012 Revised: May 26, 2012 Accepted: July 18, 2012 Published online: December 14, 2012 Abstract Thanks to the development of minimally invasive surgery, the last 20 years have witnessed a change in the treatment algorithm of benign esophageal disorders. Today a laparoscopic operation is the treatment of choice for esophageal achalasia and for most patients with gastroesophageal reflux disease. Because the pathogenesis of achalasia is unknown, treatment is palliative and aims to improve esophageal emptying by decreasing the functional obstruction at the level of the gastro-esophageal junction. The refinement of minimally invasive techniques accompanied by large, multiple randomized control trials with long-term outcome has allowed the laparoscopic Heller myotomy and partial fundoplication to become the treatment of choice for achalasia compared to endoscopic procedures, including endoscopic botulinum toxin injection and pneumatic dilatation. Patients with suspected gastroesophageal reflux need to undergo a thorough preoperative workup. After establishing diagnosis, treatment for gastroesophageal reflux should be individualized to patient characteristics and a decision about an operation made jointly between surgeon and patient. The indications for surgery have changed in the last twenty years. In the past, surgery was often considered for patients who did not respond well to acid reducing medications. Today, the best candidate for surgery is the patient who has excellent control of symptoms with proton pump inhibitors. The minimally invasive approach to antireflux surgery has allowed surgeons to control reflux in a safe manner, with excellent long term outcomes. Like achalasia and gastroesophageal reflux, the treatment of patients with paraesophageal hernias has also seen a major evolution. The laparoscopic approach has been shown to be safe, and durable, with good relief of symptoms over the long-term. The most significant controversy with laparoscopic paraesophageal hernia repair is the optimal crural repair. This manuscript reviews the evolution of these techniques Baishideng. All rights reserved. Key words: Gastroesophageal reflux disease; Esophageal achalasia; Hiatal hernia; Laparoscopic fundoplication; Laparoscopic Heller myotomy Peer reviewers: Liang-Shun Wang, Professor, Shuang-Ho Hospital-Taipei Medical University, No.291, Jhongjheng Rd., Jhonghe City, Taipei 235, Taiwan, China; Eric S Hungness, MD, Assistant Professor, Department of Surgery, Northwestern University Feinberg School of Medicine, 676 N. St. Clair St., Suite 650, Chicago, IL , United States; Dr. Satoru Motoyama, Department of Surgery, Akita University Graduate School of Medicine, Akita , Japan Bello B, Herbella FA, Allaix ME, Patti MG. Impact of minimally invasive surgery on the treatment of benign esophageal disorders. World J Gastroenterol 2012; 18(46): Available from: URL: v18/i46/6764.htm DOI: i December 14, 2012 Volume 18 Issue 46

86 Bello B et al. Minimally invasive treatment and esophageal disorders INTRODUCTION The last two decades have seen a shift in the treatment algorithm of benign esophageal disorders, largely due to the introduction and development of minimally invasive surgery. In the early 1990s, it became clear that a minimally invasive approach for esophageal achalasia, gastroesophageal reflux disease, and paraesophageal hernias, provided outcomes comparable to those achieved by open techniques but with less postoperative pain, shorter hospital stay and earlier return to work [1-3]. The late 1990s and last 10 years saw the evolution of the laparoscopic approach thanks to better instrumentation and expertise. As more of these procedures were performed, more long-term data became available allowing improved analysis of the technique and the outcome. For example, in the early 1990s a left thoracoscopic Heller myotomy was considered the surgical procedure of choice for achalasia, yet now the standard of care is a laparoscopic Heller myotomy with partial fundoplication as this operation allows better relief of dysphagia and a lower incidence of postoperative reflux [4-8]. In a similar fashion, both total fundoplication and partial fundoplication were initially considered equivalent for the treatment of gastroesophageal reflux disease, however, long-term follow-up indicates that a laparoscopic total fundoplication is superior [9]. In addition, a laparoscopic approach to paraesophageal hernias offers fewer complications and shorter hospital stay than to the open approach [10]. In this review, we focus on the impact of minimally invasive surgery on the treatment of achalasia, gastroesophageal reflux disease (GERD), and paraesophageal hernias. ACHALASIA Esophageal achalasia is a primary esophageal motility disorder characterized by lack of esophageal peristalsis and inability of the lower esophageal sphincter (LES) to relax properly in response to swallowing. The goal of treatment is to relieve the functional obstruction caused by the LES, therefore allowing emptying of food from the esophagus into the stomach by gravity. The treatment of this disease is based on a delicate balance between elimination of the functional obstruction caused by the LES and the need to prevent gastroesophageal reflux into an aperistaltic esophagus, with risk of developing complications such as strictures, Barrett s esophagus, and even adenocarcinoma [11-14]. Open era In 1913, Heller first described the simultaneous performance of two myotomies, on the anterior and posterior sides of the esophagus, to treat patients with achalasia. A decade later, Zaaijer would restrict the procedure to a single anterior myotomy. For decades, Heller myotomy was the standard treatment for esophageal achalasia. In the 1970s and 1980s, pneumatic dilatation became the primary form of treatment as it was believed that the results were equivalent to those of a myotomy but this approach avoided the postoperative pain and the long recovery time which followed surgery. Very few Heller myotomies were performed during this era, and they were mostly reserved for patients whose dysphagia did not improve with balloon dilatation or whose esophagus was perforated during a dilatation [15]. During this era, the myotomy was performed by either a left thoracotomy or a laparotomy. Ellis described his results using a left transthoracic approach to perform a Heller myotomy [6]. His myotomy extended for only 5 mm onto the gastric wall, the rationale being that a short myotomy relieved dysphagia but avoided reflux, therefore obviating the need for a fundoplication. His results showed relief of dysphagia in about 90% of patients and symptomatic reflux in 5% [12]. However, when gastroesophageal reflux was objectively measured postoperatively by manometry and ph monitoring, abnormal esophageal acid exposure was found in 29% of patients [16]. These findings underscored the importance of objective measurement of reflux in achalasia patients. A trans-abdominal approach was used mostly in Europe and South America. Bonavina et al [17] from Italy reported the results of trans-abdominal Heller myotomy and Dor fundoplication in 206 patients operated between 1976 and The operation yielded excellent or good results in 94% of patients. Abnormal postoperative gastroesophageal reflux, as measured by ph monitoring, was present in 8.6% of patients only. Minimally invasive era A shift in the treatment algorithm of achalasia has slowly occurred in the last decade due to the excellent outcome of minimally invasive techniques. In 1992, we reported our initial experience with a thoracoscopic approach [1], following the technique first described by Shimi et al [18]. Using the guidance provided by intraoperative endoscopy, we performed a left thoracoscopic myotomy which extended for only 5 mm onto the gastric wall. Like Ellis [12], the rationale for a shorter myotomy was to relieve dysphagia while trying to avoid postoperative reflux. The hospital stay was short, postoperative discomfort was minimal, and the recovery was fast [1]. Long-term follow-up showed relief of dysphagia in almost 90% of patients [19]. Despite these good results, some shortcomings of the thoracoscopic technique soon became apparent. This approach required a double lumen tube for one lung ventilation and a chest tube. In addition, the exposure of the gastroesophageal junction was limited by the diaphragm. Most striking though was that a thoracoscopic myotomy was associated with reflux in up to 60% of patients when studied by postoperative ph monitoring [19]. A significant amount of postoperative reflux after thoracoscopic Heller myotomy was also demonstrated by others [20,21]. These limitations of the thoracoscopic approach were the key reasons for the switch to a laparoscopic approach. Is a fundoplication necessary? A laparoscopic Heller myotomy alone is also associated with a high incidence of postoperative reflux, with the 6765 December 14, 2012 Volume 18 Issue 46

87 Bello B et al. Minimally invasive treatment and esophageal disorders risk of severe esophageal damage. In several retrospective studies, objective evidence of reflux was demonstrated in about 60% of patients postoperatively [22,23]. Subsequent randomized control trials confirmed this observation and suggested that a fundoplication decreases the incidence of this problem. In 2003, Falkenback et al [24] reported the results of a prospective and randomized trial comparing myotomy alone vs myotomy with Nissen fundoplication. Using postoperative ph monitoring, they found abnormal reflux in 25% of patients who had a myotomy and fundoplication as compared to 100% of patients who had a myotomy alone. Twenty percent of patients in the myotomy alone group subsequently developed Barrett s esophagus [24]. Another randomized trial compared Heller myotomy alone vs Heller myotomy and Dor fundoplication. Pathologic gastroesophageal reflux was demonstrated by ph monitoring in 48% of patients after Heller myotomy alone but in 9.5% only when a Dor fundoplication was added [25]. Relief of dysphagia was similar in the 2 groups. Both the total (Nissen) and partial (Dor) fundoplications prevent reflux in the majority of patients. The above trials provide Level 1 evidence that a fundoplication should be performed at the time of a laparoscopic Heller myotomy. Partial vs total fundoplication In patients with gastroesophageal reflux, a laparoscopic total fundoplication is the procedure of choice, even when esophageal peristalsis is weak [9]. In contrast, because of the absence of peristalsis in achalasia, a total fundoplication may cause too much resistance at the level of the gastroesophageal junction, therefore impeding the emptying of food from the esophagus into the stomach. This can eventually cause persistent or recurrent dysphagia. There have been long-term studies demonstrating that postoperative dysphagia is initially significantly decreased after a Heller myotomy with a total fundoplication [24,26,27]. Yet, most surgeons have abandoned the use of a total fundoplication and switched to a partial fundoplication based on other long-term data showing that dysphagia eventually recurs in most patients that have a total fundoplication. For example, Duranceau et al [28] initially reported excellent results with a Heller myotomy and total fundoplication, as dysphagia improved in most patients and there was no symptomatic reflux. Ten years later, however, they noted that symptoms had recurred in 82% of patients probably due to complete decompensation during the follow-up period [29]. A prospective, randomized trial comparing long-term results of laparoscopic Heller myotomy and Dor fundoplication vs laparoscopic Heller myotomy and floppy-nissen fundoplication for achalasia recently demonstrated that there was a statistically significant difference in dysphagia rates (2.8% vs 15%, P < ). Although both techniques achieved long-term reflux control, the recurrence rate of dysphagia was significantly higher among patients who underwent a Nissen fundoplication [30]. These data provide Level 1 evidence supporting the use of a partial fundoplication with a Heller myotomy as the preferred choice of fundoplication for achalasia. Partial fundoplication: Anterior vs posterior Currently, deciding between which partial fundoplication is best, either a posterior (Toupet) or an anterior fundoplication (Dor) after Heller myotomy, is controversial. Some groups favor a posterior fundoplication as it might be more effective in preventing reflux and because it might keep the edges of the myotomy separate [4,5,31-33]. One retrospective study demonstrated a lower rate of recurrent dysphagia in the Toupet fundoplication group compared to the Dor fundoplication group (3% vs 17%). However, conclusions from this study are limited since the comparison groups were not equivalent: the followup period for the Dor fundoplication group was longer and the length of the myotomy was longer in the Toupet fundoplication group [33]. Our preference is for a Dor fundoplication because it is simpler to perform, in that there is no need for a posterior dissection. In addition, it adds the advantage of covering the esophageal mucosa. It certainly is the procedure of choice if there is any suspicion of injury to the mucosa or if a perforation has occurred. Many studies have demonstrated that a laparoscopic Heller myotomy with anterior fundoplication significantly relieves the symptoms of achalasia in about 90%, while limiting gastroesophageal reflux [6-8,32,34-37]. Recently presented at the Society of American Gastrointestinal and Endoscopic Surgeons annual meeting in 2011 were the findings of a prospective, randomized, and multicenter study comparing laparoscopic Heller myotomy and Dor fundoplication and laparoscopic Heller myotomy and Toupet fundoplication. Follow-up monitoring by ph monitoring at six months showed no statistical significance between a Dor fundoplication and Toupet fundoplication [38]. The last two decades have witnessed a shift in the treatment algorithm for esophageal achalasia. The refinement of minimally invasive techniques accompanied by large, multiple randomized control trials with long-term outcome has allowed the laparoscopic Heller myotomy and partial fundoplication to become the treatment of choice for achalasia. GERD The pathophysiology of GERD is multifactorial and complex. It is well established that the lower LES plays a key role in the antireflux mechanism. This is evidence that the striated muscles of the crus fail to exert its synergistic action with the LES when a hiatal hernia is present, thus, causing reflux [39]. In addition, esophageal dysmotility can also play a crucial role: 40% to 50% of patients with GERD have low amplitude of peristalsis or an abnormal propagation of peristaltic waves [40]. As a consequence, acid clearance is impaired, potentially caus December 14, 2012 Volume 18 Issue 46

88 Bello B et al. Minimally invasive treatment and esophageal disorders ing more mucosal injury. Furthermore, it has been demonstrated that a mixed reflux of both gastric and duodenal juices plays a role in the pathogenesis of GERD, particularly in patients with Barrett s esophagus. This has a profound impact on treatment considering that proton pump inhibitors only control acid secretion and not duodenal reflux [41]. The ph of the refluxate is changed, yet the underlying problem has not been addressed: the competence of the lower esophageal sphincter. The laparoscopic 360 fundoplication addresses this very issue, which can block either type of refluxate. Open era Nissen [42] first described an antireflux procedure with a 360 wrap in Reflux symptoms improved, yet there were problems with dysphagia and inability to belch. Multiple modifications and different types of partial fundoplications were then introduced over the years. In 1986, DeMeester et al [43] described several modifications to operative technique that would prove to be valuable. Increasing the bougie size seemed to reduce the incidence of postoperative dysphagia. Similar results were obtained by shortening the length of the fundoplication. Finally, a more extensive mobilization of the fundus increased the incidence of complete distal esophageal sphincter relaxation. Laparoscopic 360 fundoplication The laparoscopic 360 fundoplication (total) was initially described by Dallemagne et al [44] in Despite some surgeons favored partial fundoplication in some settings, it was shown that laparoscopic partial fundoplication was not as effective in controlling reflux as a laparoscopic total fundoplication [9]. A 360 fundoplication using minimally invasive techniques is associated with a low morbidity, a short hospital stay, and excellent outcomes [45,46]. The operation controls reflux by improving esophageal motility, both in terms of LES competence and quality of esophageal peristalsis [9,47]. Long-term studies have shown that fundoplication controls symptoms in 93% of patients after 5 years and 89% after 10 years [48]. Postoperative dysphagia has been described in approximately 8% of patients. However, this usually resolves in most patients within a few months and rarely requires any intervention [9]. Clarifying indications The indications for surgery have changed in the last twenty years. Surgery was often considered for patients who did not respond well to acid reducing medications. However, the paradigm has changed. Today, the best candidate for surgery is the patient who has excellent control of symptoms with proton pump inhibitors [49]. We feel that laparoscopic 360 fundoplication is indicated in the following circumstances: (1) when heartburn and regurgitation are not completely controlled by medicationst; (2) when it is thought that cough is induced by reflux. For instance, Mainie et al [50] demonstrated that patients resistant to proton pump inhibitors (PPIs) with non-acid or acid reflux demonstrated by multichannel intraluminal impedance-ph monitoring and with a positive symptom index, can be treated successfully by a laparoscopic 360 fundoplication); (3) poor patient compliance with medical treatment; (4) cost of medical therapy is prohibitive (most insurance companies in the United States will pay for one PPI pill per day only, with many patients prescribed more frequent dosing); (5) postmenopausal women with osteoporosis. It has been shown that PPIs and H2 blockers may increase the risk of hip and femur fractures because of decreased calcium absorption [51] ; and (6) young and very symptomatic patients in whom life-long medical treatment is not advisable. Finally, in a recently published meta-analysis of medical vs surgical management for GERD, Wileman et al [52] have shown that, in adults, laparoscopic fundoplication is more effective than medical management for the treatment of GERD in the short to medium term. Surgery, however, carries some risks and its application should be individualized as the decision to undergo fundoplication should be based on patient and surgeon preference. The minimally invasive approach to antireflux surgery has allowed surgeons to control reflux in a safe manner without troublesome side effects. Long term outcomes are excellent and risk is minimal. Nonetheless, patients with suspected reflux need to undergo a thorough preoperative evaluation. After establishing diagnosis, treatment for gastroesophageal reflux should be individualized to patient characteristics and a decision about an operation made jointly between surgeon and patient. PARAESOPHAGEAL HERNIA Paraesophageal hernias are rare and only make up a small fraction of hiatal hernias. Yet because of their association with serious morbidity and potential mortality, knowing how to treat these patients is essential. Open era Like achalasia and gastroesophageal reflux, the treatment of patients with paraesophageal hernias has also seen a major evolution. Thanks to the advent of minimally invasive techniques, a laparoscopic repair has slowly replaced an open approach via a laparotomy or left thoracotomy. In the 1980s, an open technique was standard of care and was shown to be effective. Ellis et al [53] demonstrated an 88% benefit from an open paraesophageal hiatal hernia repair via laparotomy. Their technique commonly included an antireflux procedure and a Stamm gastrotomy. Average length of stay was 9.5 d and the complication rate was 24%. Minimally invasive era The last two decades have seen the emergence of laparoscopy for treatment of paraesophageal hernias. Though it is a technically challenging operation, the laparoscopic approach has been shown to be safe, durable, with good 6767 December 14, 2012 Volume 18 Issue 46

89 Bello B et al. Minimally invasive treatment and esophageal disorders relief of symptoms over the long-term, decreased postoperative pain, and a rapid return to normal activities. The majority of patients also undergo an antireflux procedure as well [54,55]. The basic principles of the operation include excising the hernia sac, mobilizing the esophagus extensively in the posterior mediastinum, closing the hiatus, and a fundoplication. Current status Despite popularization of the laparoscopic approach, many controversies remain. The majority of surgeons advocate complete excision of the hernia sac for several reasons. A sac remnant could potentially act as a potential lead point for a recurrence or interfere with the crural repair or fundoplication. Others argue that removing the sac can potentially cause injury to the mediastinum and pleura and that it is not necessary. Aly et al [56] showed that sac excision was not routinely performed and had no significant effect on quality of life measures and postop barium swallow radiographs. The most significant controversy with laparoscopic paraesophageal hernia repair though is recurrence rate. In several large volume studies, hiatal hernia recurrence rate is reported as high as 28%-44% for the laparoscopic repair [56-59]. These rates are significantly higher than for open repair. However, prior open series rarely measured outcomes objectively as they routinely do now. When repairing paraesophageal hernias, there can be a significant amount of tension with a primary repair, which ultimately may lead to recurrence. Due to the significant use of synthetic mesh for tension free repairs inguinal hernias, these were soon applied to paraesophageal hernias repairs. One randomized control trial showed a significant reduction in recurrence rates at the one year follow-up period [60]. However, there was a significantly higher rate of dysphagia and potential for erosion into the esophagus or stomach [60,61]. Synthetic mesh is rarely used now. In 2006, Oelschlager et al [62] published a multicenter, prospective randomized trial assessing biologic prosthesis for paraesophageal hernias as a buttress with a primary repair. They used small intestinal submucosa (SIS), an acellular xenograft consisting primarily of porcine collagen. Their findings at six month follow up were promising. The primary outcome measure was a greater than 2 cm recurrent hernia on upper gastrointestinal (UGI) series. At six months, patients who had the biologic prosthesis buttress repair had a 9% recurrence rate compared to 24% in the group who had a primary repair alone of the hiatus. The long-term data from that study were recently presented at the 2010 American College of Surgeons Annual Clinical Congress. Unlike the short-term data, there was no difference in recurrence in long-term follow up. With a median follow-up of 58 mo, there was no statistical difference in symptom control, quality of life measures or UGI recurrence rates between the primary repair alone vs primary repair with a SIS buttress. They concluded that both types of repair gave long and durable relief, but the benefit of reducing hiatal hernia recurrence in the short term had diminished by the five year mark [63]. There have been new biologic mesh materials and redesigning of existing materials that will need to be studied in the future. CONCLUSION Despite controversies for laparoscopic paraesophageal hernia repairs, there are clear advantages when compared to open repair. Hospital stays are usually around two days and much shorter than before, pain is improved, and patients have less complications. More long-term data will be needed to assess the optimal crural repair. REFERENCES 1 Pellegrini C, Wetter LA, Patti M, Leichter R, Mussan G, Mori T, Bernstein G, Way L. Thoracoscopic esophagomyotomy. 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90 Bello B et al. Minimally invasive treatment and esophageal disorders 14 Devaney EJ, Iannettoni MD, Orringer MB, Marshall B. Esophagectomy for achalasia: patient selection and clinical experience. Ann Thorac Surg 2001; 72: Sauer L, Pellegrini CA, Way LW. The treatment of achalasia. A current perspective. Arch Surg 1989; 124: ; discussion Streitz JM, Ellis FH, Williamson WA, Glick ME, Aas JA, Tilden RL. Objective assessment of gastroesophageal reflux after short esophagomyotomy for achalasia with the use of manometry and ph monitoring. J Thorac Cardiovasc Surg 1996; 111: ; discussion Bonavina L, Nosadini A, Bardini R, Baessato M, Peracchia A. Primary treatment of esophageal achalasia. Long-term results of myotomy and Dor fundoplication. Arch Surg 1992; 127: ; discussion Shimi S, Nathanson LK, Cuschieri A. Laparoscopic cardiomyotomy for achalasia. J R Coll Surg Edinb 1991; 36: Patti MG, Arcerito M, De Pinto M, Feo CV, Tong J, Gantert W, Way LW. 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Surg Endosc 2005; 19: Falkenback D, Johansson J, Oberg S, Kjellin A, Wenner J, Zilling T, Johnsson F, Von Holstein CS, Walther B. Heller s esophagomyotomy with or without a 360 degrees floppy Nissen fundoplication for achalasia. Long-term results from a prospective randomized study. Dis Esophagus 2003; 16: Richards WO, Torquati A, Holzman MD, Khaitan L, Byrne D, Lutfi R, Sharp KW. Heller myotomy versus Heller myotomy with Dor fundoplication for achalasia: a prospective randomized double-blind clinical trial. Ann Surg 2004; 240: ; discussion Frantzides CT, Moore RE, Carlson MA, Madan AK, Zografakis JG, Keshavarzian A, Smith C. Minimally invasive surgery for achalasia: a 10-year experience. J Gastrointest Surg 2004; 8: Rossetti G, Brusciano L, Amato G, Maffettone V, Napolitano V, Russo G, Izzo D, Russo F, Pizza F, Del Genio G, Del Genio A. A total fundoplication is not an obstacle to esophageal emptying after heller myotomy for achalasia: results of a long-term follow up. Ann Surg 2005; 241: Duranceau A, LaFontaine ER, Vallieres B. Effects of total fundoplication on function of the esophagus after myotomy for achalasia. Am J Surg 1982; 143: Topart P, Deschamps C, Taillefer R, Duranceau A. Longterm effect of total fundoplication on the myotomized esophagus. Ann Thorac Surg 1992; 54: ; discussion Rebecchi F, Giaccone C, Farinella E, Campaci R, Morino M. Randomized controlled trial of laparoscopic Heller myotomy plus Dor fundoplication versus Nissen fundoplication for achalasia: long-term results. Ann Surg 2008; 248: Hunter JG, Trus TL, Branum GD, Waring JP. Laparoscopic Heller myotomy and fundoplication for achalasia. Ann Surg 1997; 225: ; discussion Vogt D, Curet M, Pitcher D, Josloff R, Milne RL, Zucker K. Successful treatment of esophageal achalasia with laparoscopic Heller myotomy and Toupet fundoplication. Am J Surg 1997; 174: Oelschlager BK, Chang L, Pellegrini CA. Improved outcome after extended gastric myotomy for achalasia. Arch Surg 2003; 138: ; discussion Yamamura MS, Gilster JC, Myers BS, Deveney CW, Sheppard BC. Laparoscopic heller myotomy and anterior fundoplication for achalasia results in a high degree of patient satisfaction. Arch Surg 2000; 135: Finley RJ, Clifton JC, Stewart KC, Graham AJ, Worsley DF. Laparoscopic Heller myotomy improves esophageal emptying and the symptoms of achalasia. Arch Surg 2001; 136: Ackroyd R, Watson DI, Devitt PG, Jamieson GG. Laparoscopic cardiomyotomy and anterior partial fundoplication for achalasia. Surg Endosc 2001; 15: Chapman JR, Joehl RJ, Murayama KM, Tatum RP, Shi G, Hirano I, Jones MP, Pandolfino JE, Kahrilas PJ. Achalasia treatment: improved outcome of laparoscopic myotomy with operative manometry. Arch Surg 2004; 139: ; discussion Rawlings A, Soper NJ, Oelschlager B, Swanstrom L, Matthews BD, Pellegrini C, Pierce RA, Pryor A, Martin V, Frisella MM, Cassera M, Brunt LM. Laparoscopic Dor versus Toupet fundoplication following Heller myotomy for achalasia: results of a multicenter, prospective, randomizedcontrolled trial. Surg Endosc 2012; 26: Patti MG, Goldberg HI, Arcerito M, Bortolasi L, Tong J, Way LW. Hiatal hernia size affects lower esophageal sphincter function, esophageal acid exposure, and the degree of mucosal injury. Am J Surg 1996; 171: Diener U, Patti MG, Molena D, Fisichella PM, Way LW. Esophageal dysmotility and gastroesophageal reflux disease. J Gastrointest Surg 2001; 5: Kauer WK, Peters JH, DeMeester TR, Ireland AP, Bremner CG, Hagen JA. Mixed reflux of gastric and duodenal juices is more harmful to the esophagus than gastric juice alone. The need for surgical therapy re-emphasized. Ann Surg 1995; 222: ; discussion Nissen R. A simple operation for control of reflux esophagitis. Schweiz Med Wochenschr 1956; 86: DeMeester TR, Bonavina L, Albertucci M. Nissen fundoplication for gastroesophageal reflux disease. Evaluation of primary repair in 100 consecutive patients. Ann Surg 1986; 204: Dallemagne B, Weerts JM, Jehaes C, Markiewicz S, Lombard R. Laparoscopic Nissen fundoplication: preliminary report. Surg Laparosc Endosc 1991; 1: Meneghetti AT, Tedesco P, Galvani C, Gorodner MV, Patti MG. Outcomes after laparoscopic Nissen fundoplication are not influenced by the pattern of reflux. Dis Esophagus 2008; 21: Tedesco P, Lobo E, Fisichella PM, Way LW, Patti MG. Laparoscopic fundoplication in elderly patients with gastroesophageal reflux disease. Arch Surg 2006; 141: ; discussion Herbella FA, Tedesco P, Nipomnick I, Fisichella PM, Patti MG. Effect of partial and total laparoscopic fundoplication on esophageal body motility. Surg Endosc 2007; 21: Dallemagne B, Weerts J, Markiewicz S, Dewandre JM, Wahlen C, Monami B, Jehaes C. Clinical results of laparoscopic fundoplication at ten years after surgery. 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91 Bello B et al. Minimally invasive treatment and esophageal disorders Nissen fundoplication. J Gastrointest Surg 1999; 3: Mainie I, Tutuian R, Agrawal A, Adams D, Castell DO. Combined multichannel intraluminal impedance-ph monitoring to select patients with persistent gastro-oesophageal reflux for laparoscopic Nissen fundoplication. Br J Surg 2006; 93: Corley DA, Kubo A, Zhao W, Quesenberry C. Proton pump inhibitors and histamine-2 receptor antagonists are associated with hip fractures among at-risk patients. Gastroenterology 2010; 139: Wileman SM, McCann S, Grant AM, Krukowski ZH, Bruce J. Medical versus surgical management for gastro-oesophageal reflux disease (GORD) in adults. Cochrane Database Syst Rev 2010; (3): CD Ellis FH, Crozier RE, Shea JA. Paraesophageal hiatus hernia. Arch Surg 1986; 121: Gangopadhyay N, Perrone JM, Soper NJ, Matthews BD, Eagon JC, Klingensmith ME, Frisella MM, Brunt LM. Outcomes of laparoscopic paraesophageal hernia repair in elderly and high-risk patients. Surgery 2006; 140: ; discussion Nason KS, Luketich JD, Qureshi I, Keeley S, Trainor S, Awais O, Shende M, Landreneau RJ, Jobe BA, Pennathur A. Laparoscopic repair of giant paraesophageal hernia results in long-term patient satisfaction and a durable repair. J Gastrointest Surg 2008; 12: ; discussion Aly A, Munt J, Jamieson GG, Ludemann R, Devitt PG, Watson DI. Laparoscopic repair of large hiatal hernias. Br J Surg 2005; 92: Mattar SG, Bowers SP, Galloway KD, Hunter JG, Smith CD. Long-term outcome of laparoscopic repair of paraesophageal hernia. Surg Endosc 2002; 16: Diaz S, Brunt LM, Klingensmith ME, Frisella PM, Soper NJ. Laparoscopic paraesophageal hernia repair, a challenging operation: medium-term outcome of 116 patients. J Gastrointest Surg 2003; 7: 59-66; discussion Andujar JJ, Papasavas PK, Birdas T, Robke J, Raftopoulos Y, Gagné DJ, Caushaj PF, Landreneau RJ, Keenan RJ. Laparoscopic repair of large paraesophageal hernia is associated with a low incidence of recurrence and reoperation. Surg Endosc 2004; 18: Granderath FA, Schweiger UM, Kamolz T, Asche KU, Pointner R. Laparoscopic Nissen fundoplication with prosthetic hiatal closure reduces postoperative intrathoracic wrap herniation: preliminary results of a prospective randomized functional and clinical study. Arch Surg 2005; 140: Trus TL, Bax T, Richardson WS, Branum GD, Mauren SJ, Swanstrom LL, Hunter JG. Complications of laparoscopic paraesophageal hernia repair. J Gastrointest Surg 1997; 1: ; discussion Oelschlager BK, Pellegrini CA, Hunter J, Soper N, Brunt M, Sheppard B, Jobe B, Polissar N, Mitsumori L, Nelson J, Swanstrom L. Biologic prosthesis reduces recurrence after laparoscopic paraesophageal hernia repair: a multicenter, prospective, randomized trial. Ann Surg 2006; 244: Oelschlager BK, Pellegrini CA, Hunter J, Brunt M, Soper N, Sheppard B, Polissar N, Neradilek MB, Mitsumori L, Rohrmann, C, Swanstrom L. Biologic prosthesis to prevent recurrence after laparoscopic paraesophageal hernia repair: long-term follow-up from a multicenter, prospective, randomized trial. Proceedings of 2010 American College of Surgeons 96th Annual Clinical Congress; 2010 Oct 3-7; Washington DC, United States S- Editor Wu X L- Editor A E- Editor Li JY 6770 December 14, 2012 Volume 18 Issue 46

92 Online Submissions: doi: /wjg.v18.i World J Gastroenterol 2012 December 14; 18(46): ISSN (print) ISSN (online) 2012 Baishideng. All rights reserved. GUIDELINES FOR BASIC SCIENCE Hepatic expression and cellular distribution of the glucose transporter family Sumera Karim, David H Adams, Patricia F Lalor Sumera Karim, David H Adams, Patricia F Lalor, Centre for Liver Research and NIHR Biomedical Research Unit, Institute of Biomedical Research, University of Birmingham, Birmingham B15 2TT, United Kingdom Author contributions: Karim S, Adams DH and Lalor PF devised and wrote the manuscript; Karim S and Lalor PF performed the experiments and analyzed data included in the review. Correspondence to: Dr. Patricia F Lalor, PhD, Lecturer, Centre for Liver Research and NIHR Biomedical Research Unit, Institute of Biomedical Research, University of Birmingham, Birmingham B15 2TT, United Kingdom. [email protected] Telephone: Fax: Received: June 20, 2012 Revised: September 10, 2012 Accepted: September 19, 2012 Published online: December 14, 2012 Abstract Glucose and other carbohydrates are transported into cells using members of a family of integral membrane glucose transporter (GLUT) molecules. To date 14 members of this family, also called the solute carrier 2A proteins have been identified which are divided on the basis of transport characteristics and sequence similarities into several families (Classes 1 to 3). The expression of these different receptor subtypes varies between different species, tissues and cellular subtypes and each has differential sensitivities to stimuli such as insulin. The liver is a contributor to metabolic carbohydrate homeostasis and is a major site for synthesis, storage and redistribution of carbohydrates. Situations in which the balance of glucose homeostasis is upset such as diabetes or the metabolic syndrome can lead metabolic disturbances that drive chronic organ damage and failure, confirming the importance of understanding the molecular regulation of hepatic glucose homeostasis. There is a considerable literature describing the expression and function of receptors that regulate glucose uptake and release by hepatocytes, the most import cells in glucose regulation and glycogen storage. However there is less appreciation of the roles of GLUTs expressed by non parenchymal cell types within the liver, all of which require carbohydrate to function. A better understanding of the detailed cellular distribution of GLUTs in human liver tissue may shed light on mechanisms underlying disease pathogenesis. This review summarises the available literature on hepatocellular expression of GLUTs in health and disease and highlights areas where further investigation is required Baishideng. All rights reserved. Key words: Hepatic; Liver; Glucose transporters; Glucose; Transport; Hepatocyte Peer reviewer: Ming Li, Associate Professor, Health Sciences Center, Tulane University, 1430 Tulane Ave Sl-83, New Orleans, LA 70112, United States Karim S, Adams DH, Lalor PF. Hepatic expression and cellular distribution of the glucose transporter family. World J Gastroenterol 2012; 18(46): Available from: URL: DOI: INTRODUCTION Provision of a regular supply of glucose and other carbohydrates for fuel is vital for human survival and these are transported into cells using members of a family of integral membrane glucose transporter (GLUT) molecules [1]. To date 14 members of this family, also called the solute carrier 2A (SLC2A) proteins have been identified which can be divided on the basis of transport characteristics (intrinsic or inducible, specificities) and sequence similarities [2] into several families (Classes 1 to 3) [3,4]. The expression of these different receptor subtypes varies between different species, tissues and cellular 6771 December 14, 2012 Volume 18 Issue 46

93 Karim S et al. Hepatic glucose transporters subtypes and each has differential sensitivities to stimuli such as insulin. The liver is a contributor to metabolic carbohydrate homeostasis and is a major site for synthesis, storage and redistribution of carbohydrates. At its simplest, after a meal hepatocyte GLUTs take up glucose from the portal bloodstream and it is converted to glycogen for storage. In a glucose-depleted state, this glycogen can then be converted back to glucose for fuel with up to 70% of total hepatic glucose production arising via this route [5]. Situations in which the balance of glucose homeostasis is upset such as diabetes or the metabolic syndrome can lead metabolic disturbances that drive chronic organ damage and failure, which confirms the importance of understanding the molecular regulation of glucose homeostasis. The liver is the major store of glycogen, regulates the availability of glucose and acute liver failure is associated with profound hypoglycemia. This has led to a large body of work investigating the expression and function of receptors that regulate glucose uptake and release by hepatocytes, the most import cells in glucose regulation and glycogen storage but there is less appreciation of the roles of GLUTs expressed by other cell types within the liver. Thus to date expression of GLUT-1, GLUT-2 [6,7], GLUT-9 [8] and GLUT-10 [9] has been documented on hepatocytes but little is known about their expression or function on other cell types. However all cells require carbohydrate to function and there is evidence that non-parenchymal cells may contribute to glucose disposal. For example sinusoidal endothelial cells bind insulin with high affinity, and endothelial insulin-responses may be rate-limiting for glucose uptake [10]. Thus a better understanding of the detailed cellular distribution of GLUTs in human liver tissue may shed light on mechanisms underlying disease pathogenesis. We begin by discussing the extrahepatic expression and functions of these proteins. EXTRAHEPATIC EXPRESSION AND FUNCTION OF CLASS Ⅰ GLUCOSE TRANSPORTERS This family contains the proteins GLUTs 1 to 4 and 14 (SLC2A1-4, 14). The gene for GLUT-1 (SLC2A1) the most ubiquitous transporter is located on chromosome 1p35-p31.3 and generates a 54 Kd protein in humans and rodents [11]. It has a high Km for glucose (Km = 1-2 mmol/l) and is mainly responsible for basal glucose and uptake [12], but can also transport other hexose carbohydrates including mannose, galactose, glucosamine, 3-O-methylglucose and 2-deoxy-d-glucose. GLUT-1 is, expressed in most cells [13] at low levels, with highest expression reported on erythrocytes, the blood brain barrier, neuronal membranes, eye, placenta and lactating mammary glands [14-16]. Murine embryonic expression also suggests a developmental role [17,18]. Over expression of GLUT-1 has been documented in a variety of tumours [19] and is associated with increased proliferation rates and increased mortality [20] leading to its use as a diagnostic/ prognostic marker in some cancers [21,22]. The GLUT-2 (SLC2A2) gene located on chromosome 3q26-1-q26.2 encodes a 524 amino acid protein. GLUT-2 can efficiently transport sugars due to its high Vmax and Km for glucose, and is well suited to managing large bi-directional fluxes of glucose in and out of cells [23]. It also transports other dietary sugars such as galactose, mannose and fructose with a high affinity for glucosamine [11,24,25]. GLUT-2 is highly expressed in the liver, pancreatic beta cells, and on the basolateral surface of kidney and small intestine epithelia [26,27] with expression regulated by sugars and hormones [23,28]. Glycogenosis in the rare autosomal recessive disorder Fanconi- Bickel Syndrome has been associated with mutations in GLUT-2 [29], and diabetes mellitus in patients with prolonged hepatitis C virus (HCV) infection has been linked to virally-induced reduction in hepatocyte expression of GLUT-2 [30]. GLUT-3 (SLC2A3) was initially identified from muscle cell cdna [31]. Expression localises to the membrane of slow twitch muscle fibres [32] and it is implicated in muscle regeneration and cell fusion [33]. However its major role is in neurons, supplying the high glucose demands in the brain [11,34] and it is increased in brain tumour cells [35]. The gene for GLUT-3 is located on chromosome 12p13.3 and encodes a 496aa protein [12] which transports glucose with a high affinity (Km = 1.8 mmol/l) and maltose, xylose, dehydroascorbic acid, mannose and galactose [25]. It is also present in fat, kidney, heart, placenta and liver at lower levels [36], and is vital for the supply of substrate to early post-implanted embryos [37]. White blood cells, which need an increased supply of glucose to fuel immune functions, express several GLUTs including GLUT-3 [34] the expression of which is decreased in diabetes [38]. GLUT-4 (SLC2A4) was cloned and sequenced by several groups in 1989 [39-41]. It is a 55kDa protein responsible for more than 50% of all body glucose uptake [42]. In the absence of insulin it is sequestered in intracellular vesicles and rapidly translocated to the plasma membrane in response to insulin. GLUT-4 transports glucose (Km = 5-6 mmol/l), dehydroascorbic acid and glucosamine [11,24]. Highest levels of expression are detected in insulin sensitive tissues such as skeletal and cardiac muscle, brown and white adipose tissue [11] and endothelial cells [43]. Expression has also been documented in monocytes, and like GLUT-3 is reduced in insulinresistant individuals [44]. Mutations in the gene have been associated with diabetes. GLUT-14 (SLC2A14) was identified and cloned by Wu X et al [45] in It is located on chromosome 12p13.3, has a high sequence similarity to GLUT-3 and may have arisen as a result of gene duplication. The protein contains sugar transporter signature motifs predicted to exhibit glucose transport activity [45]. Two splice variants have been identified in the testis [45]. Mutations of GLUT-14 and its drosophila homologue have been associated with Alzheimers disease in genome wide as December 14, 2012 Volume 18 Issue 46

94 Karim S et al. Hepatic glucose transporters sociation studies in patients and insect models [46,47] and may explain the reported brain-specific dyregulation of glucose metabolism seen in this condition. EXTRAHEPATIC EXPRESSION AND FUNCTION OF CLASS Ⅱ TRANSPORTERS This family contains the transporters GLUT-5, GLUT-7, GLUT-9 and GLUT-11. GLUT-5 (SLC2A5) mrna is detected mainly in the small intestine were it is found at both the apical and basolateral membranes and functions to absorb dietary fructose (Km = 6 mmol/l) [11,48,49]. It is also expressed at lower levels in the human kidney, microglial cells, adipocytes, muscle, brain, and testes [49,50], and in common with other transporters, expression is increased in human malignant tumours [51]. The protein exhibits no activity for glucose transport in humans or mouse [11,52] and its localization is not regulated by insulin [50,53]. There is a growing interest in fructose consumption and its link with the metabolic syndrome, type 11 diabetes and obesity [49] since consuming foods and beverages which contain excessive amounts of fructose has been linked to nonalcoholic fatty liver disease (NAFLD) [54]. The thiazolidinedione drug pioglitazone [55], which is used to treat type Ⅱ diabetes, decreases GLUT-5 mrna (52%) and protein (40%) in muscle fibres of type II diabetic subjects. The GLUT-7 (SLC2A7), which was originally cloned from a human intestinal cdna library [56], has considerable sequence similarity to GLUT-5 [57] and is involved in uptake of sugars via facilitative diffusion mechanisms. Like GLUT-5 it has substrate specificity for both glucose and fructose and a key Ile-314 residue confers hexose specificity and is essential for fructose transport [58]. GLUT-7 mrna is detected in the small and large intestines at the brush border membrane of enterocytes [11,49] ; it is also detected in the prostate and testis [56]. Interestingly, disparities between the localisation of expression within the small intestine and glucose and fructose substrate availabilities suggest that alternate ligands may exist [57]. GLUT-9 (SLC2A9) shares sequence homology [59,60] and substrate specificities [58] with GLUT-5, GLUT-7 and GLUT-11 and, together with GLUT-2, is important for glucose-sensing by pancreatic B-cells [61]. Expression is localised to liver, kidneys, leukocytes [62], pancreas [61], placenta, lung [63], testis and adrenal gland. Two alternate isoforms have been identified, termed GLUT-9a and GLUT-9b [64,65], and alternative splicing results in differential subcellular localisation. Both isoforms have also been reported to transport urate with high affinity [66], and polymorphisms in the GLUT-9 gene are linked with an increased predisposition to gout [67]. Some polymorphisms have also been associated with an increased incidence of diabetes in Chinese populations [68]. GLUT- 9a expression increases in pregestational and gestational diabetes, and GLUT-9b is increased by insulin [59]. In mouse, three isoforms of this transporter are reported, and similarly elevated in diabetes [8]. Three distinct isoforms of GLUT-11 (SLC2A11) have been identified in humans, with distinct but overlapping tissue expression patterns. Thus GLUT-11-A is expressed in skeletal muscle, kidney and heart, GLUT-11-B in adipose tissue, kidney and placenta, and GLUT-11-C in pancreas, heart, adipose tissue and skeletal muscle [69-72]. Muscle expression is localised to slow twitch fibres [73] and appears to be involved in myeloma cell viability and proliferation [74]. All three variants of GLUT-11 exhibit transport activity for both glucose and fructose but not galactose when expressed in Xenopus oocytes [58,70]. EXTRAHEPATIC EXPRESSION AND FUNCTION OF CLASS Ⅲ TRANSPORTERS This family constitutes the evenly numbered transporters GLUT-6, GLUT-8, GLUT-10, GLUT-12 and GLUT-13. GLUT-6 (SCL2A6) [62] is widely expressed in normal and malignant tissue. mrna has been detected in peripheral leucocytes, brain [72] and spleen [11] as well as in pancreas, testis, colon [62] and adipose tissue [75]. Subcellular protein expression varies with plasma membrane localisation in renal collecting tubule cells and cytoplasmic localisation in germinal cells of the testis and smooth muscle. GLUT-6 has significant sequence identity with GLUT-3 and may have arisen through insertion of GLUT-3 sequence into another gene on chromosome 5 [76]. GLUT-8 (SLC2A8) is a high capacity intracellular GLUT [77] composed of 447 amino acids containing an N-terminal dileucine motif that permits trafficking via adaptor proteins to different organelles [77,78]. Expression is highest in the testis and [79], following insulin stimulation increases in the mid-piece of mature spermatozoa and translocates to the acrosome where the spermatozoa take up glucose to drive motility and the acrosome reaction. GLUT-8 may compensate for a lack of GLUT-4 in spermatozoa [80,81] and the preimplantation blastocyst, which demonstrates insulin stimulated glucose uptake via GLUT-8 translocation [82]. GLUT-8 is also found in some insulin receptive tissues including adipose tissue, muscle, brain, adrenal glands, spleen, heart and the liver [62,83,84], but not adipocytes [75] or neuronal cells [85]. GLUT-10 (SLC2A10) is a 541aa protein in humans and 513aa in zebrafish [11,86], which transports both glucose and galactose with high affinity [87]. It is expressed in the brain, lungs, adipose tissue [88], heart, placenta, and skeletal muscle with highest expression in the liver and pancreas [9,87]. The GLUT-10 gene, located on chromosome 20q [89] has been linked with type Ⅱ diabetes [9,90]. However other studies do not show any association with a diabetic phenotype [91,92]. Development of the cardiovascular system and TGFb signalling are linked to GLUT-10 function [93] and mutations are associated with altered angiogenesis and arterial tortuosity syndrome [93,94] as a consequence of a loss of regulation of smooth muscle mitochondrial antioxidants production in the absence of functional GLUT-10 [89] December 14, 2012 Volume 18 Issue 46

95 Karim S et al. Hepatic glucose transporters Table 1 Summary of reported hepatic expression of glucose transporters isoforms Class GLUT isoform Hepatic Subcellular expression/localisation expression Protein/ mrna Ref. Class Ⅰ GLUT1 Yes Sinusoidal membrane of hepatocytes, protein restricted to hepatocytes proximal Both [110,113,115,116,120] to the hepatic venule, also expressed on endothelial cells, kupffer cells and cholangiocytes; hepatocyte expression in HCC GLUT2 Yes Hepatocytes Protein [ ] GLUT3 Yes Hepatocytes, bile canalicular membrane more enriched than sinusoidal membrane Protein [115,116,132] GLUT4 Yes Stellate cells mrna [115,116,134] GLUT14 No Class Ⅱ GLUT5 Yes Normal liver tissue hepatocytes (cytoplasmic) Both [51,115] GLUT7 No [140] GLUT9 Yes Majority of expression in hepatocytes of normal liver and HCC with cytoplasmic Protein [51] expression in pericentral areas GLUT11 Yes mrna [115] Class Ⅲ GLUT6 Yes mrna [76] GLUT8 Yes Perivenous hepatocytes Both [115,143] GLUT10 Yes mrna [86,115] GLUT12 Yes mrna [98] GLUT13 No The table summarizes the evidence presented within this review to highlight the reported hepatocellular expression of glucose transporter (GLUT) isoforms at message and protein level. Pertinent references are cited in the extreme right hand column. mrna: Micro RNA; HCC: Hepatocellular carcinoma. GLUT-12 (SLC2A12) was originally identified in the MCF-7 breast cancer epithelial cell line [95]. It is expressed in insulin sensitive tissues in humans and rodents including adipose tissue, skeletal muscle (major expression in type 1 oxidative fibres) and heart [72,88,96-98] as well as human chondrocytes [99]. GLUT-12 is also found in placenta, small intestine, heart and tumours with a high metabolic and capacity glucose utilisation [ ]. In normal human muscle GLUT-12 undergoes PI3 kinase dependent translocation from an intracellular region to the plasma membrane [104]. Its expression in insulin sensitive tissues, and evidence that overexpression of GLUT-12 in mice improves glucose clearance rate and whole body insulin sensitivity [105] confirm that this transporter is insulinsensitive. The GLUT-13 (SLC2A13) gene encodes a 629 amino acid protein, located on chromosome 12q12. It is a H + /myo-inositol co-transporter [11,106,107] also known as HMIT [108] in neuronal cells [106,108]. Although there are no known reports of glucose activity for GLUT-13 [11], the rat gene contains motifs which are important for glucose transport activity ( LIVER SPECIFIC EXPRESSION OF GLUCOSE TRANSPORTER MOLECULES The data reviewed above reveal the widespread distribution and diverse function of extrahepatic transporter proteins but much less is known about their expression and function it the liver. Surprisingly, there are few studies documenting changes in expression and function in disease. Defining local expression of GLUTs in tissue will shed light on disease pathogenesis. For example, diabetes is associated with altered expression of GLUT-1, GLUT-2, GLUT-3 and GLUT-8 and GLUT-9 (reviewed in [8] ) and abnormal GLUT-1 expression on tumour endothelium in HCC has prognostic and diagnostic significance [ ]. A good example is the finding that diabetes in HCV is a consequence of virally induced downregulation of GLUT-1 and GLUT-2 on hepatocytes [30]. Similarly, transport of key substrates such as fructose has been linked to NAFLD [54]. Dysregulated glucose homeostasis and insulin resistance in NAFLD is associated with chronic organ damage affecting multiple hepatic cell types. The expression levels of transporters is not only regulated by insulin and glucose levels but also by cytokines including interleukin-6, which is increased in obesity and diabetes and can amplify insulin resistance via effects on GLUT-4 [112]. The hexose transporters also play important roles in the function of cholangiocytes [113], endothelial cells and stellate cells [114]. Thus we summarise the current state of knowledge regarding hepatocellular expression of the GLUT family of proteins, in order to highlight their potential role in tissue homeostasis and disease (Table 1). HEPATIC EXPRESSION OF CLASS Ⅰ TRANSPORTERS The widespread expression of GLUT-1 includes the liver although the precise cellular distribution remains controversial. Because hepatocytes are capable of gluconeogenesis their need for glucose uptake is modest. GLUT-1 is expressed on the sinusoidal membrane of rat and porcine [115,116] hepatocytes, and may be expressed to a greater extent than GLUT-2 during early post-natal development [117]. Expression of both GLUT-1 and GLUT-2 by foetal hepatocytes allows for efficient glycogenesis at low plasma glucose concentrations [118]. In adult animals, expression is strongest in the central acinar zones [119]. Transcription and microsomal expression of GLUT-1 is detected in periportal and perivenular hepatocytes but membrane localisation is restricted to hepatocytes 6774 December 14, 2012 Volume 18 Issue 46

96 Karim S et al. Hepatic glucose transporters Relative expression b GLUT1 GLUT2 GLUT3 GLUT4 GLUT5 GLUT6 GLUT7 GLUT8 GLUT9 GLUT10 GLUT11 GLUT12 GLUT13 Figure 1 Expression of glucose transporter glucose transporters 1-13 micro RNA in normal human livers. This figure contains unpublished data generated by the authors. Livers were collected from patients at the Liver Unit, Queen Elizabeth Hospital, Birmingham, United Kingdom with appropriate informed written consent and local ethics committee approval. Micro RNA was extracted using standard protocols and integrity was confirmed using an Agilent 2100 Bioanalyser. Transcriptome analysis was carried out for all samples using Agilent Human Whole Genome Oligo Arrays (G4112F) in accordance with Agilent one colour microarray gene expression analysis protocol. Results are expressed as means of five normal livers ± SE, and were run on triplicate plates. Data were normalized to pooled endogenous controls and differential expression is represented as power 2 - CT. b P < 0.01 vs glucose transporters (GLUT) 2. proximal to the hepatic venule [120] under basal conditions. Our own microarray analysis of human normal livers confirms expression of GLUT-1 and GLUT-2 in total liver mrna (Figure 1). In hepatocellular carcinoma, variable cytoplasmic GLUT-1 is detected and is has been used to distinguish between cholangiocarcinomas and hepatocellular carcinomas (HCC) [109] and has even been proposed as a therapeutic target for HCC [110]. Exposure of rodents to alcohol and high fat feeding results in increased GLUT-1 and decreased GLUT-2 expression in hepatocytes which presumably reflects changes in energy metabolism in response to the dietary changes [121]. Non-parenchymal cells, which cannot carry out gluconeogenesis, rely on glucose uptake rather than endogenous generation. GLUT-1 is the dominant receptor on both endothelial cells and Kupffer cells and levels increase in response to even brief exposure to LPS [122]. Interestingly, acute liver failure has been associated with increased GLUT-1 expression on cerebral vasculature in response to elevated circulating ammonia levels [123]. Cholangiocytes demonstrate basolateral expression of GLUT-1 [113] which facilitates absorption of glucose from bile. GLUT-2 fulfils the major glucose transport role in hepatocytes [36,124] (Figure 1). The protein localises to the sinusoidal plasma membrane of normal [23,125] and malignant hepatocytes. Historical reports suggest a Km for glucose transport of up to 66 mmol/l in intact rat hepatocytes [126] although contribution from other transporters likely contributes in this study since others report lower values between 10 mmol/l and 20 mmol/l [23]. GLUT-2 promotes rapid glucose efflux following gluconeogenesis. In the fasting state the liver produces glucose via glycogenesis or glycogenolysis with the conversion of glucose 6 phosphate into glucose preceding release via GLUT-2 [23]. However in the fed state glucose and insulin levels rise and inhibit endogenous glucose production through effects on enzymes involved in gluconeogenesis. This is associated with removal of membrane GLUT-2 and a subsequent fall in GLUT-2 mediated release [23]. Excess glucose is stored as glycogen or converted to lipids and hepatocyte GLUT-2 and the insulin receptor are internalised together into endosomes in response to insulin [23,127,128]. In mice lacking GLUT-2 the rate of hepatic glucose production is not impaired indicating the presence of a facilitated diffusion-independent mechanism for glucose release [129]. Thus the major role of hepatocyte GLUT-2 is to regulate efflux rather than uptake of glucose. However, in obesity insulin resistance drives an increase in GLUT-2 levels that may further exacerbate metabolic dysfunction in NAFLD [130]. Much less is known about the hepatic expression and function of GLUT-3. GLUT-3 is expressed in porcine livers [115] and localised to the plasma membrane of rat hepatocytes. Expression is focussed on the bile canalicular membrane rather than the sinusoidal membrane [116]. Mice with GLUT-3 haploinsufficiency develop obesity and insulin resistance associated with hepatic steatosis, possibly as a consequence of foetal glucose insufficiency [131]. GLUT-3 expression is increased on both primary and metastatic hepatic tumours, which might reflect an increased need for glucose uptake in cancer [132]. Low levels of GLUT-3 have been reported in the human liver [36] and are supported by our microarray analysis but detailed human studies are lacking and little is known about changes in disease. Whilst the liver is generally considered to lack significant expression of GLUT-4 [133], a recent study reports expression of GLUT-4 mrna in porcine liver [115]. Al December 14, 2012 Volume 18 Issue 46

97 Karim S et al. Hepatic glucose transporters though there is little evidence for expression in hepatocytes, GLUT-4 has been detected in sinusoidal endothelial cells and stellate cells where it can mediate glucose uptake by semicarbazide sensitive amine oxidase mediated effects on insulin receptor signalling [134] which explains our findings of expression at mrna level in humans (Figure 1). Expression on stellate cells is enhanced by leptin signalling [114] leading to HSC activation that may contribute to fibrogenesis in NAFLD. In contrast, in murine models of diet-induced obesity GLUT-4 mrna is decreased in the liver [135] and cirrhosis is associated with decreased extrahepatic GLUT-4 mrna [42]. Deletion of skeletal muscle GLUT-4 results in redirection of excess circulating glucose to the liver where is becomes fuel for conversion to lipid storage [ ]. Thus glucose homeostasis is maintained by a complex relationship between intra- and extra hepatic levels of GLUT-4 regulated by metabolic activity and dietary intake. To date there are no published reports concerning expression of GLUT-14 in the liver. HEPATIC EXPRESSION OF CLASS Ⅱ TRANSPORTERS GLUT-5 protein has been detected in human hepatocytes [51] although low to undetectable RNA levels in pigs [115] imply species-specific differences in GLUT-5 expression. Hepatic metastases from lung and breast cancer are GLUT-5 positive [132] but under normal conditions liver expression is minimal (Figure 1). A mechanistic link between elevations in GLUT-5 expression in small intestine and alterations in hepatic metabolism [139] has been suggested. GLUT-7 was initially reported as a hepatic microsomal GLUT found in the endoplasmic reticulum, which facilitated the release of glucose formed in the process of gluconeogenesis and glycogenolysis for export into the blood [36,140]. However this has recently been challenged by studies showing that neither human nor rat livers contain GLUT-7 mrna [141] and our data in Figure 1, suggesting that the previous findings were due to a cloning artefact. Definitive studies need to be performed to clarify the situation. GLUT-9 has been detected in the cytoplasm of pericentral hepatocytes in normal human liver and in HCC [51]. The receptor appears to be functional for glucose transport because plasma membrane expression of GLUT-9 correlates with glucose influx in HepG2 cells [142] and GLUT-9 inactivation in mouse hepatocytes leads to hyperuricosuria [143]. Although GLUT-11 mrna has been detected in porcine liver [115], there are no studies documenting expression of GLUT-11 in the human liver. HEPATIC EXPRESSION OF CLASS Ⅲ TRANSPORTERS Little is known about the hepatic expression of the recently identified Class Ⅲ transporters although our microarray data (Figure 1) is indicative of some degree of expression. Presence of mrna for GLUT-6 has been described in hepatoma cell lines but has not been detected in normal human liver [76]. GLUT-8 mrna has been detected in perivenous hepatocytes in pig [115] and mouse [144] livers where it may regulate glycolytic flux. Mice with type Ⅰ diabetes show decreased expression whereas expression increases in insulin resistance and type Ⅱ diabetes suggesting that expression is regulated by insulin [144]. Hepatic expression of GLUT-10 has been reported in pigs [115] and zebrafish [86] but we are unaware of any data in humans. GLUT-12 mrna has been documented in all bovine tissues including the liver where levels are low compared to spleen and skeletal muscle [98], but again detailed cellular expression data is currently lacking. There are no known reports of GLUT-13 expression in the liver. CONCLUSION Systemic carbohydrate homeostasis is maintained by a complex relationship between organs such as the pancreas, intestine, muscle and liver. Intra- and extra hepatic levels of GLUT molecules are regulated in part by metabolic activity, dietary intake, and disease state. For example, diabetes is associated with altered expression of GLUT-1, GLUT-2, GLUT-3 and GLUT-8 and GLUT-9 [8] and abnormal GLUT-1 expression on tumour endothelium in HCC [ ] permits efficient glucose uptake by tumour cells even at low blood glucose concentrations. Chronic fructose intake drives glucose and glycogen storage, lipogenesis and production of lipogenic intermediates as well as promoting production of very-low-density lipoproteins [145]. This suggests that the reported hyperlipidaemic and hyperuricaemic effects of fructose, coupled with macrovesicular steatosis and lobular inflammation patterns [146] characteristic of human NAFLD seen in rodents fed high fructose diets, may be enhanced in the context of altered expression of fructose transporters within the hepatic parenchyma and especially so for individuals with high fructose intake or pre-existing hyperlipidaemia or metabolic syndrome. New data is increasingly suggesting the merits of targeting members of the GLUT family therapeutically. Thus overexpression of GLUT-1 in tumours, particularly those with poor prognosis has been suggested as a possible means to selectively inhibit tumour cell metabolism [147], although expression of GLUT-1 red blood cells will likely preclude use therapeutically. Similarly targeting of GLUT-3, which is involved in neovascularisation in glioblastoma has been suggested to prevent resistance to conventional therapy [148], and GLUT-4 is of particular interest in the context of diabetes and insulin resistance, with efforts underway to design therapeutics to enable appropriate glucose uptake independently of insulin stimulation [149]. Alterations in hepatic expression of GLUT transporters have been described in response to 6776 December 14, 2012 Volume 18 Issue 46

98 Karim S et al. Hepatic glucose transporters insulin resistance and hyperlipidaemia, alcohol consumption, viral infection and carcinogenesis, with diverse functions including biliary transport, fibrogenesis, urate transport and angiogenesis executed by family members in extraparenchymal cells. Combined with the central role of the liver in regulation of circulating carbohydrate therefore, future definition of the spatial, temporal and disease-specific expression of GLUTs within the liver microenvironment is key to understanding disease pathogenesis and potential hepatic complications of systemic inhibition. REFERENCES 1 Augustin R. The protein family of glucose transport facilitators: It s not only about glucose after all. IUBMB Life 2010; 62: Bell GI, Kayano T, Buse JB, Burant CF, Takeda J, Lin D, Fukumoto H, Seino S. Molecular biology of mammalian glucose transporters. Diabetes Care 1990; 13: Olson AL, Pessin JE. 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102 Karim S et al. Hepatic glucose transporters 134 Weston CJ, Adams DH. Hepatic consequences of vascular adhesion protein-1 expression. J Neural Transm 2011; 118: Hoffler U, Hobbie K, Wilson R, Bai R, Rahman A, Malarkey D, Travlos G, Ghanayem BI. Diet-induced obesity is associated with hyperleptinemia, hyperinsulinemia, hepatic steatosis, and glomerulopathy in C57Bl/6J mice. Endocrine 2009; 36: Ciaraldi TP, Abrams L, Nikoulina S, Mudaliar S, Henry RR. Glucose transport in cultured human skeletal muscle cells. Regulation by insulin and glucose in nondiabetic and noninsulin-dependent diabetes mellitus subjects. J Clin Invest 1995; 96: Garvey WT, Maianu L, Zhu JH, Brechtel-Hook G, Wallace P, Baron AD. Evidence for defects in the trafficking and translocation of GLUT4 glucose transporters in skeletal muscle as a cause of human insulin resistance. J Clin Invest 1998; 101: Petersen KF, Dufour S, Savage DB, Bilz S, Solomon G, Yonemitsu S, Cline GW, Befroy D, Zemany L, Kahn BB, Papademetris X, Rothman DL, Shulman GI. The role of skeletal muscle insulin resistance in the pathogenesis of the metabolic syndrome. Proc Natl Acad Sci USA 2007; 104: Sakar Y, Nazaret C, Lettéron P, Ait Omar A, Avenati M, Viollet B, Ducroc R, Bado A. Positive regulatory control loop between gut leptin and intestinal GLUT2/GLUT5 transporters links to hepatic metabolic functions in rodents. PLoS One 2009; 4: e Waddell ID, Zomerschoe AG, Voice MW, Burchell A. Cloning and expression of a hepatic microsomal glucose transport protein. Comparison with liver plasma-membrane glucose-transport protein GLUT 2. Biochem J 1992; 286 ( Pt 1): Burchell A. A re-evaluation of GLUT 7. Biochem J 1998; 331 (Pt 3): Takanaga H, Chaudhuri B, Frommer WB. GLUT1 and GLUT9 as major contributors to glucose influx in HepG2 cells identified by a high sensitivity intramolecular FRET glucose sensor. Biochim Biophys Acta 2008; 1778: Preitner F, Bonny O, Laverrière A, Rotman S, Firsov D, Da Costa A, Metref S, Thorens B. Glut9 is a major regulator of urate homeostasis and its genetic inactivation induces hyperuricosuria and urate nephropathy. Proc Natl Acad Sci USA 2009; 106: Gorovits N, Cui L, Busik JV, Ranalletta M, Hauguel de- Mouzon S, Charron MJ. Regulation of hepatic GLUT8 expression in normal and diabetic models. Endocrinology 2003; 144: Mayes PA. Intermediary metabolism of fructose. Am J Clin Nutr 1993; 58: 754S-765S 146 Kawasaki T, Igarashi K, Koeda T, Sugimoto K, Nakagawa K, Hayashi S, Yamaji R, Inui H, Fukusato T, Yamanouchi T. Rats fed fructose-enriched diets have characteristics of nonalcoholic hepatic steatosis. J Nutr 2009; 139: Evans A, Bates V, Troy H, Hewitt S, Holbeck S, Chung YL, Phillips R, Stubbs M, Griffiths J, Airley R. Glut-1 as a therapeutic target: increased chemoresistance and HIF- 1-independent link with cell turnover is revealed through COMPARE analysis and metabolomic studies. Cancer Chemother Pharmacol 2008; 61: Le Calvé B, Rynkowski M, Le Mercier M, Bruyère C, Lonez C, Gras T, Haibe-Kains B, Bontempi G, Decaestecker C, Ruysschaert JM, Kiss R, Lefranc F. Long-term in vitro treatment of human glioblastoma cells with temozolomide increases resistance in vivo through up-regulation of GLUT transporter and aldo-keto reductase enzyme AKR1C expression. Neoplasia 2010; 12: Morgan BJ, Chai SY, Albiston AL. GLUT4 associated proteins as therapeutic targets for diabetes. Recent Pat Endocr Metab Immune Drug Discov 2011; 5: S- Editor Gou SX L- Editor A E- Editor Xiong L 6781 December 14, 2012 Volume 18 Issue 46

103 Online Submissions: doi: /wjg.v18.i World J Gastroenterol 2012 December 14; 18(46): ISSN (print) ISSN (online) 2012 Baishideng. All rights reserved. GUIDELINES FOR CLINICAL PRACTICE Utility of faecal calprotectin analysis in adult inflammatory bowel disease Lyn A Smith, Daniel R Gaya Lyn A Smith, Daniel R Gaya, Inflammatory Bowel Diseases Service, Gastroenterology Unit, Glasgow Royal Infirmary, Glasgow G4 0SF, United Kingdom Author contributions: Smith LA performed the literature review and wrote the article; Gaya DR contributed to the writing of the article and had final approval of the version to be published. Correspondence to: Dr. Daniel R Gaya, Consultant Gastroenterologist, Inflammatory Bowel Diseases Service, Gastroenterology Unit, Glasgow Royal Infirmary, Glasgow G4 0SF, United Kingdom. [email protected] Telephone: Fax: Received: June 19, 2012 Revised: August 23, 2012 Accepted: September 19, 2012 Published online: December 14, 2012 Abstract The inflammatory bowel diseases (IBD), Crohn s disease and ulcerative colitis, are chronic relapsing, remitting disorders. Diagnosis, along with assessment of disease activity and prognosis present challenges to managing clinicians. Faecal biomarkers, such as faecal calprotectin, are a non-invasive method which can be used to aid these decisions. Calprotectin is a calcium and zinc binding protein found in the cytosol of human neutrophils and macrophages. It is released extracellularly in times of cell stress or damage and can be detected within faeces and thus can be used as a sensitive marker of intestinal inflammation. Faecal calprotectin has been shown to be useful in the diagnosis of IBD, correlates with mucosal disease activity and can help to predict response to treatment or relapse. With growing evidence supporting its use, over the last decade this faecal biomarker has significantly changed the way IBD is managed Baishideng. All rights reserved. Key words: Faecal calprotectin; Inflammatory bowel disease; Crohn s disease; Ulcerative colitis; Faecal biomarker Peer reviewers: Xiaofa Qin, MD, PhD, Department of Surgery, University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Newark, NJ 07103, United States; Adrian G Cummins, Associate Professor, Department of Gastroenterology and Hepatology, The Queen Elizabeth Hospital, 28 Woodville Road, Woodville South, SA 5011, Australia Smith LA, Gaya DR. Utility of faecal calprotectin analysis in adult inflammatory bowel disease. World J Gastroenterol 2012; 18(46): Available from: URL: com/ /full/v18/i46/6782.htm DOI: org/ /wjg.v18.i INTRODUCTION It is desirable to have simple diagnostic tests and disease markers for use in the assessment and follow up of chronic diseases such as inflammatory bowel disease (IBD). These markers should be easy to perform, acceptable to both patients and health workers, economical, ideally non-invasive and have a high sensitivity and specificity. Calprotectin, first described in 1980 [1], is a protein found in the cytosol of neutrophils and macrophages composed of two subunits S100A8 and S100A9. It can be detected in plasma, urine, cerebrospinal fluid, faeces, saliva, synovial fluid and colonic biopsies [2]. It is stable in faeces for up to seven days at room temperature and has a homogenous distribution in faeces [3], properties which lend it to testing spot faecal samples. There has been recent emphasis of the involvement of the innate immune system in the pathogenesis of IBD [4]. Calprotectin is classed as a damage associated molecular pattern protein (DAMP) having antimicrobial protective properties. DAMPs are released by the innate immune system from damaged or activated cells, initiating and perpetuating the immune response. The extracellular release of calprotectin during times of cell stress/damage makes it an accurate marker of intestinal inflammation. As early as 1992 it was shown that faecal calprotectin 6782 December 14, 2012 Volume 18 Issue 46

104 Smith LA et al. Faecal calprotectin in adult IBD is elevated in patients with both ulcerative colitis (UC) and Crohn s disease (CD) [3] and this has been confirmed by subsequent studies [5-9]. Calprotectin levels in faeces correlate with faecal excretion of 111-indium labelled leucocytes, deemed to be the gold standard for measuring intestinal inflammation (r = 0.80, P < ) [5]. Disadvantages of this gold standard test compared with calprotectin are the exposure to radiation, cost and the three day collection of faeces. Faecal calprotectin levels have also been shown to correlate well with radiolabelled white cell scanning, another method of assessing intestinal inflammation, in adults with CD [10]. Spot faecal samples of < 5 g have been shown to be as reliable as 24 h collection samples for measuring calprotectin levels [3] indicating that calprotectin is evenly distributed throughout the faeces. An elevated faecal calprotectin is not specific for IBD. Any inflammatory process within the gastrointestinal tract will result in the activation of the innate immune response and release of calprotectin. Faecal calprotectin concentration has been shown in studies to be elevated in many conditions including infection, colorectal cancer, untreated coeliac disease, microscopic colitis and diverticulitis [11-13]. Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to cause significant increases in faecal calprotectin levels within seven days due to NSAIDs induced intestinal inflammation with endoscopic correlation [14,15]. Proton pump inhibitors (PPIs) have been associated with significantly elevated faecal calprotectin levels, regardless of reason for PPI [16]. Initially faecal calprotectin concentration was reported in mg/l, but more recent assays (post 2000) usually report faecal calprotectin concentration as μg/g. To compare these results, faecal calprotectin concentrations obtained using assays pre-2000 need to be multiplied by a factor of five. USE OF FAECAL CALPROTECTIN IN DIAGNOSIS OF IBD Diagnosis of IBD has historically been based on a combination of clinical history and examination, blood parameters, radiology and endoscopy. The addition of a faecal biomarker able to reduce the need for invasive endoscopic procedures or exposure to radiation is advantageous. Limburg et al [12], in 2000, published a study of 110 patients attending for colonoscopy for the investigation of chronic diarrhoea showing that increased faecal calprotectin levels were significantly (P = ) associated with the presence of colorectal inflammation (CD, UC, microscopic colitis or diverticulitis). Within the colonic inflammation subgroup, calprotectin concentrations were highest amongst subjects with IBD. The negative predictive value of faecal calprotectin in this dataset was 93%. IBD and irritable bowel syndromes (IBS) can present in a similar clinical fashion with symptoms such as diarrhoea and abdominal pain. Routine colonoscopy in these patients is costly, invasive and has associated morbidity and mortality. Serum markers of inflammation such as C reactive protein (CRP) and erythrocyte sedimentation rate (ESR) in isolation are not sufficiently sensitive or specific for the diagnosis of IBD [7]. The use of faecal calprotectin to distinguish between IBD and IBS has been analysed in several studies. In 2000 Tibble et al [7] presented results of a prospective study of 220 consecutive patients in whom the principal differential diagnosis was that of either IBS or CD. They excluded patients with UC on sigmoidoscopy and biopsy. A diagnosis of CD was made from a combination of radiological, endoscopic and histological investigations. A diagnosis of IBS was made on basis of normal investigations and a compatible history fulfilling the Rome criteria. All patients subsequently diagnosed with CD had significantly higher faecal calprotectin concentrations than those with IBS. The investigators found that using a cut-off point of 30 mg/l faecal calprotectin had a 100% sensitivity and 97% specificity in discriminating between active CD and IBS. Schoepfer et al [17] looked at the accuracy of faecal biomarkers alone and in combination with the IBD antibodies, antineutrophil cytoplasmic antibody (ANCA) and anti-saccharomyces Cerevisiae manna antibody (ASCA), in discriminating IBD from IBS. They found that the overall accuracy of faecal calprotectin for discriminating between IBD and IBS was 89% (sensitivity 83%, specificity 100%). There was only a marginal increase in overall accuracy when faecal calprotectin was combined with IBD antibodies to 91%. Faecal calprotectin has been studied as a tool to predict abnormal small-bowel radiology [18]. The study looked at 73 consecutive patients attending for small bowel follow through whose presenting symptoms were consistent with a possible diagnosis of IBD. The control group consisted of 25 patients with IBS, 25 normal volunteers and 25 patients with active CD. A faecal calprotectin level above 60 μg/g predicted all abnormal barium follow through results. The negative predictive value of a single calprotectin result below 60 μg/g of stool was 100% compared with 91% each for erythrocyte sedimentation rate cut off of 10 mm and C-reactive protein of 6 mg/l. Somewhat in contrast to this Sipponen et al [19] found that faecal calprotectin had a low utility for predicting the presence of small bowel CD on wireless capsule endoscopy, sensitivity was low at 59% with a moderate specificity of 71% using a cut-off of 50 μg/g. A recent meta-analysis analysed 30 prospective studies comparing the diagnostic precision of faecal calprotectin against a histological diagnosis [20]. Summary receiver operating characteristic curve analysis showed a sensitivity of 0.95 (95% CI: ), specificity of 0.91 (95% CI: ), and an area under the curve (AUC) of 0.95 for the diagnosis of IBD. The diagnostic precision of faecal calprotectin for IBD was higher in children than adults with better accuracy at a cut-off level of 100 μg/g vs 50 μg/g. This meta-analysis also showed that faecal calprotectin was superior to CRP, ESR, ASCA, perinuclear anti-neutrophil cytoplasmic an December 14, 2012 Volume 18 Issue 46

105 Smith LA et al. Faecal calprotectin in adult IBD tibodies and anti-escherichia coli outer membrane porin C antibody in diagnosis of IBD. Thus it can be stated that a normal faecal calprotectin result, in the absence of red flag symptoms and in the context of positive Rome criteria, is associated with a high likelihood of subsequent non-organic diagnosis and further endoscopic or radiological evaluation may be avoided in such patients. A meta-analysis published in 2010 to assess whether the use of faecal calprotectin reduces the number of unnecessary endoscopic procedures in the investigation of suspected IBD showed that screening with faecal calprotectin would result in a 67% reduction in the number of adults requiring endoscopy. The downside of this screening strategy is delayed diagnosis in 6% of adults because of a false negative test result [13]. Faecal calprotectin appears to better reflect disease activity in UC rather than CD [21] but faecal calprotectin has not been found to be useful in distinguishing UC from CD. Quail et al [22] looked at faecal calprotectin concentrations in Scottish children with a diagnosis of IBD; there was no statistical difference in calprotectin concentrations between CD and non-crohn s patients (UC or IBD type unspecified). FAECAL CALPROTECTIN IN DISEASE ACTIVITY AND RESPONSE TO TREATMENT In IBD, the presence of active gut inflammation is associated with migration of leucocytes, including neutrophils, to the gut mucosa [23]. As a result the faecal stream contains increased levels of these inflammatory proteins including calprotectin. Faecal calprotectin has been shown to differentiate quiescent from active disease in both patients with CD and UC [10,24-26]. Correlation of faecal calprotectin tends to be higher with endoscopic activity than clinical activity indices [24,27] and indeed some studies have demonstrated no significant correlation between faecal calprotectin and clinical indices [10,28]. In general faecal calprotectin correlates better with colonic CD rather than ileal disease [27-31] and an inflammatory rather than a structuring/penetrating phenotype [27,31]. Sipponen et al [24] showed that in active disease (CD endoscopic index of severity, CDEIS 3), faecal calprotectin concentrations were significantly higher in colonic than in ileal CD. Also, in limited ileal disease faecal calprotectin failed to correlate with endoscopic activity. In UC Ricanek et al [32] showed that the median faecal calprotectin concentration was higher in patients with extensive and left sided disease distribution compared with proctitis (740 μg/g, 2106 μg/g, 86 μg/g respectively; P = and P = 0.009). There was no significant difference in faecal calprotectin concentration between extensive and left sided disease distribution. There have been several studies looking at the use of faecal calprotectin to predict or monitor response to treatment. In a study looking at 11 patients with relapsing IBD [33] (11 CD and 27 UC) faecal calprotectin was analysed at inclusion and after 8 wk of treatment (end of study). Treatment was individualised medical therapy. A normalised faecal calprotectin concentration at 8 weeks predicted a complete response in 100% patients. There was a significant decline in faecal calprotectin levels (P < 0.001) in patients with UC responding to treatment defined as normalisation of clinical and endoscopic scores. Within the small subgroup of patients with CD although 81% of patients achieved a complete clinical response defined clinically as a Harvey Bradshaw Index [34] (HBI) 5 there was no significant decline in calprotectin levels. This study was limited by small numbers and also the lack of endoscopic evidence of disease activity or remission in CD patients, it is possible that these patients had ongoing subclinical inflammation. In fact it has been shown that in patients with steroid induced clinical remission faecal calprotectin levels can remain elevated [35,36]. This finding is in keeping with earlier studies showing incomplete mucosal healing in patients treated with corticosteroids [37]. Sipponen et al [35] were able to show a significant decrease in faecal calprotectin (P = 0.005) in patients with CD who responded both clinically and endoscopically (using CDEIS) to an individualised escalation of treatment. There was no significant change in faecal calprotectin concentration in patients without endoscopic response. Faecal calprotectin may be able to predict colectomy in patients with acute severe UC. Ho et al [38] showed that in patients with acute severe UC requiring inpatient treatment with intravenous corticosteroids faecal calprotectin was significantly higher in patients who failed to respond to medical therapy and required colectomy than those who did not (P = 0.04). The AUC was 0.65 (P = 0.04) for faecal calprotectin to predict colectomy with a maximum likelihood ratio of 9.23 at a cut-off of μg/g (specificity of 97.4%). Overall in the study faecal calprotectin concentrations were high with 86% of patients having levels of > 500 μg/g (median 1020 μg/g). Faecal calprotectin can be used to monitor response to biological therapy. Palmon et al [39] showed that faecal calprotectin concentration decreases significantly at week 2 after an infliximab (IFX) infusion in 17 patients with CD on maintenance IFX therapy. Calprotectin levels were noted to rise back to baseline values by week 4 again despite a low median HBI. There was no endoscopic assessment of disease activity in this study. The rise in faecal calprotectin at week 4 may once again indicate a subclinical recurrence of mucosal inflammation. Sipponen et al [40] assessed the role of faecal calprotectin in monitoring clinical, using the CD activity index (CDAI) and CDEIS response to anti-tumor necrosis factor-α (TNF-α) therapy (IFX or adalumimab) in 15 patients with CD. Following 12 wk of treatment faecal calprotectin levels declined significantly from baseline level (P = 0.001) and changes in faecal calprotectin correlated to endoscopic appearances as scored using CDEIS (Spear December 14, 2012 Volume 18 Issue 46

106 Smith LA et al. Faecal calprotectin in adult IBD Table 1 Correlation of faecal calprotectin with endoscopic findings, histology and clinical indices, when available Ref. Patient population Endoscopic assessment score Correlation coefficient with endoscopy (r ) Correlation coefficient with histology Correlation with clinical index (r ) Røseth et al [6] UC MAYO 0.57 NA NA D'Incà et al [8] CD SES-CD 0.48 r = 0.12 NA D'Incà et al [8] UC MAYO 0.51 r = 0.32 NA Sipponen et al [24] CD CDEIS 0.73 NA 0.397(CDAI) Schoepfer et al [27] CD SES-CD 0.75 NA NA Jones et al [28] CD SES-CD 0.45 NA 0.23 (CDAI) Sipponen et al [31] CD SES-CD (exchange ileal only disease) 0.32 (CDAI) Sipponen et al [40] CD CDEIS (exchange ileal only disease) NA Langhorst et al [48] UC MAYO 0.49 NA NA Langhorst et al [48] CD SES-CD 0.35 NA NA Hanai et al [63] UC Matts 0.81 NA 0.68 (CDAI) Schoepfer et al [64] UC Rachmilewitz 0.83 NA NA NA: Not available; MAYO: Mayo score [65] ; SES-CD: Simplified endoscopic activity score for Crohn s disease (CD) [66] ; CDEIS: Crohn s disease endoscopic index of severity [67] ; CDAI: Crohn s disease activity index [68] ; Matts: Matts grading [69] ; UC: Ulcerative colitis. man s rank correlation r = 0.561, P = 0.03) suggesting that faecal calprotectin is a useful non-invasive marker of mucosal response to anti-tnf-α treatment. PREDICTING MUCOSAL HEALING Historically clinical practice has considered interpretation of symptoms and the use of scoring systems such as the CDAI, HBI and the Rachmilewitz UC activity index [41] to determine treatment success in IBD. These indices however tend to reflect patient well-being and quality of life rather than the degree of mucosal inflammation [10,28]. In both CD and UC there is evidence that mucosal healing is associated with sustained remission and reduced need for surgery [42,43] and following ileal resection the endoscopic appearance of the neoterminal ileum mucosa at 1 year post surgery has been shown to predict symptomatic relapse [44]. Thus mucosal healing is evolving into the new goal of IBD treatment. Røseth et al [45] have demonstrated that normalisation of faecal calprotectin concentration corresponds to endoscopic mucosal healing. Seventeen patients with CD and 28 with UC clinically in remission who had faecal calprotectin concentrations of < 50 mg/l underwent endoscopic assessment of their lower GI tract and macroscopic mucosal appearances were assessed. Biopsies were also taken to assess histological inflammation. All but one of these patients with faecal calprotectin < 50 mg/l had inactive mucosal disease on colonoscopy. Several subsequent studies have went on to show that concentration of faecal calprotectin correlates with both histological and endoscopic disease activity in IBD and these are summarised in Table 1. Furthermore, several of the studies included in Table 1 show that correlation of faecal calprotectin with endoscopic appearances is stronger than correlation with clinical indices [28,40]. In contrast to the evidence shown in Table 1, Denis et al [46] failed to find a significant correlation between CDEIS and faecal calprotectin concentration. This was a small study of 28 patients with CD who had CDAI > 150 but a normal serum CRP. This lack of correlation may reflect the population studied as more than half of the patients had isolated ileal disease and overall disease activity was low (median CDEIS 3.4). Interestingly one study in paediatric patients with IBD showed that calprotectin concentration correlated more closely with histological inflammation rather than endoscopic findings suggesting that faecal calprotectin may be more sensitive than macroscopic endoscopic appearances in evaluating disease activity status [47]. USE OF FAECAL CALPROTECTIN TO PREDICT RELAPSE Being able to identify patients at high risk of relapse, and those with sub-clinical intestinal inflammation, may allow adjustment of their treatment strategy thus preventing clinical relapse. A non-invasive method of identifying this would reduce cost and risk of morbidity and mortality to patients. As sensitivity and specificity of serum markers of inflammation correlate poorly with intestinal inflammation their ability to predict disease relapse is poor [48,49]. Several studies have looked at the use of faecal calprotectin to predict relapse in patients with IBD and have demonstrated significant differences in faecal calprotectin concentration in relapsers compared with nonrelapsers. A summary of these studies is shown in Table 2. Interestingly, faecal calprotectin appears less useful for predicting relapse in patients with ileal CD compared with patients with UC or colonic/ileocolonic CD [29,30]. The major outlier in these datasets is the study by Laharie et al [50] which looked at the use of faecal calprotectin to predict relapse specifically in 65 patients with CD treated with IFX induction regimen and then maintained on immunomodulator alone. There was no significant difference in faecal calprotectin concentration between those patients who relapsed by 14 wk post induction and those who did not even when the analysis was restricted to patients with pure colonic disease. No endoscopic 6785 December 14, 2012 Volume 18 Issue 46

107 Smith LA et al. Faecal calprotectin in adult IBD Table 2 The use of faecal calprotectin to predict relapse Ref. Patient population Cut-off calprotectin level Sensitivity for relapse (%) Specificity for relapse (%) Increased risk of relapse Costa et al [21] UC 150 μ/l fold CD fold D'Incà et al [29] UC 130 mg/kg fold CD fold García-Sánchez et al [30] CD + UC 120 μ/g Ileal CD 223 μ/g Tibble et al [49] CD + UC 50 mg/l (250 μ/g) Laharie et al [50] CD post IFX 130 μ/g μ/g Kallel et al [70] Colonic CD 340 μ/g fold Gisbert et al [71] CD + UC 150 μ/g UC: Ulcerative colitis; CD: Crohn s disease; IFX: Infliximab. evaluation was performed after IFX induction so although the study did show a median drop in faecal calprotectin concentration of 340 μg/g following induction there may have been ongoing subclinical inflammation. Another limitation of this study is that disease relapse was defined on clinical grounds alone. Mao et al [51] performed a meta-analysis of the predictive capacity of faecal calprotectin in IBD relapse. Analysing 6 studies they found a pooled sensitivity of 78% and specificity of 73%. Capacity to predict relapse was comparable between CD and UC. Due to the small number of patients the predictive value of faecal calprotectin in ileal only CD patients was not assessed. FAECAL CALPROTECTIN POST-SURGERY More than 80% of CD patients require surgery within 10 years of diagnosis and by 3-5 years after surgery around a third of patients will have had a clinical relapse [44,52]. The role of faecal calprotectin in predicting post-surgical recurrence of CD has been assessed. One study assessed 39 patients with CD undergoing bowel resection [53]. The majority of patients (67%) had ileocolonic disease. Measurements of faecal calprotectin, CDAI and ultrasound examination were performed at 3 mo post surgery. Endoscopy was performed at 1 year regardless of patient symptoms and was considered the gold standard for recurrence of disease. All patients were clinically in remission at 3 mo. Using a cut-off level of 200 mg/l, in predicting endoscopic post-surgical recurrence, faecal calprotectin has a sensitivity of 63% and specificity of 75% leading the authors to suggest that patients with elevated faecal calprotectin levels at 3 mo post surgery should be assessed endoscopically for early recurrence. Lamb et al [54] prospectively followed 13 patients for 12 mo post ileocaecal resection for symptomatic CD. Faecal calprotectin was seen to normalise after uncomplicated surgery at 2 mo and remained within normal limits in 8 patients who remained clinically in remission. In the remaining 5 patients there was an increase in faecal calprotectin concentration after initial normalisation associated with disease recurrence or post operative intra-abdominal collections. The authors conclusion from this small number of patients was that patients with low levels of faecal calprotectin after resection who had symptoms were unlikely to have mucosal inflammation. It should be pointed out thought that there was no scheduled endoscopic evaluation of the gut mucosa during the 12 mo follow up period. Pouchitis is an inflammatory condition with significant associated morbidity common in patients post restorative proctocolectomy [55]. Diagnosis can be difficult histologically due to the patchy distribution of inflammation. Faecal calprotectin has been shown to reliably differentiate between inflamed and non-inflamed pouches and correlates with severity of pouchitis [55-57] thus may reduce the need for endoscopic evaluation of the pouch in some patients. USE OF FAECAL CALPROTECTIN TO AID DECISIONS OF WITHDRAWAL OF TREATMENT There has been focus of late on when and in which patients it is appropriate to withdraw anti TNF-α therapy driven by costs and concerns about long term safety. Faecal calprotectin levels may assist in this decision making. Louis et al [58] published results of a prospective study (STORI) of CD patients who had been in steroid free remission on IFX for at least six months. Relapse after IFX withdrawal was associated with various risk factors including a faecal calprotectin concentration of 300 μg/g. Other risk factors for relapse identified on multivariate analysis included male sex, absence of surgical resection, leucocyte counts > 6 10/L, haemoglobin 145 g/l and hscrp 5.0 mg/l. Patients with no more than 2 of the above risk factors had a reduced risk of relapse within 1 year (15% compared with 43.9% overall). POINT-OF-CARE FAECAL CALPROTECTIN TESTING Faecal calprotectin concentration is most commonly 6786 December 14, 2012 Volume 18 Issue 46

108 Smith LA et al. Faecal calprotectin in adult IBD measured using an enzyme-linked immunosorbent assay (ELISA) technique in clinical laboratories and several ELISA kits are available. Recently point-of-care or bedside faecal calprotectin tests have become available which may be advantageous in clinical circumstances such as primary care or when a more rapid result is required. These tests can be performed in less than 30 min. Correlation between faecal calprotectin concentrations measured using an ELISA technique and a point-of-care test has been shown to be good in recent initial studies [59-62]. CONCLUSION Faecal calprotectin is now playing a major role in the investigation and diagnosis of patients presenting to the physician with lower gastrointestinal symptoms and can obviate the need for costly invasive investigations in selected patients. Perhaps more excitingly faecal calprotectin is dramatically changing the way IBD is managed. There is a growing body of evidence that faecal calprotectin correlates well with mucosal disease activity and this in turn makes it useful in assessing activity, monitoring response to treatment, predicting relapse and aiding in the difficult decision of whether or not to withdraw biological therapy to a degree than clinical scoring indices or other non-invasive serological markers cannot. REFERENCES 1 Fagerhol MK, Dale I, Andersson T. A radioimmunoassay for a granulocyte protein as a marker in studies on the turnover of such cells. Bull Eur Physiopathol Respir 1980; 16 Suppl: Johne B, Fagerhol MK, Lyberg T, Prydz H, Brandtzaeg P, Naess-Andresen CF, Dale I. Functional and clinical aspects of the myelomonocyte protein calprotectin. Mol Pathol 1997; 50: Røseth AG, Fagerhol MK, Aadland E, Schjønsby H. Assessment of the neutrophil dominating protein calprotectin in feces. A methodologic study. Scand J Gastroenterol 1992; 27: Kono H, Rock KL. How dying cells alert the immune system to danger. Nat Rev Immunol 2008; 8: Røseth AG, Schmidt PN, Fagerhol MK. Correlation between faecal excretion of indium-111-labelled granulocytes and calprotectin, a granulocyte marker protein, in patients with inflammatory bowel disease. Scand J Gastroenterol 1999; 34: Røseth AG, Aadland E, Jahnsen J, Raknerud N. Assessment of disease activity in ulcerative colitis by faecal calprotectin, a novel granulocyte marker protein. Digestion 1997; 58: Tibble J, Teahon K, Thjodleifsson B, Roseth A, Sigthorsson G, Bridger S, Foster R, Sherwood R, Fagerhol M, Bjarnason I. A simple method for assessing intestinal inflammation in Crohn s disease. Gut 2000; 47: D Incà R, Dal Pont E, Di Leo V, Ferronato A, Fries W, Vettorato MG, Martines D, Sturniolo GC. Calprotectin and lactoferrin in the assessment of intestinal inflammation and organic disease. Int J Colorectal Dis 2007; 22: Costa F, Mumolo MG, Bellini M, Romano MR, Ceccarelli L, Arpe P, Sterpi C, Marchi S, Maltinti G. Role of faecal calprotectin as non-invasive marker of intestinal inflammation. Dig Liver Dis 2003; 35: Gaya DR, Lyon TDB, Duncan A, Neilly JB, Han S, Howell J, Liddell C, Stanley AJ, Morris AJ, Mackenzie JF. Faecal calprotectin in the assessment of Crohn s disease activity. Q J Med 2005; 98: Summerton CB, Longlands MG, Wiener K, Shreeve DR. Faecal calprotectin: a marker of inflammation throughout the intestinal tract. Eur J Gastroenterol Hepatol 2002; 14: Limburg PJ, Ahlquist DA, Sandborn WJ, Mahoney DW, Devens ME, Harrington JJ, Zinsmeister AR. Fecal calprotectin levels predict colorectal inflammation among patients with chronic diarrhea referred for colonoscopy. Am J Gastroenterol 2000; 95: Van Rheenen PF, Van de Vijver E, Fidler V. Faecal calprotectin for screening of patients with suspected inflammatory bowel disease: diagnostic meta-analysis. BMJ 2010; 342: c Meling TR, Aabakken L, Røseth A, Osnes M. Faecal calprotectin shedding after short-term treatment with non-steroidal anti-inflammatory drugs. Scand J Gastroenterol 1996; 31: Tibble JA, Sigthorsson G, Foster R, Scott D, Fagerhol MK, Roseth A, Bjarnason I. High prevalence of NSAID enteropathy as shown by a simple faecal test. Gut 1999; 45: British Society of Gastroenterology annual meeting March Abstracts. Gut 2002; 50 Suppl 2: A Schoepfer AM, Trummler M, Seeholzer P, Seibold-Schmid B, Seibold F. Discriminating IBD from IBS: comparison of the test performance of fecal markers, blood leukocytes, CRP, and IBD antibodies. Inflamm Bowel Dis 2008; 14: Dolwani S, Metzner M, Wassell JJ, Yong A, Hawthorne AB. Diagnostic accuracy of faecal calprotectin estimation in prediction of abnormal small bowel radiology. Aliment Pharmacol Ther 2004; 20: Sipponen T, Haapamäki J, Savilahti E, Alfthan H, Hämäläinen E, Rautiainen H, Koskenpato J, Nuutinen H, Färkkilä M. Fecal calprotectin and S100A12 have low utility in prediction of small bowel Crohn s disease detected by wireless capsule endoscopy. Scand J Gastroenterol 2012; 47: von Roon AC, Karamountzos L, Purkayastha S, Reese GE, Darzi AW, Teare JP, Paraskeva P, Tekkis PP. Diagnostic precision of fecal calprotectin for inflammatory bowel disease and colorectal malignancy. Am J Gastroenterol 2007; 102: Costa F, Mumolo MG, Ceccarelli L, Bellini M, Romano MR, Sterpi C, Ricchiuti A, Marchi S, Bottai M. Calprotectin is a stronger predictive marker of relapse in ulcerative colitis than in Crohn s disease. Gut 2005; 54: Quail MA, Russell RK, Van Limbergen JE, Rogers P, Drummond HE, Wilson DC, Gillett PM. Fecal calprotectin complements routine laboratory investigations in diagnosing childhood inflammatory bowel disease. Inflamm Bowel Dis 2009; 15: Vermeire S, Van Assche G, Rutgeerts P. Laboratory markers in IBD: useful, magic, or unnecessary toys? Gut 2006; 55: Sipponen T, Savilahti E, Kolho KL, Nuutinen H, Turunen U, Färkkilä M. Crohn s disease activity assessed by fecal calprotectin and lactoferrin: correlation with Crohn s disease activity index and endoscopic findings. Inflamm Bowel Dis 2008; 14: Langhorst J, Elsenbruch S, Mueller T, Rueffer A, Spahn G, Michalsen A, Dobos GJ. Comparison of 4 neutrophilderived proteins in feces as indicators of disease activity in ulcerative colitis. Inflamm Bowel Dis 2005; 11: Xiang JY, Ouyang Q, Li GD, Xiao NP. Clinical value of fecal calprotectin in determining disease activity of ulcerative colitis. World J Gastroenterol 2008; 14: Schoepfer AM, Beglinger C, Straumann A, Trummler M, 6787 December 14, 2012 Volume 18 Issue 46

109 Smith LA et al. Faecal calprotectin in adult IBD Vavricka SR, Bruegger LE, Seibold F. Fecal calprotectin correlates more closely with the Simple Endoscopic Score for Crohn s disease (SES-CD) than CRP, blood leukocytes, and the CDAI. Am J Gastroenterol 2010; 105: Jones J, Loftus EV, Panaccione R, Chen LS, Peterson S, Mc- Connell J, Baudhuin L, Hanson K, Feagan BG, Harmsen SW, Zinsmeister AR, Helou E, Sandborn WJ. Relationships between disease activity and serum and fecal biomarkers in patients with Crohn s disease. Clin Gastroenterol Hepatol 2008; 6: D Incà R, Dal Pont E, Di Leo V, Benazzato L, Martinato M, Lamboglia F, Oliva L, Sturniolo GC. Can calprotectin predict relapse risk in inflammatory bowel disease? Am J Gastroenterol 2008; 103: García-Sánchez V, Iglesias-Flores E, González R, Gisbert JP, Gallardo-Valverde JM, González-Galilea A, Naranjo- Rodríguez A, de Dios-Vega JF, Muntané J, Gómez-Camacho F. Does fecal calprotectin predict relapse in patients with Crohn s disease and ulcerative colitis? J Crohns Colitis 2010; 4: Sipponen T, Kärkkäinen P, Savilahti E, Kolho KL, Nuutinen H, Turunen U, Färkkilä M. Correlation of faecal calprotectin and lactoferrin with an endoscopic score for Crohn s disease and histological findings. Aliment Pharmacol Ther 2008; 28: Ricanek P, Brackmann S, Perminow G, Lyckander LG, Sponheim J, Holme O, Høie O, Rydning A, Vatn MH. Evaluation of disease activity in IBD at the time of diagnosis by the use of clinical, biochemical, and fecal markers. Scand J Gastroenterol 2011; 46: Wagner M, Peterson CG, Ridefelt P, Sangfelt P, Carlson M. Fecal markers of inflammation used as surrogate markers for treatment outcome in relapsing inflammatory bowel disease. World J Gastroenterol 2008; 14: ; discussion Harvey RF, Bradshaw JM. A simple index of Crohn s-disease activity. Lancet 1980; 1: Sipponen T, Björkesten CG, Färkkilä M, Nuutinen H, Savilahti E, Kolho KL. Faecal calprotectin and lactoferrin are reliable surrogate markers of endoscopic response during Crohn s disease treatment. Scand J Gastroenterol 2010; 45: Kolho KL, Raivio T, Lindahl H, Savilahti E. Fecal calprotectin remains high during glucocorticoid therapy in children with inflammatory bowel disease. Scand J Gastroenterol 2006; 41: Modigliani R, Mary JY, Simon JF, Cortot A, Soule JC, Gendre JP, Rene E. Clinical, biological, and endoscopic picture of attacks of Crohn s disease. Evolution on prednisolone. Groupe d Etude Thérapeutique des Affections Inflammatoires Digestives. Gastroenterology 1990; 98: Ho GT, Lee HM, Brydon G, Ting T, Hare N, Drummond H, Shand AG, Bartolo DC, Wilson RG, Dunlop MG, Arnott ID, Satsangi J. Fecal calprotectin predicts the clinical course of acute severe ulcerative colitis. Am J Gastroenterol 2009; 104: Digestive Disease Week and the 107th Annual Meeting of the American Gastroenterological Association Institute, May 20-25, 2006, Los Angeles, California, USA. Abstracts. Gastroenterology 2006; 130: A Sipponen T, Savilahti E, Kärkkäinen P, Kolho KL, Nuutinen H, Turunen U, Färkkilä M. Fecal calprotectin, lactoferrin, and endoscopic disease activity in monitoring anti-tnfalpha therapy for Crohn s disease. Inflamm Bowel Dis 2008; 14: Rachmilewitz D. Coated mesalazine (5-aminosalicylic acid) versus sulphasalazine in the treatment of active ulcerative colitis: a randomised trial. BMJ 1989; 298: Frøslie KF, Jahnsen J, Moum BA, Vatn MH. Mucosal healing in inflammatory bowel disease: results from a Norwegian population-based cohort. Gastroenterology 2007; 133: Baert F, Moortgat L, Van Assche G, Caenepeel P, Vergauwe P, De Vos M, Stokkers P, Hommes D, Rutgeerts P, Vermeire S, D Haens G. Mucosal healing predicts sustained clinical remission in patients with early-stage Crohn s disease. Gastroenterology 2010; 138: ; quiz Rutgeerts P, Geboes K, Vantrappen G, Beyls J, Kerremans R, Hiele M. Predictability of the postoperative course of Crohn s disease. Gastroenterology 1990; 99: Røseth AG, Aadland E, Grzyb K. Normalization of faecal calprotectin: a predictor of mucosal healing in patients with inflammatory bowel disease. Scand J Gastroenterol 2004; 39: Denis MA, Reenaers C, Fontaine F, Belaïche J, Louis E. Assessment of endoscopic activity index and biological inflammatory markers in clinically active Crohn s disease with normal C-reactive protein serum level. Inflamm Bowel Dis 2007; 13: Bunn SK, Bisset WM, Main MJ, Gray ES, Olson S, Golden BE. Fecal calprotectin: validation as a noninvasive measure of bowel inflammation in childhood inflammatory bowel disease. J Pediatr Gastroenterol Nutr 2001; 33: Langhorst J, Elsenbruch S, Koelzer J, Rueffer A, Michalsen A, Dobos GJ. Noninvasive markers in the assessment of intestinal inflammation in inflammatory bowel diseases: performance of fecal lactoferrin, calprotectin, and PMNelastase, CRP, and clinical indices. Am J Gastroenterol 2008; 103: Tibble JA, Sigthorsson G, Bridger S, Fagerhol MK, Bjarnason I. Surrogate markers of intestinal inflammation are predictive of relapse in patients with inflammatory bowel disease. Gastroenterology 2000; 119: Laharie D, Mesli S, El Hajbi F, Chabrun E, Chanteloup E, Capdepont M, Razaire S, de Lédinghen V, Zerbib F. Prediction of Crohn s disease relapse with faecal calprotectin in infliximab responders: a prospective study. Aliment Pharmacol Ther 2011; 34: Mao R, Xiao YL, Gao X, Chen BL, He Y, Yang L, Hu PJ, Chen MH. Fecal calprotectin in predicting relapse of inflammatory bowel diseases: a meta-analysis of prospective studies. Inflamm Bowel Dis 2012; 18: Bernell O, Lapidus A, Hellers G. Risk factors for surgery and recurrence in 907 patients with primary ileocaecal Crohn s disease. Br J Surg 2000; 87: Orlando A, Modesto I, Castiglione F, Scala L, Scimeca D, Rispo A, Teresi S, Mocciaro F, Criscuoli V, Marrone C, Platania P, De Falco T, Maisano S, Nicoli N, Cottone M. The role of calprotectin in predicting endoscopic post-surgical recurrence in asymptomatic Crohn s disease: a comparison with ultrasound. Eur Rev Med Pharmacol Sci 2006; 10: Lamb CA, Mohiuddin MK, Gicquel J, Neely D, Bergin FG, Hanson JM, Mansfield JC. Faecal calprotectin or lactoferrin can identify postoperative recurrence in Crohn s disease. Br J Surg 2009; 96: Thomas P, Rihani H, Røseth A, Sigthorsson G, Price A, Nicholls RJ, Bjarnason I. Assessment of ileal pouch inflammation by single-stool calprotectin assay. Dis Colon Rectum 2000; 43: Pakarinen MP, Koivusalo A, Natunen J, Ashorn M, Karikoski R, Aitola P, Rintala RJ, Kolho KL. Fecal calprotectin mirrors inflammation of the distal ileum and bowel function after restorative proctocolectomy for pediatric-onset ulcerative colitis. Inflamm Bowel Dis 2010; 16: Johnson MW, Maestranzi S, Duffy AM, Dewar DH, Forbes A, Bjarnason I, Sherwood RA, Ciclitira P, Nicholls JR. Faecal calprotectin: a noninvasive diagnostic tool and marker of severity in pouchitis. Eur J Gastroenterol Hepatol 2008; 20: Louis E, Mary JY, Vernier-Massouille G, Grimaud JC, Bouhnik Y, Laharie D, Dupas JL, Pillant H, Picon L, Veyrac M, 6788 December 14, 2012 Volume 18 Issue 46

110 Smith LA et al. Faecal calprotectin in adult IBD Flamant M, Savoye G, Jian R, Devos M, Porcher R, Paintaud G, Piver E, Colombel JF, Lemann M. Maintenance of remission among patients with Crohn s disease on antimetabolite therapy after infliximab therapy is stopped. Gastroenterology 2012; 142: e5; quiz e31 59 Damms A, Bischoff SC. Validation and clinical significance of a new calprotectin rapid test for the diagnosis of gastrointestinal diseases. Int J Colorectal Dis 2008; 23: Wassell J, Wallage M, Brewer E. Evaluation of the Quantum Blue rapid test for faecal calprotectin. Ann Clin Biochem 2012; 49: Kok L, Elias SG, Witteman BJ, Goedhard JG, Muris JW, Moons KG, de Wit NJ. Diagnostic accuracy of point-of-care fecal calprotectin and immunochemical occult blood tests for diagnosis of organic bowel disease in primary care: the Cost-Effectiveness of a Decision Rule for Abdominal Complaints in Primary Care (CEDAR) study. Clin Chem 2012; 58: Sydora MJ, Sydora BC, Fedorak RN. Validation of a pointof-care desk top device to quantitate fecal calprotectin and distinguish inflammatory bowel disease from irritable bowel syndrome. J Crohns Colitis 2012; 6: Hanai H, Takeuchi K, Iida T, Kashiwagi N, Saniabadi AR, Matsushita I, Sato Y, Kasuga N, Nakamura T. Relationship between fecal calprotectin, intestinal inflammation, and peripheral blood neutrophils in patients with active ulcerative colitis. Dig Dis Sci 2004; 49: Schoepfer AM, Beglinger C, Straumann A, Trummler M, Renzulli P, Seibold F. Ulcerative colitis: correlation of the Rachmilewitz endoscopic activity index with fecal calprotectin, clinical activity, C-reactive protein and blood leukocytes. Inflamm Bowel Dis 2009; 15: Schroeder KW, Tremaine WJ, Ilstrup DM. Coated oral 5-aminosalicylic acid therapy for mildly to moderately active ulcerative colitis. A randomized study. N Engl J Med 1987; 317: Daperno M, D Haens G, Van Assche G, Baert F, Bulois P, Maunoury V, Sostegni R, Rocca R, Pera A, Gevers A, Mary JY, Colombel JF, Rutgeerts P. Development and validation of a new, simplified endoscopic activity score for Crohn s disease: the SES-CD. Gastrointest Endosc 2004; 60: Mary JY, Modigliani R. Development and validation of an endoscopic index of the severity for Crohn s disease: a prospective multicentre study. Groupe d Etudes Thérapeutiques des Affections Inflammatoires du Tube Digestif (GE- TAID). Gut 1989; 30: Best WR, Becktel JM, Singleton JW, Kern F. Development of a Crohn s disease activity index. National Cooperative Crohn s Disease Study. Gastroenterology 1976; 70: Matts SG. The value of rectal biopsy in the diagnosis of ulcerative colitis. Q J Med 1961; 30: Kallel L, Ayadi I, Matri S, Fekih M, Mahmoud NB, Feki M, Karoui S, Zouari B, Boubaker J, Kaabachi N, Filali A. Fecal calprotectin is a predictive marker of relapse in Crohn s disease involving the colon: a prospective study. Eur J Gastroenterol Hepatol 2010; 22: Gisbert JP, Bermejo F, Pérez-Calle JL, Taxonera C, Vera I, McNicholl AG, Algaba A, López P, López-Palacios N, Calvo M, González-Lama Y, Carneros JA, Velasco M, Maté J. Fecal calprotectin and lactoferrin for the prediction of inflammatory bowel disease relapse. Inflamm Bowel Dis 2009; 15: S- Editor Gou SX L- Editor A E- Editor Xiong L 6789 December 14, 2012 Volume 18 Issue 46

111 Online Submissions: doi: /wjg.v18.i World J Gastroenterol 2012 December 14; 18(46): ISSN (print) ISSN (online) 2012 Baishideng. All rights reserved. REVIEW Have guidelines addressing physical activity been established in nonalcoholic fatty liver disease? Carmine Finelli, Giovanni Tarantino Carmine Finelli, Center of Obesity and Eating Disorder, Stella Maris Mediterraneum Foundation, Chiaromonte, Potenza, Italy Giovanni Tarantino, Department of Clinical and Experimental Medicine Federico II University Medical School of Naples, Naples, Italy Author contributions: Both authors equally contributed to the design and draft of manuscript. Correspondence to: Giovanni Tarantino, MD, Professor, Department of Clinical and Experimental Medicine Federico II University Medical School of Naples, Via Sergio Pansini, 5, Naples, Italy. [email protected] Telephone: Fax: Received: March 30, 2012 Revised: June 29, 2012 Accepted: July 9, 2012 Published online: December 14, 2012 Research, Kurume University School of Medicine, 67 Asahimachi, Kurume , Japan; Reza Malekzadeh, Professor, Director, Digestive Disease Research Center, Tehran University of Medical Sciences, Shariati Hospital, Kargar Shomali Avenue, Tehran, Iran; Raquel Rocha, MD, Professor Adjunto, Department of Sciences of Nutrition, School of Nutrition, Federal University of Bahia, Avenida Araújo Pinho, 32., Canela, Salvador, BA Cep , Brazil Finelli C, Tarantino G. Have guidelines addressing physical activity been established in nonalcoholic fatty liver disease? World J Gastroenterol 2012; 18(46): Available from: URL: DOI: Abstract The purpose of this review was to highlight, in relation to the currently accepted pathophysiology of non-alcoholic fatty liver disease (NAFLD), the known exercise habits of patients with NAFLD and to detail the benefits of lifestyle modification with exercise (and/or physical activity) on parameters of metabolic syndrome. More rigorous, controlled studies of longer duration and defined histopathological end-points comparing exercise alone and other treatment are needed before better, evidence-based physical activity modification guidelines can be established, since several questions remain unanswered Baishideng. All rights reserved. Key words: Non-alcoholic fatty liver disease; Non-alcoholic steatohepatitis; Physical activity; Diet Peer reviewers: Eyvind J Paulssen, MD, PhD, Department of Gastroenterology, University Hospital of North Norway, PO Box 83, N-9038 Tromsø, Norway; Takumi Kawaguchi, MD, PhD, Department of Digestive Disease Information and INTRODUCTION Compared with our ancestors, Western societies today lead a lifestyle that is much more sedentary, probably as a result of cultural changes stemming from disregarded traditional customs and modern usages. Taking into account differences in body size, our energy expenditure per kilogram of body weight has been estimated to be 40% less than that of our prehistoric ancestors [1]. Current estimates suggest that 7 out of 10 adults are inactive or lack adequate conditioning [2], and this lack of adequate exercise, combined with dietary indiscretion, has contributed to the worldwide epidemic of obesity and non-alcoholic fatty liver disease (NAFLD). Within the United States, data suggest that 64.5% of the adult population is now overweight or obese, with a worldwide prevalence of 40%-60% [3-5]. Obesity, combined with host factors such as diet, sedentary lifestyle and genetic predisposition, has been directly associated with increases in the prevalence of insulin resistance, type 2 diabetes (T2D) and the metabolic syndrome. The hepatic manifestation of the metabolic syndrome, NAFLD, has also increased in prevalence, and is now considered to be around 20%-30% in Western countries. Among 6790 December 14, 2012 Volume 18 Issue 46

112 Finelli C et al. NAFLD and physical activity morbidly obese patients undergoing bariatric surgery, approximately 90% have NAFLD and 36%-37% have the more aggressive form of fatty liver, non-alcoholic steatohepatitis (NASH) [6,7]. In patients with NAFLD, advancing age, increasing weight, the number of features of the metabolic syndrome and the degree of insulin resistance have all been independently associated with NASH severity [8]. Evidence-based treatment options for NAFLD are currently lacking. Recent data suggest that the thiazolidinedione class of insulin sensitisers may be efficacious, but widespread utilisation of these agents awaits further investigation [9]. Evidence also supports a role for weight loss, achieved through exercise. HORMONAL AND NON-HORMONAL REGULATORS OF GLUCOSE, LIPID AND ENERGY METABOLISM IN NAFLD The majority of NAFLD patients are overweight or obese and have underlying insulin, and probably leptin, resistance that results in dysregulated energy metabolism. The regulation of glucose and lipid metabolism involves a complex interplay between adipose tissue, skeletal muscle and the liver. While our knowledge of the pathogenesis of hepatic steatosis has undoubtedly increased over the last decade, many uncertainties remain, and it remains the subject of intense investigation. Hepatic steatosis derives from several possible sources including: (1) increased free fatty acid (FFA) delivery to the liver as a result of dietary fat intake and increased lipolysis within insulin-resistant adipose tissue; (2) increased hepatic de novo lipogenesis (DNL); (3) decreased FFA oxidation; and (4) decreased triacylglycerol export from the liver in the form of very low-density lipoprotein. The largest contributor to hepatic steatosis in patients with NAFLD is increased FFA influx to the liver (60%), followed by DNL (26%) [10]. Increased hepatic lipid supply In insulin-resistant states, principally obesity and T2D, adipose tissue hormone-sensitive lipase activity is not fully suppressed by insulin, resulting in enhanced lipolysis and non-esterified fatty acid flux into the systemic circulation (Figure 1). The precise mechanism of adipocyte insulin resistance in obesity remains a subject of controversy, but an emerging body of data suggest that an altered adipocytokine milieu in visceral fat resulting from macrophage infiltration is important [11]. This milieu is characterised by increased expression of pro-inflammatory cytokines, such as tumour necrosis factor-α and interleukin-6 (IL-6), that can directly inhibit insulin, signalling, and decreased expression of adiponectin, an anti-steatosis and insulin-sensitising adipocyte-derived cytokine (adipocytokine) in both skeletal muscle and liver [12]. Hepatic lipid synthesis and oxidation Energy metabolism within the liver is tightly regulated. Two transcription factors, sterol regulatory elementbinding protein (SREBP-1) and carbohydrate response element-binding protein (ChREBP), are intimately involved in hepatic glucose and lipid metabolism, and their activity is increased in animal models of NAFLD [13,14]. The former is induced by insulin and high-fat diets, and regulates glycolytic and lipogenic gene expression resulting in increased DNL and a concomitant decrease in FFA oxidation resulting from malonyl-coa-induced inhibition of carnitine palmitoyl transferase-1 reducing mitochondrial FFA uptake [15]. ChREBP exerts similar effects on glycolytic and lipogenic gene expression and also increases the expression of genes involved in triglyceride synthesis. In contrast to SREBP-1, ChREBP is upregulated by glucose, which increases its nuclear translocation and its DNA binding/transcriptional activity [16]. Inhibition of ChREBP in ob/ob leptin-deficient mice reduces hepatic steatosis by decreasing lipogenesis and enhancing FFA β-oxidation, with a concomitant decrease in circulating plasma triglycerides and FFA resulting in the restoration of hepatic, skeletal muscle and adipose tissue insulin sensitivity [14]. This study provides further evidence linking fat accumulation to insulin resistance in both hepatic and non-hepatic tissues, principally skeletal muscle [17,18]. Insulin resistance Skeletal muscle is the primary site for glucose disposal via insulin-dependent pathways, accounting for about 75% of whole-body insulin-stimulated glucose uptake [19]. Insulin binding to the insulin receptor on the myocyte plasma membrane results in autophosphorylation of the receptor, allowing insulin receptor substrate (IRS)-1 adaptor protein to bind and undergo tyrosine phosphorylation (Figure 2A). IRS-1-associated phosphatidylinositol 3-kinase (PI3K) activity is increased, which results in downstream activation of protein kinase B, leading to enhanced glucose uptake into the cell, via increased glucose transporter (GLUT)-4 translocation from the cytosol to the cell membrane, and up-regulation of glycogen synthesis and glucose oxidation [18]. In obese individuals, there is reduced insulin-stimulated glucose disposal in skeletal muscle, most probably due to increased intramyocellular lipid content (Figure 2B) [18]. The increased concentration of FFA (or more probably their esterification product, diacylglycerol (DAG), activates the serine/threonine kinase PKCh, which phosphorylates IRS-1 on critical serine sites, thereby inhibiting its tyrosine phosphorylation and subsequently the activation of PI3K and the resulting glucose uptake, glycogen synthesis and glycolysis. Other cytokine-regulated serine/threonine kinases including IkB kinase-b (IKKb) and JNK-1 may also be involved since the inhibition of IKKb via exercise [20] or salicylates results in improved insulin sensitivity [21]. As discussed above, hyperglycaemia resulting from skeletal muscle insulin resistance in obesity leads to increased hepatic fat synthesis, reduced fat oxidation and steatosis via activation of ChREBP. Moreover, the accumulation of FFA/DAG in the liver results in hepatic 6791 December 14, 2012 Volume 18 Issue 46

113 Finelli C et al. NAFLD and physical activity Circulating insulin FFA Hormone sensitive lipase FFA delivery SREBP Lipogenic genesis DNL Triglyceride Adipose tissue Altered adipocitokine action ATP ChREBP Liver TNF-α Circulating glucose Adiponectin (VLDL) Figure 1 Mechanisms contributing to glucose and lipid dysmetabolism. In the setting of insulin resistance, there is increased adipose tissue hormone-sensitive lipase activity that results in enhanced lipolysis and increased non-esterified fatty acid (NEFA) delivery to the liver. NEFAs are preferentially esterified to triglycerides. Additionally, hyperinsulinaemia leads to increased sterol regulatory element patients with NAFLD binding protein (SREBP) expression, resulting in increased de novo lipogenesis (DNL) and decreased fatty acid oxidation. Carbohydrate response element-binding protein (ChREBP) is induced by hyperglycaemia and leads to further increases in DNL. Decreased hepatic lipid transport may also occur, in part via altered synthesis of apolipoprotein B, leading to decreased very low-density lipoprotein (VLDL) production. TNF-α: Tumour necrosis factor-α; FFA: Free fatty acid. A Insulin Glucose Insulin Glucose B AMPK Glucose AMP/ATP Glycogen synthesis Glucose oxidation Insulin receptor IRS-1/2 FFA/DAG P13K PKCh GLUT-4 Akt Figure 2 Mechanisms for free fatty acid-induced skeletal muscle insulin resistance. A: Normal glucose uptake into skeletal muscle occurs via binding of insulin to the insulin receptor, resulting in receptor autophosphorylation and subsequent binding and tyrosine phosphorylation of insulin receptor substrate (IRS)-1. Subsequently, phosphatidylinositol 3-kinase (PI3K) is activated and results in downstream activation of protein kinase B, leading to glucose transporter (GLUT)-4 translocation to the myocyte plasma membrane and glucose uptake into the cell; B: Increased intramyocellular lipid content leads to the activation of protein kinase Ch (PKCh) which results in serine phosphorylation of IRS-1, thereby inhibiting its tyrosine phosphorylation. This prevents the activation of PI3K and GLUT-4 translocation to the cell surface. Thus glucose entry into the cell is inhibited. AMPK: AMP-activated protein kinase; DAG: Diacylglycerol; FFA: Free fatty acid; Akt: Protein Kinase B. insulin resistance, via PKCe-induced IRS-2 serine phosphorylation, which reduces insulin s inhibitory effect on gluconeogenesis, contributing further to hyperglycaemia [18,22]. Leptin resistance Lack of response to the adipocytokine leptin (leptin resistance) rather than insulin resistance may be an important factor in the pathogenesis of hepatic steatosis. The evidence for leptin resistance in NAFLD, though indirect, is compelling. Leptin exerts a number of antisteatosis effects on the liver, including inhibition of lipid synthesis via reduced expression of stearoyl CoA desaturase (SCD)-1 [23] and enhanced FFA oxidation, via up-regulation of peroxisome proliferator-activated receptor-α (PPAR-α) [24], and yet patients with NAFLD 6792 December 14, 2012 Volume 18 Issue 46

114 Finelli C et al. NAFLD and physical activity have increased serum leptin concentrations [25]. Recent studies in genetically leptin resistant (ZDF) rats have dissociated the direct anti-steatotic effects of leptin from any indirect effects via improved hepatic insulin sensitivity [26]. The mechanism of leptin resistance in obesity is unclear; however, it may be enhanced by the ingestion of fructose which leads to an inhibition of STAT-3, abrogating leptin-mediated PPAR-α activation resulting in decreased fatty acid oxidation, increased SREBP-1 expression and increased hepatic triglyceride content [27]. Further inhibition of STAT-3 may occur as a result of up-regulation of suppressor of cytokine signalling-3 (SOCS-3) by adipose tissue or hepatocyte-derived inflammatory cytokines [15,28]. AMP-activated protein kinase Of particular relevance to modify NAFLD natural history, AMP-activated protein kinase (AMPK) has recently emerged as a key orchestrator of both hormonal and non-hormonal regulators of energy metabolism in liver, skeletal muscle and adipose tissue. Activated by an increase in the AMP/ATP ratio, AMPK activity is enhanced by physiological processes that induce metabolic stress and either decrease ATP production (ischaemia, hypoxia) or increase its consumption (exercise) [29]. Once activated, AMPK acts to increase ATP-generating cellular events while turning off energy-consuming processes to restore energy balance. In the liver, the activation of AMPK by exercise [30], starvation, adiponectin [31], leptin (via inhibition of SCD-1), the biguanide metformin [32] or the thiazolidinedione class of insulin sensitisers [33] suppresses expression of SREBP-1 [34], ChREBP [35] and acetyl-coa carboxylase [36] resulting in decreased DNL and increased FFA oxidation. Hepatic gluconeogenesis is also inhibited. Within skeletal muscle, exercise, leptin or drug related AMPK activation enhances glucose uptake via direct non-insulin-dependent GLUT-4 translocation, and increases pyruvate oxidation [32]. In adipose tissue, hormone-sensitive lipase activation is suppressed, resulting in decreased lipolysis [32]. ChREBP, a newly discovered transcription factor, plays an essential role in glucose-induced L-pyruvate kinase (L-PK) [37] gene transcription by binding to the carbohydrate-responsive element of L-PK promoter [38,39]. It is well known that glucose metabolism is inhibited by fatty acids, which serve as an alternative fuel source and thus conserve glucose. This phenomenon has been termed the fat sparing effect on glucose [37,40]. Kawaguchi et al [41] investigated the mechanism by which feeding high fat diets results in decreased activity of ChREBP in the liver. It s strongly suggested that the fatty acid inhibition of glucose-induced l-pk transcription resulted from AMPK phosphorylation of ChREBP at Ser(568), which inactivated the DNA binding activity. AMPK was activated by the increased AMP that was generated by the fatty acid activation. AMPK is a metabolic master switch mediating adaptation of the cell to variations in nutritional environment [42]. Its activity is stimulated by increases in intracellular AMP-to-ATP ratio in response to stresses such as exercise, hypoxia, and glucose deprivation. AMPK has acute effects on energy metabolism pathways and longterm effects involving changes in gene expression. AMPK is a major therapeutic target for the treatment of diabetes. The effect of a short-term overexpression of AMPK specifically in the liver by adenovirus-mediated transfer of a gene encoding a constitutively active form of AMPKα2 (AMPKα2-CA) has been investigated [43]. The short-term overexpression of AMPKα2-CA in the liver results in a metabolic switch from glucose to lipid metabolism. The lower plasma glucose concentrations in Ad AMPKα2-CA-infected mice lead to an increase in hepatic lipid utilization, resulting in a decrease in white adipose mass. The concomitant accumulation of hepatic triglycerides leads to the generation of ketone bodies, which are required as alternative substrates to supply energy to peripheral tissues in conditions of low glucose availability. Another study investigated the effects of fasting and refeeding on AMPK and ChREBP mrna, protein and activity levels; as well as the expression of lipogenic genes involved in regulating lipid synthesis in broiler chicken (Gallus gallus) liver [44]. In general, evidence was found for coordinate transcriptional regulation of lipogenic program genes in broiler chicken liver, but specific regulatory roles for AMPK and ChREBP in that process remain to be further characterized. Moreover, thioredoxin-interacting protein (TXNIP) regulates critical biological processes including inflammation, stress and apoptosis. TXNIP is upregulated by glucose and is a critical mediator of hyperglycemia-induced beta-cell apoptosis in diabetes. In contrast, the saturated long-chain fatty acid palmitate, although toxic to the beta-cell, inhibits TXNIP expression. The mechanisms involved in the opposing effects of glucose and fatty acids on TXNIP expression are unknown. Shaked et al [45] showed that AMPK is an important regulator of Txnip transcription via modulation of ChREBP activity. The divergent effects of glucose and fatty acids on TXNIP expression result in part from their opposing effects on AMPK activity. In light of the important role of TXNIP in beta-cell apoptosis, its inhibition by fatty acids can be regarded as an adaptive/protective response to glucolipotoxicity. The finding that AMPK mediates nutrient regulation of TXNIP may have important implications for the pathophysiology and treatment of diabetes. EFFECT OF EXERCISE ALONE ON OBESITY, VISCERAL FAT AND INSULIN RESISTANCE Exercise physiology and the salutatory effects on weight loss, fat reduction and insulin sensitivity have been described in great detail. These beneficial effects are now considered to reflect, at least in part, the effect of exer December 14, 2012 Volume 18 Issue 46

115 Finelli C et al. NAFLD and physical activity cise on the activation of AMPK. In obese non-diabetics, exercise has been shown to reduce the risk of developing T2D by up to 46% [33]. Physical training, consisting of 20 min cycling or running, 20 min swimming at submaximal heart rate, followed by 20 min of warm up/cool down three times per week for 4 wk, resulted in a significant reduction in body weight and percentage body fat, and this was associated with improved whole-body glucose uptake, decreased fasting insulin concentrations and increased circulating adiponectin and mrna expression in muscle. Among patients with T2D, increasing exercise led to a reduction in fasting plasma glucose [34]. The intensity of exercise needed to show improvement in metabolic profiles has been studied by several investigators. O Donovan and colleagues evaluated the effects of 24 wk of moderate intensity exercise, defined as cycling three times weekly at 60% VO2max to burn 400 kcal, vs high-intensity exercise, defined as cycling three times weekly at 80% VO2max to burn 400 kcal, versus no exercise, on insulin sensitivity, triglycerides and glucose concentration [35]. Training at 60% VO2max was as effective as training at 80% VO2max when 400 kcal were expended per session, suggesting that moderate exercise, expending 400 kcal per session, three times per week is sufficient to improve insulin sensitivity. The overall energy expenditure achieved per work-out session appears to be more important than the intensity of the exercise. This is supported by two recent studies performed in obese patients [36,46]. Daily exercise for 12 wk, performed at not greater than 70% VO2max (about 80% maximum heart rate) on a treadmill to achieve 700 kcal energy expenditure (about 60 min) resulted in an 8% body weight loss and was associated with significant reductions in abdominal obesity, visceral fat, waist circumference and insulin resistance [36]. Furthermore, this study showed that exercise without weight loss also reduced both abdominal and visceral fat. Ray et al [46] also demonstrated that daily aerobic exercise for min, starting at 60%-65% maximum heart rate and increasing to 80%-85% maximum heart rate (about 70% VO2max) over 4 wk improved visceral fat content and this correlated with improved glucose metabolism and loss of insulin resistance. These encouraging results were seen with only a 3% weight loss over this time period. The effects of aerobic vs restrictive exercise have also been debated. A Turkish study examined the effects of aerobic exercise, defined as walking briskly for 15 min and exercising on a stationary bicycle for min three times per week the first month, exercising min four times per week the second month, and min five times per week the third month, in a group of 20 obese women compared with a restrictive exercise utilizing a stationary exercise unit in a similar group of women over a 3-mo period [47]. While improvement in body mass index (BMI), fasting glucose and postprandial glucose were seen in both groups, reduced fat mass (as measured by bioelectric impedance), decreased low-density lipoprotein and insulin resistance were seen only in the aerobic exercise group. In another study in 39 older obese men, aerobic or restrictive exercise training 3 d per week for 6 mo resulted in similar improvement in whole-body glucose disposal [48]. Weight loss remains fundamental to the management of NAFLD, but is mistakenly perceived as the primary rationale for promoting physical activity (PA) participation. However, obesity management is not simply a function of weight loss. Outside the context of liver disease, it is well established that exercise enhances insulin sensitivity, reduces progression to T2D, and favorably modifies serum lipids independent of weight loss [49,50]. When combined with the observation that high fitness and habitual PA are associated with improved functional capacity, quality-of-life measures, well-being, and reduced allcause mortality [51], the importance of incorporating PA therapy, beyond assisting weight loss, becomes apparent. At present, there is an overall paucity of evidence concerning the benefits of PA as treatment for NAFLD, even though PA is certainly useful in NAFLD-associated diseases such as obesity, T2D and cardiovascular disease. What is available shows a conclusive benefit of PA when coupled with energy restriction when weight loss is achieved, and it is encouraging for an independent benefit in the absence of weight loss. Although weight loss remains fundamental, patients should be counseled on the spectrum of benefits conferred by regular PA. Management should include assessment of cardiorespiratory fitness and PA levels, and the setting of lifestyle goals based on adoption of regular exercise, with a focus on the attainment of sustainable PA habits. The dose (intensity and volume) of PA required to reduce liver fat remains unclear. Furthermore, from the present evidence, it is difficult to discern the relative importance of structured exercise and fitness vs less structured PA. Although several examples of a hepatic benefit from low-dose PA therapy have been cited [52-55], in the absence of robust data and knowledge of the long-term sustainability of such outcomes, it would seem reasonable to promote the current public health recommendations for health promotion, disease prevention, and weight management (Table 1). This recommends that individuals accumulate min or more of moderate intensity (about 45%-70% of VO2max) exercise on most days of the week [51]. If weight loss is the goal, exercise confers a reduction in body weight in an apparent dose-response fashion with exercise volume, even when prescribed without associated restriction of energy intake [56]. Greater amounts of exercise may be needed for most individuals to induce significant weight loss or prevent weight being regained in the long term. The consensus suggests that little weight loss is achieved with < 150 min of exercise per week, modest (2-3 kg) losses are attainable with > 150 min/wk (with an energy equivalent of kcal/wk), and moderate weight loss (5-7.5 kg) often results from min/wk ( kcal) of aerobic 6794 December 14, 2012 Volume 18 Issue 46

116 Finelli C et al. NAFLD and physical activity Table 1 Recommendations for physical activity in non-alcoholic fatty liver disease Patients should be appropriately screened for contraindications prior to initiating exercise testing or therapy Physical fitness assessment via exercise testing. Physical activity level assessment by subjective (questionnaire/diary) or objective (e.g., accelerometer) means Accumulate min or more of moderate intensity rhythmic exercise using large muscle groups on at least 5 d/wk Moderate intensity physical activity between 150 and 250 min/wk for preventing weight gain Physical activity > 250 min/wk for clinically significant weight loss Moderate-to-high intensity resistance training 3 d/wk for enhancing insulin sensitivity activity [56]. These targets can be achieved using a variety of exercise modalities, with the outcome of cardiorespiratory fitness being a reliable and easily quantifiable endpoint measure of structured aerobic exercise. Although there is currently no longitudinal evidence available concerning its benefit in NAFLD, progressive resistance training may be useful for the management of obesityrelated comorbidities, particularly insulin resistance [56]. The benefits of nonstructured leisure-time PA, including reduced sedentary time, are becoming increasingly recognized and have, in some studies, shown efficacy in improving cardiometabolic risk and promoting weight loss [56,57]. Clear guidelines for such lifestyle PA are lacking, and reliable measurement, particularly of intensity, is more difficult. PA habits and adherence can be estimated by questionnaires, pedometers, and accelerometers (reviews of which can be found elsewhere) [58], and the latter may further promote adherence to PA [58]. Hybrid training of voluntary and electrical muscle contractions Hybrid exercise is an exercise method that combines electrically stimulated and volitional contraction. This technique produces resistance against the motion of a volitionally contracting muscle by means of a force generated by an electrically stimulated antagonist [59-62]. In particular, hybrid exercise resists utilizes electrically stimulated eccentric contractions and concentric volitional contractions with reciprocal limb movements. Both the volitionally activated agonist and the electrically stimulated antagonist contract during joint motion. The result is that both muscles are trained and that a longitudinal compressive load is placed on the bone. This technique requires minimal external stabilization as compared with conventional weight training. Matsuse et al [59] have shown that such hybrid exercise increased the extension torque of the elbow joint by about 30% and the cross-sectional areas (CSA) of the proximal upper extremity muscles by about 15% over a 12-wk period. In addition, Iwasaki et al [60] demonstrated that 6 wk of hybrid exercise effectively increased the extension of the knee joint by 19-33%. Takano et al [61] demonstrated that 12 wk of treatment increased extension torque by 39% and the CSA of quadriceps muscle by 9% in elderly subjects. However, the mechanism by which hybrid exercise achieves these increases in muscular strength and bulk is still unknown. Muscle atrophy occurs as a consequence of denervation, injury, joint immobilization, bed rest, glucocorticoid treatment, sepsis, cancer and aging [63]. Unfortunately, there is no effective treatment for muscle atrophy. The maintenance of muscle mass is controlled by a balance between protein synthesis and protein degradation pathways, which is thought to shift toward protein degradation during atrophy [63]. Recently, a signaling pathway that increases protein synthesis was shown to promote muscle hypertrophy, thereby overcoming muscle atrophy [64,65]. The Hybrid Training (HYBT) method utilizing combined electrical stimulation and voluntary muscle contraction has been developed as a muscle training method [66]. It has already been shown that the method is technically sound and clinically effective in healthy young subjects. The HYBT method increases muscle strength and mass and is as effective as the weight machine training (WMT), an effective method for improving muscle strength and hypertrophy in elderly people [67,68]. However, a critical problem is that the equipment for WMT is large in size and takes space. In addition, unlike the WMT, the HYBT device, which is portable and not large in size, is so easy to handle that it can be placed at the bedside. Therefore, the HYBT may become a safe, effective method of muscle training for elderly people [66]. Although skeletal muscle regulates glucose metabolism, partly by releasing IL-6, the effects of hybrid training on glucose metabolism remain unclear. Kawaguchi et al [69] showed the effects of hybrid training on glucose metabolism and serum IL-6 levels in elderly people. This study showed the safety and good adherence of hybrid training for lower extremities in elderly people. Furthermore, hybrid training decreased fasting blood glucose and serum IL-6 levels in elderly people. Kawaguchi et al [70] investigated the therapeutic efficacy of hybrid training in patients with NAFLD. Physical inactivity is a risk factor for the development of NAFLD. HYBT of voluntary and electrical muscle contractions improved hepatic steatosis and reduced insulin resistance and serum IL-6 levels in NAFLD patients who are resistant to lifestyle counseling. Possible role of myokine in patients with NAFLD Skeletal muscle has recently been identified as an organ that produces and releases cytokines, which have been named myokines. Given that skeletal muscle is the largest organ in the human body, our discovery that contracting skeletal muscle secretes proteins sets a novel paradigm: skeletal muscle is an endocrine organ producing and releasing myokines in response to contraction which can influence metabolism in other tissues and organs. With the discovery that exercise provokes an in December 14, 2012 Volume 18 Issue 46

117 Finelli C et al. NAFLD and physical activity crease in a number of cytokines, a possible link between skeletal muscle contractile activity and immune changes was established. For most of the last century, researchers sought a link between muscle contraction and humoral changes in the form of an exercise factor, which could be released from skeletal muscle during contraction and mediate some of the exercise-induced metabolic changes in other organs such as the liver and adipose tissue. It has been suggested that cytokines or other peptides that are produced, expressed, and released by muscle fibers and exert either paracrine or endocrine effects should be classified as myokines [71]. The nervous, endocrine, and immune systems all contribute to the maintenance of homeostasis. Interestingly, although these individual systems operate independently to a certain degree, each with their own collection of highly specific cells and regulatory factors, they also depend on each other for normal development and function. It appears that skeletal muscle has the capacity to express several myokines. To date the list includes IL-6, IL-8 and IL-15 [71]. Contractile activity plays a role in regulating the expression of many of these cytokines in skeletal muscle [71]. The discovery that IL-6 is released from contracting skeletal muscle has generated much interest among the scientific community because this finding is somewhat paradoxical. On one hand, IL-6 is markedly produced and released in the postexercise period when insulin action is enhanced, but on the other hand, IL-6 has been associated with obesity and reduced insulin action. Given the controversy, this review focuses on the metabolic roles of IL-6. Despite the fact that acute IL-6 treatment may enhance glucose uptake and fat oxidation in skeletal muscle, there are, nonetheless, a number of studies both in vitro [72-75] and in rodents in vivo [76-78] that demonstrate that IL-6 is capable of inducing insulin resistance. It appears that most, if not all, in vivo studies seem to suggest that IL-6 induces insulin resistance via adverse effects on the liver. Subjecting lean mice to chronically elevated IL-6 for 5 days causes hepatic insulin resistance [75], while treating either ob/ob (leptin-deficient) mice [77] or liver-inducible kappa kinase transgenic mice that display hepatic insulin resistance [79] with IL-6 neutralizing antibodies improves hepatic insulin resistance. The IL-6-induced insulin resistance appears due to increased SOCS proteins (SOCS-3) expression [75], since it is thought that SOCS-3 may directly inhibit the insulin receptor [80]. However, even the negative effect of SOCS-3 on insulin action has recently been brought into question. Liver specific activator of transcription 3 (STAT3) knockout mice that express low levels of hepatic SOCS-3 protein, paradoxically are unable to suppress hepatic glucose production after intracerebral ventricular insulin infusion [81]. Moreover, the prevention of IL-6 signaling either by neutralizing antibodies or by genetic deletion of IL-6 markedly reduces insulin-induced phosphorylation of hepatic STAT3 [81]. These results suggest that the local production of IL-6 is important for the phosphorylation of hepatic STAT3 induced by the brain insulin action. In a separate study, liver specific SOCS-3 knockout mice exhibited obesity and systemic insulin resistance with age [82]. Furthermore, in this recent study, insulin signaling was reduced in skeletal muscle [82], suggesting that deletion of the SOCS-3 gene in the liver modulates insulin sensitivity in other organs. Possibly, the most convincing data to suggest that IL-6 may be antiobesogenic is the observation that IL-6 knockout mice develop mature onset obesity and glucose intolerance [83] ; however, even this observation is unclear [84]. Whether IL-6 has positive effects on obesity and insulin action is clearly unresolved and requires further work. However, IL-6 unquestionably has a poor prognosis for certain inflammatory diseases [85], and due to the immunoreactive nature of IL-6, it is clear that rhil-6 treatment may not be a wise therapeutic treatment strategy in human disease. This is most likely due to the previously described trans-signaling of IL-6. The soluble IL-6 receptor controls the transition from the acute to the chronic phase in many proinflammatory diseases such as peritonitis [86], a transition that can be inhibited by treatment with a soluble gp130 receptor fragment that neutralizes the trans-signaling process [86]. Therefore, other cytokines that signal through the gp130 receptor, but which do not activate trans-signaling of IL-6, such as ciliary neurotrophic factor, show some therapeutic promise as an antiobesity therapy [87]. NAFLD and its subsequent complications create a significant health burden, and currently there is no effective treatment strategy. The biochemical mechanisms that underlie NAFLD are unclear at this time, but there is evidence that insulin resistance is a major contributing factor. In addition, circulating concentrations of inflammatory cytokines - myokines (e.g., IL-6) as well as decreased antiinflammatory factors (e.g., adiponectin, IL-10) are not only implicated in the development of insulin resistance and T2D, but are also related to NAFLD. Such inflammatory mechanisms are fundamental in the progression of NAFLD toward higher risk cirrhotic states. Regular exercise can reverse insulin resistance, suppress low-grade systemic inflammation, and attenuate inflammatory markers associated with NAFLD. Thus, exercise has the potential to become an effective treatment and prevention modality for NAFLD and NASH. CONCLUSION Our knowledge of the pathological consequences of lack of adequate exercise on adipose tissue, skeletal muscle and the liver is improving, and this will help establish more specific guidelines for the proper exercise regimens that will improve underlying metabolic pathways and ultimately decrease the incidence and severity of NAFLD. Moderate exercise, preferably a combination of aerobic and restrictive, performed 3-4 times per week, expending about 400 calories each time seems adequate to augment improvement in the metabolic profiles of patients with 6796 December 14, 2012 Volume 18 Issue 46

118 Finelli C et al. NAFLD and physical activity NAFLD. PA has long been considered a cornerstone of a healthy lifestyle. Although its protective role in cardiovascular and metabolic diseases is well established [88], its place and importance in NAFLD still requires scientific support and clarification. In a study, an inverse association was found between cardio-respiratory fitness categories and the prevalence of NAFLD. Whereas fitness and BMI were independent of each other in their associations with the prevalence of NAFLD, the addition of waist circumference to the regression model attenuated the association [89]. This is in line with the fact that abdominal obesity has been shown to be a major risk factor for NAFLD, of greater importance than BMI [90,91], and is consistent with previous studies demonstrating that exercise-induced weight loss is associated with a preferential reduction in abdominal fat [92,93], and that, at any given weight, individuals who exercise more have less visceral fat than those who are sedentary [94]. The suggested effect of PA on NAFLD may stem from other mechanisms as well. Exercise alone, in the absence of any change in body weight or composition, may enhance insulin sensitivity and glucose homeostasis [95,96]. PA appears to result in insulin-receptor up-regulation in muscle tissue and hence increased delivery of glucose and insulin to the muscles [97]. Exercise also has a beneficial effect on FFA metabolism by enhancing whole-body lipid oxidation [98]. Hepatic triglyceride accumulation was shown to decrease with exercise intervention [99] and hepatic FFA uptake was lower in trained compared with untrained subjects [100]. From the perspective of NAFLD patients, weekly or daily performance of walking, swimming, or cycling might seem as simple as jumping of the cliff. Thus, it seems that among NAFLD patients, even small increments in regular PA can improve liver enzymes; encouraging information that can be provided to patients. Time spent sedentary, measured objectively by individually calibrated heart rate monitoring, predicted higher levels of fasting insulin, independent of the amount of time spent at moderate- and vigorous-intensity activity levels. This highlights the importance of reducing sedentary time in order to improve metabolic status, in addition to the benefits associated with a physically active lifestyle [101]. Environmental factors that discourage PA include an environment that encourages automobile use rather than walking (like lack of sidewalks), and that has few cues to promote activity and numerous cues that discourage activity (television, computers, etc.) [102,103]. It should be emphasized that associations between air pollution and a multitude of health effects are now well established. Given ubiquitous exposure to some level of air pollution, the attributable health burden can be high, particularly for susceptible populations affected by cardiorespiratory problems when walking, running at a slow or leisurely pace, or cycling [104]. More rigorous, controlled studies, of longer duration and defined histopathological end-points comparing exercise alone and other treatment are needed before better, evidence-based PA modification guidelines can be established, since several questions remain unanswered. Does PA modification work equally well in men vs women? 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120 Finelli C et al. NAFLD and physical activity 51 Billinger SA, Coughenour E, Mackay-Lyons MJ, Ivey FM. Reduced cardiorespiratory fitness after stroke: biological consequences and exercise-induced adaptations. Stroke Res Treat 2012; 2012: Zelber-Sagi S, Ratziu V, Oren R. Nutrition and physical activity in NAFLD: an overview of the epidemiological evidence. World J Gastroenterol 2011; 17: Kistler KD, Brunt EM, Clark JM, Diehl AM, Sallis JF, Schwimmer JB. Physical activity recommendations, exercise intensity, and histological severity of nonalcoholic fatty liver disease. Am J Gastroenterol 2011; 106: ; quiz Al-Mamari A. Atherosclerosis and physical activity. Oman Med J 2009; 24: Thoma C, Day CP, Trenell MI. Lifestyle interventions for the treatment of non-alcoholic fatty liver disease in adults: a systematic review. J Hepatol 2012; 56: Donnelly JE, Blair SN, Jakicic JM, Manore MM, Rankin JW, Smith BK. American College of Sports Medicine Position Stand. Appropriate physical activity intervention strategies for weight loss and prevention of weight regain for adults. Med Sci Sports Exerc 2009; 41: Zelber-Sagi S, Nitzan-Kaluski D, Goldsmith R, Webb M, Zvibel I, Goldiner I, Blendis L, Halpern Z, Oren R. Role of leisure-time physical activity in nonalcoholic fatty liver disease: a population-based study. Hepatology 2008; 48: Biddle SJ, Gorely T, Pearson N, Bull FC. An assessment of self-reported physical activity instruments in young people for population surveillance: Project ALPHA. Int J Behav Nutr Phys Act 2011; 8: 1 59 Matsuse H, Shiba N, Umezu Y, Nago T, Tagawa Y, Kakuma T, Nagata K, Basford JR. Muscle training by means of combined electrical stimulation and volitional contraction. Aviat Space Environ Med 2006; 77: Iwasaki T, Shiba N, Matsuse H, Nago T, Umezu Y, Tagawa Y, Nagata K, Basford JR. Improvement in knee extension strength through training by means of combined electrical stimulation and voluntary muscle contraction. Tohoku J Exp Med 2006; 209: Takano Y, Haneda Y, Maeda T, Sakai Y, Matsuse H, Kawaguchi T, Tagawa Y, Shiba N. Increasing muscle strength and mass of thigh in elderly people with the hybrid-training method of electrical stimulation and volitional contraction. Tohoku J Exp Med 2010; 221: Matsuse H, Iwasa C, Imaishi K, Nago T, Tagawa Y, Kakuma T, Shiba N. Hybrid-training method increases muscle strength and mass in the forearm without adverse effect of hand function in healthy male subjects. Kurume Med J 2011; 57: Kachaeva EV, Shenkman BS. Various jobs of proteolytic enzymes in skeletal muscle during unloading: facts and speculations. J Biomed Biotechnol 2012; 2012: Trendelenburg AU, Meyer A, Jacobi C, Feige JN, Glass DJ. TAK-1/p38/nNFκB signaling inhibits myoblast differentiation by increasing levels of Activin A. Skelet Muscle 2012; 2: 3 65 Douillard A, Galbes O, Begue G, Rossano B, Levin J, Vernus B, Bonnieu A, Candau R, Py G. Calpastatin overexpression in the skeletal muscle of mice prevents clenbuterol-induced muscle hypertrophy and phenotypic shift. Clin Exp Pharmacol Physiol 2012; 39: Maddocks M, Murton AJ, Wilcock A. Improving muscle mass and function in cachexia: non-drug approaches. Curr Opin Support Palliat Care 2011; 5: Nocera J, Buford TW, Manini TM, Naugle K, Leeuwenburgh C, Pahor M, Perri MG, Anton SD. The impact of behavioral intervention on obesity mediated declines in mobility function: implications for longevity. J Aging Res 2011; 2011: Domański M, Ciechanowski K. Sarcopenia: a major challenge in elderly patients with end-stage renal disease. J Aging Res 2012; 2012: Kawaguchi T, Shiba N, Takano Y, Maeda T, Sata M. Hybrid training of voluntary and electrical muscle contractions decreased fasting blood glucose and serum interleukin-6 levels in elderly people: a pilot study. Appl Physiol Nutr Metab 2011; 36: Kawaguchi T, Shiba N, Maeda T, Matsugaki T, Takano Y, Itou M, Sakata M, Taniguchi E, Nagata K, Sata M. Hybrid training of voluntary and electrical muscle contractions reduces steatosis, insulin resistance, and IL-6 levels in patients with NAFLD: a pilot study. J Gastroenterol 2011; 46: Pedersen BK, Akerström TC, Nielsen AR, Fischer CP. Role of myokines in exercise and metabolism. J Appl Physiol 2007; 103: Lagathu C, Bastard JP, Auclair M, Maachi M, Capeau J, Caron M. Chronic interleukin-6 (IL-6) treatment increased IL-6 secretion and induced insulin resistance in adipocyte: prevention by rosiglitazone. Biochem Biophys Res Commun 2003; 311: Athyros VG, Tziomalos K, Karagiannis A, Anagnostis P, Mikhailidis DP. Should adipokines be considered in the choice of the treatment of obesity-related health problems? Curr Drug Targets 2010; 11: Das SK, Balakrishnan V. Role of cytokines in the pathogenesis of non-alcoholic Fatty liver disease. Indian J Clin Biochem 2011; 26: Senn JJ, Klover PJ, Nowak IA, Zimmers TA, Koniaris LG, Furlanetto RW, Mooney RA. Suppressor of cytokine signaling-3 (SOCS-3), a potential mediator of interleukin-6-dependent insulin resistance in hepatocytes. J Biol Chem 2003; 278: Alexandrov IM, Ivshina M, Jung DY, Friedline R, Ko HJ, Xu M, O Sullivan-Murphy B, Bortell R, Huang YT, Urano F, Kim JK, Richter JD. Cytoplasmic polyadenylation element binding protein deficiency stimulates PTEN and Stat3 mrna translation and induces hepatic insulin resistance. PLoS Genet 2012; 8: e Braunersreuther V, Viviani GL, Mach F, Montecucco F. Role of cytokines and chemokines in non-alcoholic fatty liver disease. World J Gastroenterol 2012; 18: Rocha VZ, Folco EJ. Inflammatory concepts of obesity. Int J Inflam 2011; 2011: Cai D, Yuan M, Frantz DF, Melendez PA, Hansen L, Lee J, Shoelson SE. Local and systemic insulin resistance resulting from hepatic activation of IKK-beta and NF-kappaB. Nat Med 2005; 11: Lebrun P, Van Obberghen E. SOCS proteins causing trouble in insulin action. Acta Physiol (Oxf) 2008; 192: Fukushima A, Loh K, Galic S, Fam B, Shields B, Wiede F, Tremblay ML, Watt MJ, Andrikopoulos S, Tiganis T. T-cell protein tyrosine phosphatase attenuates STAT3 and insulin signaling in the liver to regulate gluconeogenesis. Diabetes 2010; 59: Yoshimura A, Suzuki M, Sakaguchi R, Hanada T, Yasukawa H. SOCS, Inflammation, and Autoimmunity. Front Immunol 2012; 3: Boden G. Obesity, insulin resistance and free fatty acids. Curr Opin Endocrinol Diabetes Obes 2011; 18: Suzuki T, Imai J, Yamada T, Ishigaki Y, Kaneko K, Uno K, Hasegawa Y, Ishihara H, Oka Y, Katagiri H. Interleukin-6 enhances glucose-stimulated insulin secretion from pancreatic beta-cells: potential involvement of the PLC-IP3- dependent pathway. Diabetes 2011; 60: Prieto J. Inflammation, HCC and sex: IL-6 in the centre of the triangle. J Hepatol 2008; 48: Jones SA, Scheller J, Rose-John S. 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121 Finelli C et al. NAFLD and physical activity resistance with ciliary neurotrophic factor. Handb Exp Pharmacol 2011; (203): Williams MA, Haskell WL, Ades PA, Amsterdam EA, Bittner V, Franklin BA, Gulanick M, Laing ST, Stewart KJ. Resistance exercise in individuals with and without cardiovascular disease: 2007 update: a scientific statement from the American Heart Association Council on Clinical Cardiology and Council on Nutrition, Physical Activity, and Metabolism. Circulation 2007; 116: Cohen JC, Horton JD, Hobbs HH. Human fatty liver disease: old questions and new insights. Science 2011; 332: Vernon G, Baranova A, Younossi ZM. Systematic review: the epidemiology and natural history of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in adults. Aliment Pharmacol Ther 2011; 34: Prasad DS, Kabir Z, Dash AK, Das BC. Abdominal obesity, an independent cardiovascular risk factor in Indian subcontinent: A clinico epidemiological evidence summary. J Cardiovasc Dis Res 2011; 2: Conn VS, Hafdahl AR, Cooper PS, Brown LM, Lusk SL. Meta-analysis of workplace physical activity interventions. Am J Prev Med 2009; 37: Ferrara CM, Goldberg AP, Ortmeyer HK, Ryan AS. Effects of aerobic and resistive exercise training on glucose disposal and skeletal muscle metabolism in older men. J Gerontol A Biol Sci Med Sci 2006; 61: Wong SL, Katzmarzyk P, Nichaman MZ, Church TS, Blair SN, Ross R. Cardiorespiratory fitness is associated with lower abdominal fat independent of body mass index. Med Sci Sports Exerc 2004; 36: Umpierre D, Ribeiro PA, Kramer CK, Leitão CB, Zucatti AT, Azevedo MJ, Gross JL, Ribeiro JP, Schaan BD. Physical activity advice only or structured exercise training and association with HbA1c levels in type 2 diabetes: a systematic review and meta-analysis. JAMA 2011; 305: Thoma C, Day CP, Trenell MI. Lifestyle interventions for the treatment of non-alcoholic fatty liver disease in adults: a systematic review. J Hepatol 2012; 56: Breen L, Philp A, Shaw CS, Jeukendrup AE, Baar K, Tipton KD. Beneficial effects of resistance exercise on glycemic control are not further improved by protein ingestion. PLoS One 2011; 6: e Hannukainen JC, Kalliokoski KK, Borra RJ, Viljanen AP, Janatuinen T, Kujala UM, Kaprio J, Heinonen OJ, Viljanen T, Haaparanta M, Iozzo P, Parkkola R, Nuutila P. Higher free fatty acid uptake in visceral than in abdominal subcutaneous fat tissue in men. Obesity (Silver Spring) 2010; 18: Tamura Y, Tanaka Y, Sato F, Choi JB, Watada H, Niwa M, Kinoshita J, Ooka A, Kumashiro N, Igarashi Y, Kyogoku S, Maehara T, Kawasumi M, Hirose T, Kawamori R. Effects of diet and exercise on muscle and liver intracellular lipid contents and insulin sensitivity in type 2 diabetic patients. J Clin Endocrinol Metab 2005; 90: Haus JM, Solomon TP, Marchetti CM, Edmison JM, González F, Kirwan JP. Free fatty acid-induced hepatic insulin resistance is attenuated following lifestyle intervention in obese individuals with impaired glucose tolerance. J Clin Endocrinol Metab 2010; 95: Kozey-Keadle S, Libertine A, Staudenmayer J, Freedson P. The Feasibility of Reducing and Measuring Sedentary Time among Overweight, Non-Exercising Office Workers. J Obes 2012; 2012: Lichtenstein AH, Appel LJ, Brands M, Carnethon M, Daniels S, Franch HA, Franklin B, Kris-Etherton P, Harris WS, Howard B, Karanja N, Lefevre M, Rudel L, Sacks F, Van Horn L, Winston M, Wylie-Rosett J. Diet and lifestyle recommendations revision 2006: a scientific statement from the American Heart Association Nutrition Committee. Circulation 2006; 114: Zivkovic AM, German JB, Sanyal AJ. Comparative review of diets for the metabolic syndrome: implications for nonalcoholic fatty liver disease. Am J Clin Nutr 2007; 86: McKone TE, Ryan PB, Ozkaynak H. Exposure information in environmental health research: current opportunities and future directions for particulate matter, ozone, and toxic air pollutants. J Expo Sci Environ Epidemiol 2009; 19: S- Editor Shi ZF L- Editor O Neill M E- Editor Xiong L 6800 December 14, 2012 Volume 18 Issue 46

122 Online Submissions: doi: /wjg.v18.i World J Gastroenterol 2012 December 14; 18(46): ISSN (print) ISSN (online) 2012 Baishideng. All rights reserved. ORIGINAL ARTICLE Ileocecal valve dysfunction in small intestinal bacterial overgrowth: A pilot study Larry S Miller, Anil K Vegesna, Aiswerya Madanam Sampath, Shital Prabhu, Sesha Krishna Kotapati, Kian Makipour Larry S Miller, Anil K Vegesna, Department of Medicine, Section of Gastroenterology, Hofstra Northshore Long Island Jewish Hospital, School of Medicine at Hofstra University, Manhasset, NY 11030, United States Aiswerya Madanam Sampath, Shital Prabhu, Sesha Krishna Kotapati, Kian Makipour, Department of Medicine, Section of Gastroenterology, Temple University Hospital, Philadelphia, PA 19140, United States Author contributions: Miller LS and Vegesna AK contributed equally to concept and design, analysis and interpretation, drafting, critical revision and final approval of the article; Sampath AM, Prabhu S and Kotapati SK contributed to analysis and interpretation, drafting of the article; Makipour K contributed to concept and design of the article; all authors contributed for final approval of the article. Supported by National Institute of Health, No. 1RO1DK A2 Correspondence to: Larry S Miller, MD, Professor of Medicine, Department of Medicine, Section of Gastroenterology, Hofstra Northshore Long Island Jewish Hospital, School of Medicine at Hofstra University, 300 community drive, Manhasset, NY 11030, United States. [email protected] Telephone: Fax: Received: June 1, 2012 Revised: August 13, 2012 Accepted: August 25, 2012 Published online: December 14, 2012 Abstract AIM: To explore whether patients with a defective ileocecal valve (ICV)/cecal distension reflex have small intestinal bacterial overgrowth. METHODS: Using a colonoscope, under conscious sedation, the ICV was intubated and the colonoscope was placed within the terminal ileum (TI). A manometry catheter with 4 pressure channels, spaced 1 cm apart, was passed through the biopsy channel of the colonoscope into the TI. The colonoscope was slowly withdrawn from the TI while the manometry catheter was advanced. The catheter was placed across the ICV so that at least one pressure port was within the TI, ICV and the cecum respectively. Pressures were continuously measured during air insufflation into the cecum, under direct endoscopic visualization, in 19 volunteers. Air was insufflated to a maximum of 40 mmhg to prevent barotrauma. All subjects underwent lactulose breath testing one month after the colonoscopy. The results of the breath tests were compared with the results of the pressures within the ICV during air insufflation. RESULTS: Nineteen subjects underwent colonoscopy with measurements of the ICV pressures after intubation of the ICV with a colonoscope. Initial baseline readings showed no statistical difference in the pressures of the TI and ICV, between subjects with positive lactulose breath tests and normal lactulose breath tests. The average peak ICV pressure during air insufflation into the cecum in subjects with normal lactulose breath tests was significantly higher than cecal pressures during air insufflation (49.33 ± 7.99 mmhg vs ± 2.14 mmhg, P = ). The average percentage difference of the area under the pressure curve of the ICV from the cecum during air insufflations in subjects with normal lactulose breath tests was significantly higher (280.72% ± 43.29% vs 100% ± 0%, P = ). The average peak ICV pressure during air insufflation into the cecum in subjects with positive lactulose breath tests was not significantly different than cecal pressures during air insufflation ± 3.52 mmhg vs ± 3.39 mmhg. The average percentage difference of the area under the pressure curve of the ICV from the cecum during air insufflation was not significantly different % ± 7.96% vs 100% ± 0%. The total symptom score for subjects with normal lactulose breath tests and subjects with positive lactulose breath tests was not statistically different (13.30 ± 4.09 vs ± 6.58). The ICV peak pressures during air insufflations were significantly higher in subjects with normal lactulose breath tests than in subjects with positive lactulose breath tests (P = 0.005). The aver December 14, 2012 Volume 18 Issue 46

123 Miller LS et al. Ileocecal valve dysfunction in bacterial overgrowth age percent difference of the area under the pressure curve in the ICV from cecum was significantly higher in subjects with normal lactulose breath tests than in subjects with positive lactulose breath tests (P = ). Individuals with positive lactulose breath tests demonstrated symptom scores which were significantly higher for the following symptoms: not able to finish normal sized meal, feeling excessively full after meals, loss of appetite and bloating. CONCLUSION: Compared to normal, subjects with a positive lactulose breath test have a defective ICV cecal distension reflex. These subjects also more commonly have higher symptom scores Baishideng. All rights reserved. Key words: Ileocecal valve; Ileocecal sphincter; Cecum; Reflex; Lactulose breath test; Small bowel bacterial overgrowth Peer reviewer: Jørgen Valeur, MD, PhD, Department of Medicine, Institute of Medicine, Haukeland University Hospital, University of Bergen, NO-5021 Bergen, Norway Miller LS, Vegesna AK, Sampath AM, Prabhu S, Kotapati SK, Makipour K. Ileocecal valve dysfunction in small intestinal bacterial overgrowth: A pilot study. World J Gastroenterol 2012; 18(46): Available from: URL: com/ /full/v18/i46/6801.htm DOI: org/ /wjg.v18.i INTRODUCTION The literature regarding the ileocecal valve (ICV) and its relationship to small intestinal bacterial overgrowth (SIBO) is limited [1-3]. Surgical section of the ileocecal ligament in dog, a procedure that suppresses the ileocolonic angle and reduces sphincter competence, increases the amount of cecoileal reflux [4,5]. Surgical removal of the ICV maximizes reflux ultimately leading to bacterial overgrowth [6]. Dinning et al [1] using temporary, side diverting, defunctioning ileostomies, recorded ICV pressures in a configuration close to that surgically produced by Quigley et al [7] in dogs, hence allowing precise positioning of the manometric assembly across the ICV for prolonged periods. In these patients, a sustained pressure of about 10 mmhg is observed either using a pull-through or sleeve recording over a 4.8 cm distance. In fasted humans, phasic activity of the ICV, unrelated to motor activity of more oral or aboral zones, is observed for 35% of the recording time [1-3]. During these waves that occur at about four to eight waves per minute, the basal tone is doubled. On the contrary, ICV tone is reduced in humans and in dogs while prolonged propagated contractions are observed on the distal ileum [1,8]. In dogs and to a lesser extent in humans, ileal motor events propagate across the ICV into the colon. For instance, Quigley et al [8] show that 50% of ileal discrete clustered contractions and 76% of ileal prolonged propagated contractions continued propagating in the proximal canine colon. Colonic distension is followed consistently by contraction of the ICV in dogs and in humans. This enhanced motility of the ICV comprises simultaneously an increase in tone together with larger amplitude ICV phasic pressure waves [1-3] while the ileal contractions are unaffected [7]. Using a technique in which radioactive tracer is instilled in the cecum it was found that backward flow from the cecum to the ileum is episodic. In dog, the volume of the refluxate is low accounting for about 7% of the total radioactivity injected to the cecum [9]. This minimal reflux rate can be explained by the competence of the canine ICV towards reflux. Surgical section of the ileocecal ligament in dog, a procedure that suppresses the ileocolonic angle and reduces sphincter competence [4,5], increases the amount of cecoileal reflux to 44% of the total radioactivity [9]. In the current study, we aimed to explore whether patients with a positive lactulose breath test (indicative of SIBO) may have an incompetent ICV leading to reflux of colonic contents into the small intestine [10]. To test this hypothesis we measured pressures within the ICV during cecal distension and compared these pressures with the results of lactulose breath tests. MATERIALS AND METHODS Study design and conduct This research study was approved by the Temple University Institutional review board on 06/27/2007. This study is registered with clinicaltrials.gov ID: NCT Written Informed consent was given by all the participants prior to their inclusion in this study. All subjects filled out a symptom questionnaire on a scale of 0 to 5 with 0 being no symptom and 5 being very severe symptoms. Eligibility requirements Subjects undergoing screening colonoscopy were included in the study. Subjects who were currently on medications such as prokinetics, antibiotics and anticholinergics were excluded from the study. Subjects with history of gastroparesis, Crohn s disease, ulcerative colitis, diseases causing diarrhea, and long standing uncontrolled diabetes were also excluded from the study. Assessments and outcome measurements All subjects were sedated with Midazolam and Fentanyl. Colonic preparation was used in all subjects (polyethylene glycol preparation). A custom make water perfused manometry catheter (Mui Scientific, Mississauga, Canada) with 4 pressure measurement ports spaced one cm apart was passed through the biopsy channel of the colonoscope and placed across the ICV with at least one port in the terminal ileum and one port in the cecum (Figure 1). The simultaneous video endoscopy and pressure readings (30 Hz) were continuously recorded on a Medical 6802 December 14, 2012 Volume 18 Issue 46

124 Miller LS et al. Ileocecal valve dysfunction in bacterial overgrowth Endoscope Manometry catheter Ileum Cecum ICV Manometry catheter Cecum Colon Appendix Figure 1 Schematic of the terminal ileum, ileocecal valve and colon. It shows the colonoscope and manometry catheter passing through the biopsy channel of the colonoscope. The 4 channel manometry catheter is placed across the ileocecal valve so that at least 1 channel is in the cecum and 1 channel in the Ileum. Cecum ICV Manometry catheter Measurement Solar System (MMS, Dover, NH) (Figure 2). Video endoscopy was used to make sure that the catheter remained in the correct position during the entire study. If the catheter position changed during the study, the catheter was repositioned according to markings on the catheter. All the air was removed from the cecum by suction through the biopsy channel of the colonoscope and baseline measurements were taken. After the baseline measurements, air was slowly and continuously instilled into the cecum through the air/water channel of the colonoscope with the air insufflation setting, set at low on endoscopy processor (Evis, Exera Ⅱ, CLV-180, Olympus America Inc, Center Valley, PA). The peak pressures were measured in all the channels at a time when the pressure in ICV reached its peak between the start of air insufflation and the end of air insufflation. Only studies in which the video playback showed the catheter to be in the correct position were used for analysis. A threshold cecal pressure of 40 mmhg was used to avoid barotrauma. If the pressure exceeded 40 mmhg the air flow into the cecum was stopped. In vivo and cadaveric animal experiments have yielded data regarding intraluminal pressure that can lead to rupture of the colon [11-16]. An adult human cadaveric cecum exposed to less than 40 mmhg of intraluminal pressure generally does not rupture, but cecum exposed to more than 150 mmhg of pressure always ruptures [11-16]. The pressures needed for perforation of the right colon and cecum are lower than those needed for perforation of the sigmoid and descending colon [12,13]. It is estimated that the upper limit of safe intraluminal human colonic pressure is 80 mmhg, because perforation can occur at pressures greater than 140 mmhg [17]. The area under the pressure curve was measured within all the channels, from the time of the beginning of the upslope of the pressures within the cecum, after the start of air insufflation, to the time when the ICV reached its peak pressure, before the end of air insufflation. All patients returned one month after the colonoscopy for a lactulose breath test using Breath Tracker Digital Microlyzer, QuinTron Instrument Company, Figure 2 Representative trace of a simultaneous 4 channel manometry and video endoscopy. A: The response of the ileocecal valve during cecal distention by air insufflation in a subject with a normal lactulose breath test. Note the increase in pressure within the ileocecal valve greater than in the cecum and ileum during distension of the cecum; B: The response of the ileocecal valve during cecal distention by air insufflation in a subject with positive lactulose breath test. Note the similar pressures within all the manometry ports (a common cavity effect). ICV: Ileocecal valve. Milwaukee, WI, United States. All subjects presented for the lactulose breath test after an overnight fast. The subjects were given 10 g of lactulose in 120 ml of water to drink and instructed to breathe into a collection bag every 15 min for 3 h after a sample of their breath was collected at baseline. All breath samples were end-expiratory and analyzed immediately. The concentrations of breath hydrogen and methane were measured in parts per million (ppm). The measurements were then graphed and analyzed [18-21]. Graphs of methane and hydrogen concentration were plotted against time. A positive lactulose breath test was defined as a double peak of hydrogen or a combination of both hydrogen and methane above 20 ppm within the first 2 h. All subjects were given a gastrointestinal questionnaire which evaluated the following habitual symptoms: nausea, vomiting, stomach fullness, not able to finish normal sized meal, feeling excessively full after meals, loss of appetite, bloating, stomach or belly visibly larger, upper abdominal pain, upper abdominal discomfort, lower abdominal pain, lower abdominal discomfort, diarrhea, gas from above, and gas from below. Statistical analysis All pressures were calculated relative to baseline colonic 6803 December 14, 2012 Volume 18 Issue 46

125 Miller LS et al. Ileocecal valve dysfunction in bacterial overgrowth A Pressure (mmhg) B Pressure (mmhg) pressures. The analysis of pressures was performed using an unpaired Student s t-test, with a two tail distribution and equal variance. The questionnaires were evaluated using non-parametric tests (Mann Whitney U-test). The analysis was performed in a blinded manner. A Spearman correlation was used to compare ICV pressures to hydrogen and methane excretion. RESULTS Pressure changes in normal subjects Ileum ICV Cecum 1 Cecum 2 Baseline Peak Post Pressure changes in subjects with SIBO Ileum ICV Cecum 1 Cecum 2 Baseline Peak Post Figure 3 Average pressure changes (baseline, peak and post) in the colon, ileocecal valve and terminal ileum during and after air insufflation. A: The average response of the ileocecal valve during cecal distention by air insufflation in subjects with normal lactulose breath tests; B: The average response of the ileocecal valve during cecal distention by air insufflation in subjects with positive lactulose breath tests. ICV: Ileocecal valve; SIBO: Small intestinal bacterial overgrowth. Nineteen subjects (56.4 years, 7 male and 12 female) underwent colonoscopy with measurements of the ICV pressures after intubation of the ICV with a colonoscope. Cecal pressures before air insufflation were considered as baseline pressures and all the pressures noted were relative to cecal pressures. If the recorded pressures were less than the baseline cecal pressures, than they were considered negative. The resting pressures in the subjects with normal lactulose breath tests were ± 1.44 mmhg and 1.18 ± 0.73 mmhg in TI and ICV respectively. The resting pressures in the subjects with positive lactulose breath tests were ± 1.07 mmhg and 1.70 ± 2.49 mmhg in TI and ICV respectively. Initial readings showed no statistical difference in the resting pressures of the TI or ICV between subjects with positive lactulose breath tests and normal lactulose breath tests. A Parts per million B Parts per million Average breath test in normal subjects H2 readings Methane readings t /min Average breath test in subjects with SIBO H2 readings Methane readings t /min Figure 4 Average lactulose breath test results. A: Normal lactulose breath tests; B: Positive lactulose breath tests. ICV: Ileocecal valve; SIBO: Small intestinal bacterial overgrowth. Subjects with normal lactulose breath tests Ten subjects (5 male and 5 female), (age: 55.8 ± 3.05 years, weight: ± 7.29 kg). The average dose of Midazolam and Fentanyl per kg was 0.06 mg and 0.73 mg. The average peak ICV pressure during air insufflation into the cecum in subjects with normal lactulose breath tests was significantly higher than cecal pressures during air insufflation (49.33 ±7.99 mmhg vs ± 2.14 mmhg, P = ). The average percentage difference of the area under the pressure curve of the ICV from the cecum during air insufflations was significant higher (280.72% ± 43.29% vs 100% (P = ). The average symptom score for all subjects with normal lactulose breath tests was ± Subjects with positive lactulose breath tests Nine subjects (2 male and 7 female), (age: 57 ± 4.92 years, weight: ± 6.17 kg). The average dose of Midazolam and Fentanyl per kg was 0.08 mg and 0.50 mg. The average peak ICV pressure during air insufflation into the cecum in subjects with positive lactulose breath tests was not significantly different than cecal pressures during air insufflation (21.23 ± 3.52 mmhg vs ± 3.39 mmhg). The average percentage difference of the area under the pressure curve of the ICV from the cecum during air insufflation was not significantly different % ± 7.96% vs 100%. The average total symptom score for all subjects with positive lactulose breath tests 6804 December 14, 2012 Volume 18 Issue 46

126 Miller LS et al. Ileocecal valve dysfunction in bacterial overgrowth Percent change from cecum Average percent change of area under the curve Normal subjects Subjects with SIBO Ileum ICV Cecum 1 Cecum 2 Figure 5 Average percent change of the area under the pressure curve during air insufflation into the cecum, in normal subjects (normal lactulose breath tests) and in subjects with small bowel bacterial over growth (positive lactulose breath tests), with respect to the cecum. ICV: Ileocecal valve; SIBO: Small intestinal bacterial overgrowth. was ± In some prior studies methane seems to influence smooth muscle function [22]. Analyzing the breath test results, 3 of the subjects were methane producers and one of these subjects was also positive for hydrogen. Six subjects were hydrogen producers. There was no significant difference in the peak ICV pressures between methane producers and hydrogen producers (23.7 ± 0.67 mmhg vs 26.8 ± 2.48 mmhg). There was only a weak correlation between the pressure measurements and the hydrogen excretion (r = 0.19) and the pressure measurements and the methane excretion (r = 0.25). Comparison of subjects with normal lactulose breath tests to subjects with positive lactulose breath tests There is no significant difference in age and weight between subjects with normal lactulose breath test and subjects with positive lactulose breath test. There is no significant difference in the drug dose/kg of Midazolam and Fentanyl between the two groups. The ICV peak pressures during air insufflations were significantly higher in the subjects with normal lactulose breath tests than in the subjects with positive lactulose breath tests (P = 0.005) (Figures 3 and 4). The average percent difference of the area under the pressure curve within the ICV from cecum during air insufflations was significantly higher in subjects with normal lactulose breath tests than in subjects with positive lactulose breath tests (P = ) (Figure 5). The cecal peak pressures during air insufflations were not significantly different in the subjects with normal lactulose breath tests compared to the subjects with positive lactulose breath tests. The total symptom scores were not significantly different in subjects with normal lactulose breath tests than in subjects with positive lactulose breath tests. However, significantly higher symptom scores were observed in the subjects with positive lactulose breath tests in the individual symptoms of: not able to finish normal sized meal (P = 0.036), feeling excessively full after meals (P = 0.029), loss of appetite (P = 0.018) and bloating (P = ). DISCUSSION In this study we explored weather patients with positive lactulose breath tests have an incompetent ICV. We tested this hypothesis by measuring ICV pressures during cecal distension in subjects with positive and negative lactulose breath tests. In designing this study consideration was given to using a sleeve sensor to measure ICV pressures. However, this was rejected in favor of a four port pressure transducer because a sleeve sensor would have measured the peak pressure recorded and would not have been able to distinguish between the terminal ileum, the ICV or the cecum. Consideration was also given to using a barostat to distend the cecum. However, it was decided that a barostat would be very cumbersome to use during routine colonoscopy and would not distend the cecum in the correct position to reproducibly increase ICV pressure. We felt that insufflation of air, into the cecum, was closer to the actual physiology of gas being produced in the colon causing distension of the cecum. We did not use the same volume of gas in each subject. Rather we used a cutoff cecal pressure. We did this for safety purposes since we did not want to go above a certain threshold pressure and risk barotrauma to the subjects. A cutoff pressure of 40 mmhg was used during cecal distension. Finally consideration was given to collection of fluid from the small intestine to assay for SIBO. However, the colonoscopies were performed after a bowel preparation, which flushes bacteria out of the small intestine and this would have resulted in false negative results. In addition the colonoscope needs to pass through the colon before it can enter the small intestine and this could have contaminated the colonoscope with bacteria and could have resulted in false positive results for SIBO. Yu et al [23] have recently published an important study in which they combined oro-cecal scintigraphy and lactulose hydrogen breath testing demonstrating that breath testing may detect oro-cecal transit, not small intestinal bacterial overgrowth in patients with irritable bowel syndrome (IBS). Thus the lactulose hydrogen breath test may measure small intestinal transit rather than SIBO in IBS-patients. However, the patient population in that study was different than in the current study and the lactulose breath test is still used around the world to diagnose SIBO. Since the results of the lactulose breath test are dependent on motility within the stomach, the small intestine and the colon, we attempted to control for variability in motility between subjects by eliminating patients with known motility disorders, such as gastroparesis and small bowel motility disorders. We eliminated subjects who were taking any medications that would affect motility, such as prokinetics, anticholinergics and narcotics. The dosages of medications given during the colonoscopies were not significantly different between 6805 December 14, 2012 Volume 18 Issue 46

127 Miller LS et al. Ileocecal valve dysfunction in bacterial overgrowth the two groups, so as not to influence motility during the colonoscopy. We found that the baseline pressure measurements showed no difference in the pressures of the TI, and ICV between the two groups. These findings are similar to the findings of Quigley et al [24], because ileocecal muscle behaves as a sphincter, we were surprised by the absence of clear tonic pressures across the ICV. However, when tested for the ICV cecal distension reflex using air insufflation into the cecum, we recorded an increase in the pressure within the ICV in subjects with normal lactulose breath tests which were significantly higher than in subjects with positive lactulose breath test. Subjects, with a positive lactulose breath test commonly had a common cavity effect (equalization of the pressures across all of the compartments) during cecal air insufflation. When the lactulose breath test is evaluated against the gold standard of jejunal aspiration with bacterial culturing for the diagnosis of SIBO the sensitivity and specificity of the breath test is much lower. We suggest a number of potential explanations for these results. First, perhaps not all SIBO is due to an incompetent ICV. For example, in cases of scleroderma in which there are diverticulum in the small intestine it has been suggested that SIBO is due to bacteria growing within the diverticulum. Second and probably more important is the fact that these cultures were taken from the jejunum which is a great distance from the ICV and TI. Reflux of colonic contents would not be expected to wash backwards beyond the ileum due to the propulsive motion of peristalsis within the small intestine, which is presumably normal in these subjects. Normal peristalsis should keep the jejunal bacterial count relatively low even when refluxing colonic contents into the ileum [14]. Although we did not design this study to determine if subjects undergoing screening colonoscopy with a positive lactulose breath test have gastrointestinal (GI) symptoms, we did find that many of the subjects in our study had GI symptoms. While these symptoms were more common in the group with positive lactulose breath tests, they were not uncommon in the group with negative lactulose breath tests and we found that there was no significant difference in the overall symptom scores between the two groups. On the other hand, significantly higher symptom scores were demonstrated in the subjects with positive lactulose breath tests for the individual symptoms of, not able to finish a normal sized meal, feeling excessively full after meals, loss of appetite and bloating. The Initial studies evaluating the treatment of IBS patients with poorly absorbed antibiotics, were based on the hypothesis that a significant proportion of these patients actually had occult SIBO [25]. A number of studies have suggested that there are beneficial effects when poorly absorbed antibiotics are used to treat patients with IBS [26,27]. Initial studies reported the presence of SIBO in up to 80% of IBS patients, on the basis of a rapid rise in breath hydrogen during lactulose breath testing. In a recent issue of the New England Journal of Medicine, Pimentel et al [28] reported the results of two large, double-blind, placebo-controlled trials of rifaximin, in patients with IBS without constipation. They found that patients in the group treated with rifaximin had adequate relief of IBS symptoms or bloating. Similarly significant results were obtained in an analysis of relief of symptoms during the 10-wk period after the end of the double-blind treatment phase. Harder et al [29] showed that gas is less well tolerated in the small intestine than in the large bowel. Based on these studies the most likely mode of action of rifaximin is a reduction in overall small bowel bacterial load or a decrease in colonic flora. This may lead to decreased bacterial fermentation and less bloating, possibly in combination with decreased secretion of bacterial products or host responses to bacterial products that contribute to the generation of symptoms. In the future, we plan to perform a study on subjects with documented irritable bowel syndrome, by the Rome Ⅲ criteria. In order to determine if a subgroup of patients with IBS symptoms is due to a defective ileocecal valve/cecal distension reflex with reflux of fecal flora or gas into the small intestine. The results of our study suggest that some GI symptoms may be due to a defective ICV cecal distension reflex which either allows gas from the colon to distend the small intestine or lead to reflux of colonic contents into the small intestine, the colonization of the small intestine with fecal flora and the development of SIBO. We hypothesize that even though a course of nonabsorbable antibiotics may clear the small intestine or the colon of colonic flora, the symptomatic effect may not be durable because the underlying defect, the defective ICV cecal distension reflex, is still present and colonic flora or colonic gas may eventually reflux back into the small intestine. The results of the current study suggest that a restoration of the demonstrated dysfunction (pharmacological, endoscopic or surgical) by preventing coloileal reflux may have a more durable effect than antibiotic therapy. Indeed, prevention of coloileal reflux by constructing an artificial ileocolonic valve was suggested by Kellogg in Bakkevold [30] demonstrated that the nipple valve anastomosis may prevent recurrence of Crohn s disease, after ileocolic resection. Pimentel et al [28] suggested that some IBS like symptoms, afflicting many millions of patients, might be due to SIBO and might respond to antibiotic treatment usually used to treat SIBO. In summary, the current study demonstrates that subjects with a positive lactulose breath test have a defective ICV cecal distension reflex. In addition it demonstrates that subjects with a defective ICV cecal distension reflex more commonly have certain GI symptoms. We propose that this pathophysiologic mechanism (defective ICV/cecal distension reflex) which may cause SIBO and IBS like symptoms due to reflux of fecal flora and the production of gas within the small intestine and/or the production of gas within the colon, with reflux of the gas into the small intestine December 14, 2012 Volume 18 Issue 46

128 Miller LS et al. Ileocecal valve dysfunction in bacterial overgrowth ACKNOWLEDGEMENTS We would like to thank Dilek Yarar for her contribution for this project. COMMENTS Background Small intestinal bacterial overgrowth (SIBO) refers to a condition in which abnormally large numbers of bacteria are present in the small intestine and the types of bacteria in the small intestine resemble the bacteria of the colon. Irritable bowel syndrome (IBS) is a disorder that leads to abdominal pain and cramping, changes in bowel movements, and other symptoms. SIBO and some forms of IBS may be due to the distension of the small intestine with gas produced by bacteria within the small intestine and/or colon. Recent studies strongly suggest that some IBS like symptoms, afflicting many millions of patients, might be due to small intestinal bacterial overgrowth SIBO. Research frontiers A percentage of patients with IBS like symptoms have relief of their symptoms when they are treated with non-absorbable antibiotics which suppress the intestinal microbiota. It is thought that suppression of the microflora, within the colon or the small intestine is the mechanism of this therapy. Innovations and breakthroughs The initial studies evaluating the treatment of IBS patients with poorly absorbed antibiotics, were based on the hypothesis that a significant proportion of these patients actually had occult SIBO. A number of studies have suggested that there are beneficial effects when poorly absorbed antibiotics are used to treat patients with IBS. Initial studies reported the presence of SIBO in up to 80% of IBS patients, on the basis of a rapid rise in breath hydrogen during lactulose breath testing. The current study is the first study to evaluate the role of ileocecal valve (ICV)/cecal distention reflex in patients suffering with small bowel bacterial overgrowth. Applications These findings imply that repair of the ICV (pharmacologic, endoscopic or surgical) or restoration of the ICV/cecal distension reflex may be used to treat patients with SIBO and in patients with IBS like symptoms in the future. Terminology The ileocecal valve is a sphincter muscle situated at the junction of the ileum (last portion of your small intestine) and the colon (first portion of your large intestine). Its function is to allow digested food materials to pass from the small intestine into your large intestine. The ileocecal valve also blocks these waste materials from backing back up into your small intestine. It is intended to be a one-way valve, only opening up to allow processed foods to pass through. Peer review The authors examined the ICV/cecal distention reflex in patients with positive lactulose breath test and in subjects with negative lactulose breath test. The authors found that subjects with a positive lactulose breath test have a defective ICV/cecal distension reflex. This defective ICV/cecal distension reflex may cause SIBO and IBS like symptoms due to reflux of fecal flora and the production of gas within the small intestine and/or the production of gas within the colon, with reflux of the gas into the small intestine. REFERENCES 1 Dinning PG, Bampton PA, Kennedy ML, Kajimoto T, Lubowski DZ, de Carle DJ, Cook IJ. Basal pressure patterns and reflexive motor responses in the human ileocolonic junction. Am J Physiol 1999; 276: G331-G340 2 Kajimoto T, Dinning PG, Gibb DB, de Carle DJ, Cook IJ. Neurogenic pathways mediating ascending and descending reflexes at the porcine ileocolonic junction. Neurogastroenterol Motil 2000; 12: Rothstein RD, DeRiso J, Ouyang A. Ileocecal sphincter contraction to colonic distension: a tachykinin-mediated spinal reflex. Am J Physiol 1990; 258: G585-G590 4 Köhler LW, Heddle R, Miedema BW, Phillips SF, Kelly KA. Response of canine ileocolonic sphincter to intraluminal acetic acid and colonic distension. Dig Dis Sci 1991; 36: Kumar D, Phillips SF. The contribution of external ligamentous attachments to function of the ileocecal junction. Dis Colon Rectum 1987; 30: Gazet RJ, Jarrett J. The ileocaeco-colic sphincter. studies in vitro in man, monkey, cat, and dog. Br J Surg 1964; 51: Quigley EM, Phillips SF, Cranley B, Taylor BM, Dent J. Tone of canine ileocolonic junction: topography and response to phasic contractions. Am J Physiol 1985; 249: G350-G357 8 Quigley EM, Phillips SF, Dent J. Distinctive patterns of interdigestive motility at the canine ileocolonic junction. Gastroenterology 1984; 87: Kumar D, Phillips SF, Brown ML. Reflux from ileum to colon in the dog. Role of external ligamentous attachments. Dig Dis Sci 1988; 33: King CE, Toskes PP. Small intestine bacterial overgrowth. Gastroenterology 1979; 76: Burt AV. Pneumatic rupture of the intestinal canal: With experimental data showing the mechanism of perforation and the pressure required. Archives of Surgery 1931; 22: Harned RK, Consigny PM, Cooper NB, Williams SM, Woltjen AJ. Barium enema examination following biopsy of the rectum or colon. Radiology 1982; 145: Kozarek RA, Earnest DL, Silverstein ME, Smith RG. Airpressure-induced colon injury during diagnostic colonoscopy. Gastroenterology 1980; 78: Luchette FA, Doerr RJ, Kelly K, Kulaylat M, Stephan RM, Hassett JM. Colonoscopic impaction in left colon strictures resulting in right colon pneumatic perforation. Surg Endosc 1992; 6: Noveroske RJ. Intracolonic pressures during barium enema examination. Am J Roentgenol Radium Ther Nucl Med 1964; 91: Sandhu KS, Cohen H. Cecal perforation following fiberoptic flexible sigmoidoscopy. Am J Med 1987; 82: Tzelepis GE, Nasiff L, McCool FD, Hammond J. Transmission of pressure within the abdomen. J Appl Physiol 1996; 81: Parodi A, Capurso G, Perri F, Cuoco L, Lauritano EC. H2- breath testing for small-intestinal bacterial overgrowth. Aliment Pharmacol Ther 2009; 29 (Suppl 1): Bures J, Cyrany J, Kohoutova D, Förstl M, Rejchrt S, Kvetina J, Vorisek V, Kopacova M. Small intestinal bacterial overgrowth syndrome. World J Gastroenterol 2010; 16: Kerlin P, Wong L. Breath hydrogen testing in bacterial overgrowth of the small intestine. Gastroenterology 1988; 95: Riordan SM, McIver CJ, Walker BM, Duncombe VM, Bolin TD, Thomas MC. The lactulose breath hydrogen test and small intestinal bacterial overgrowth. Am J Gastroenterol 1996; 91: Pimentel M, Lin HC, Enayati P, van den Burg B, Lee HR, Chen JH, Park S, Kong Y, Conklin J. Methane, a gas produced by enteric bacteria, slows intestinal transit and augments small intestinal contractile activity. Am J Physiol Gastrointest Liver Physiol 2006; 290: G1089-G Yu D, Cheeseman F, Vanner S. Combined oro-caecal scintigraphy and lactulose hydrogen breath testing demonstrate that breath testing detects oro-caecal transit, not small intestinal bacterial overgrowth in patients with IBS. Gut 2011; 60: Quigley EM, Borody TJ, Phillips SF, Wienbeck M, Tucker RL, Haddad A. Motility of the terminal ileum and ileocecal sphincter in healthy humans. Gastroenterology 1984; 87: Kerckhoffs AP, Visser MR, Samsom M, van der Rest ME, de Vogel J, Harmsen W, Akkermans LM. Critical evaluation of diagnosing bacterial overgrowth in the proximal small 6807 December 14, 2012 Volume 18 Issue 46

129 Miller LS et al. Ileocecal valve dysfunction in bacterial overgrowth intestine. J Clin Gastroenterol 2008; 42: Koo HL, DuPont HL. Rifaximin: a unique gastrointestinalselective antibiotic for enteric diseases. Curr Opin Gastroenterol 2010; 26: Singh VV, Toskes PP. Small bowel bacterial overgrowth: presentation, diagnosis, and treatment. Curr Gastroenterol Rep 2003; 5: Pimentel M, Lembo A, Chey WD, Zakko S, Ringel Y, Yu J, Mareya SM, Shaw AL, Bortey E, Forbes WP. Rifaximin therapy for patients with irritable bowel syndrome without constipation. N Engl J Med 2011; 364: Harder H, Serra J, Azpiroz F, Passos MC, Aguadé S, Malagelada JR. Intestinal gas distribution determines abdominal symptoms. Gut 2003; 52: Bakkevold KE. Nipple valve anastomosis for preventing recurrence of Crohn disease in the neoterminal ileum after ileocolic resection. A prospective pilot study. Scand J Gastroenterol 2000; 35: S- Editor Gou SX L- Editor Kerr C E- Editor Lu YJ 6808 December 14, 2012 Volume 18 Issue 46

130 Online Submissions: doi: /wjg.v18.i World J Gastroenterol 2012 December 14; 18(46): ISSN (print) ISSN (online) 2012 Baishideng. All rights reserved. ORIGINAL ARTICLE Schizandra arisanensis extract attenuates cytokinemediated cytotoxicity in insulin-secreting cells Yi-Shin Hsu, Yao-Haur Kuo, Hui-Ling Cheng, Peter R Flatt, Hui-Kang Liu Yi-Shin Hsu, Yao-Haur Kuo, Hui-Kang Liu, Division of Herbal Drugs and Natural Products, National Research Institute of Chinese Medicine, Taipei 112, Taiwan, China Yao-Haur Kuo, Hui-Ling Cheng, Graduate Institute of Integrated Medicine, China Medical University, Taichung 404, Taiwan, China Peter R Flatt, School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland BT52 1SA, United Kingdom Author contributions: Hsu YS and Liu HK were responsible for all bio-assays, research design, data analysis and manuscript writing; Kuo YH and Cheng HL were responsible for crude extract preparation, compound preparation and chemical analysis, and part of the manuscript writing; Flatt PR contributed to manuscript drafting and discussions on the results. Supported by National Science Council, No. NSC B , No. NSC B MY2; and National Research Institute of Chinese Medicine, No. NRICM95-DHM-04 Correspondence to: Dr. Hui-Kang Liu, Division of Herbal Drugs and Natural Products, National Research Institute of Chinese Medicine, Li-Nong Street, Section 2, Taipei 112, Taiwan, China. [email protected] Telephone: Fax: Received: July 10, 2012 Revised: September 18, 2012 Accepted: September 22, 2012 Published online: December 14, 2012 Abstract AIM: To explore the bioactivity of an ethanolic extract of Schizandra arisanensis (SA-Et) and isolated constituents against interleukin-1β and interferon-γ-mediated β cell death and abolition of insulin secretion. METHODS: By employing BRIN-BD11 cells, the effects of SA-Et administration on cytokine-mediated cell death and abolition of insulin secretion were evaluated by a viability assay, cell cycle analysis, and insulin assay. The associated gene and protein expressions were also measured. In addition, the bioactivities of several peak compounds collected from the SA-Et were tested against cytokine-mediated β cell death. RESULTS: Our results revealed that SA-Et dose-dependently ameliorated cytokine-mediated b cell death and apoptosis. Instead of suppressing inducible nitric oxide synthase/nitric oxide cascade or p38mapk activity, suppression of stress-activated protein kinase/c-jun NH2-terminal kinase activity appeared to be the target for SA-Et against the cytokine mix. In addition, SA-Et provided some insulinotropic effects which re-activated the abolished insulin exocytosis in cytokine-treated BRIN-BD11 cells. Finally, schiarisanrin A and B isolated from the SA-Et showed a dose-dependent protective effect against cytokine-mediated β cell death. CONCLUSION: This is the first report on SA-Et ameliorating cytokine-mediated b cell death and dysfunction via anti-apoptotic and insulinotropic actions Baishideng. All rights reserved. Key words: Schizandra arisanensis ; C19 homolignans; Type 1 diabetes; Insulin-secreting cells; Interleukin-1β; Interferon-γ Peer reviewers: Fabrizio Montecucco, MD, Assistant Professor, Division of Cardiology, Department of Internal Medicine, University of Geneva, Avenue de la Roseraie 64, 1211 Geneva, Switzerland; Qiuwei Pan, PhD, Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Room L-462 s Gravendijkwal 230, 3015 CE Rotterdam, Netherlands Hsu YS, Kuo YH, Cheng HL, Flatt PR, Liu HK. Schizandra arisanensis extract attenuates cytokine-mediated cytotoxicity in insulin-secreting cells. World J Gastroenterol 2012; 18(46): Available from: URL: com/ /full/v18/i46/6809.htm DOI: org/ /wjg.v18.i INTRODUCTION The onset of type 1 diabetes occurs when approximately 60%-90% of insulin secreting cells are lost and/or are dysfunctional due to b-cell directed autoimmunity [1] December 14, 2012 Volume 18 Issue 46

131 Hsu YS et al. b-cell protective effects of SA-Et During the progression of autoimmunity, heavy infiltrations of mononuclear cells lead to substantial damage to b cells by generating locally high concentrations of proinflammatory cytokines, perforin, and FasL-Fas interactions within the micro-environment of islets [2,3]. As a result, deleterious outcomes include excessive proinsulin secretion, the abolition of glucose-induced insulin secretion, and ultimately b cell death [4]. Currently, scientists have developed a wide range of approaches to prevent or intervene in type 1 diabetes by stimulating b-cell proliferation, attenuating the cytotoxic effects of inflammatory agents, or modulating autoimmune response, and so on [1]. While many clinical trials are ongoing, the application of herbal medicine remains to be fully explored. In cell culture systems, exposing b cells to an interleukin (IL)-1β and interferon (IFN)-γ mixture can mimic the consequences of an immune attack in type 1 diabetes, which affects pro-insulin secretion [5] and induces β cell death [6]. By employing such a platform, potential β cell protective herbal material can be identified. Schizandra arisanensis (SA-Et) is one of the schizandraceous plants from Taiwan. This plant also contains various C18 dibenzocylcooctadiene lignans and C19 homolignans [7,8]. The indications for this herb in traditional Chinese medicine include diabetes, hepatitis, immunomodulation, and cancer [7,9-11]. Therefore, in the present study, we investigated the potential b cell protective bioactivity of the ethanol extract of SA-Et, including its isolated constituents against cytokine-mediated β cell cytotoxicity and dysfunction. MATERIALS AND METHODS Plant material and reagents Stems of SA-Et were collected and authenticated by Dr. Kuo YH in October 2005 in Chiayi County, Taiwan. A voucher specimen (No. NRICM ) was deposited at the National Research Institute of Chinese Medicine. The following were used as positive controls in the various biological assays performed; recombinant cytokines (IL-1β and IFN-γ; PeproTech, NJ, United States); epigallocatachin gallate (EGCG; Fluka, MO, United States); nitro-l-arginine methyl ester (L-NAME), glucose, KCl and CaCl2 (Sigma-Aldrich Corp., MO, United States). These reagents were obtained at the highest purity available (> 97%) from the suppliers indicated. Preparation of the ethanol extract of SA-Et Dried stems of SA-Et were ground and then extracted with 95% (v/v) EtOH at 45 three times for 48 h each time. Under reduced pressure, the combined ethanol extracts were concentrated to an ethanolic extract residue of SA-Et. DMSO was employed as the dissolving reagent, and aliquots were prepared and stored at -20 before the biological assays were carried out. High-performance liquid chromatographic analysis of the SA-Et A filtered volume (100 μl) of the SA-Et solution (1.0 mg/ml in methanol) was prepared and injected into a reverse-phase high-performance liquid chromatography (HPLC) system. The HPLC analysis was performed using a Waters HPLC (cont 600 Pump, 996 photodiode array detector, 600 controller, and 717 plus autosampler; Milford, Massachusetts, United States), using a reversephase RP-18 column (4.6 mm 250 mm i.d.). The solvent system used was a gradient of water and CH3CN. The gradient system was as follows: 0-60 min, 45%-60% CH3CN, and min, 60%-75% CH3CN; with elution performed at a solvent flow rate of 1 ml/min. The detection chromatogram was recorded at 215 nm. Isolation of C19 homolignan and C18 lignan compounds Peak compounds including schiarisanrin A (peak 5), schiarisanrin B (peak 1), schiarisanrin C (peak 4), schiarisanrin E (peak 3), and macelignan (peak 6) were obtained from the SA-Et followed by the procedures described below. Isolation of taiwanschirin A (peak 2) was based on a previous method [12]. Five grams of SA-Et was partitioned with water and n-hexane, to give the n-hexane residue (SA-Et-H). Further separations of SA-Et-H (100 mg) were performed on a Waters HPLC, using a preparative Cosmosil 5C18 AR-Ⅱ column (250 mm 10 mm i.d.; Nacalai Tesque, Kyoto, Japan), employing a gradient system [water (A) and CH3CN (B), mobile phase was as follows: 0-2 min, 45%-50% (B); 2-20 min, 50%-55% (B), min, 55%-60% (B), min, 60%-70% (B), and min, 70%-100% (B)], at a flow rate of 3 ml/min, under 215 nm UV, to yield schiarisanrin A (4.6 mg), schiarisanrin B (4.1 mg), schiarisanrin C (5.6 mg), schiarisanrin E (6.3 mg), macelignan (11.5 mg), and schizanrin A (3.6 mg), which were identified by comparison with authentic samples and reported spectroscopic data (UR, infrared, and nuclear magnetic resonance) in the literature [8,12]. Their purity was over 75% as analyzed by HPLC. Cytotoxicity test BRIN-BD11 cells were routinely cultured in RPMI-1640 containing 10% (v/v) fetal bovine serum and 2 g/l glucose [13]. For the cytotoxicity test, BRIN-BD11 cells were seeded into 24-well plates at the density of cells/ well. After overnight attachment, cells were incubated with cytokines according to the figure legends. The viability at the end of treatment was measured by a neutral red assay as described previously [14]. The absorbance under control conditions was set to 100% viability. Cell cycle analysis At the end of treatment, cells were trypsinized and collected by centrifugation. Following a phosphate-buffered saline (PBS) wash, cell pellets were resuspended in ethanol overnight at -20. Fixed cells were then washed with PBS before being resuspended in 1% (v/v) Triton X-100 and incubated for 15 min. The cells were then resuspended in propidium iodide (PI) staining solution, consisting of 20 μg/ml PI, 50 μg/ml RNase A, and % (v/v) Triton X-100 in PBS, and incubated at 4 for 30 min 6810 December 14, 2012 Volume 18 Issue 46

132 Hsu YS et al. b-cell protective effects of SA-Et Table 1 Sequence of primer sets for conventional reverse transcription-polymerase chain reaction b-actin Forward Reverse Glucokinase Forward Reverse Insulin Forward Reverse inos Forward Reverse inos: inducible nitric oxide synthase. 5 -CGTAAAGACCTCTATGCCAA-3 5 -AGCCATGCCAAATGTGTCAT-3 5 -AAGGGAACTACATCGTAGGA-3 5 -CATTGGCGGTCTTCATAGTA-3 5 -TGCCCAGGCTTTTGTCAAACAGCACCTT-3 5 -CTCCAGTGCCAAGGTCTGAA-3 5 -TTTTCACGACACCCTTCACC-3 5 -GACCTGATGTTGCCACTGTTAG-3 with protection from light. Finally, the cells were passed through a mesh prior to cell cycle analysis by employing FACSan (BD Bioscience, San Jose, California, United States). Western blot analysis This procedure followed a previous methodology [15]. In brief, cells were washed with ice-cold PBS and scraped into ice-cold lysis buffer. Cell debris was removed by centrifugation. Equal amounts of protein (40 g) were subjected to separation on sodium dodecylsulfate 10% polyacrylamide gels. Following transfer to nitrocellulose membranes, blots were blocked with 5% (w/v) non-fat milk in Tris-buffered saline containing 0.1% (v/v) Tween 20 for 1 h, and incubated with primary antibodies at 4 overnight prior to incubation for 1 h at room temperature with the secondary antibody. Finally, results were visualized after development of films with the aid of an enhanced chemiluminescence kit (Amersham Biosciences, Uppsala, Sweden). Measurement of gene expression Total RNA was extracted using the TRI-reagent according to the manufacturer s instructions. Total RNA (1 μg) was reverse-transcribed to generate templates. Complementary (c) DNA at 50 ng was employed for the polymerase chain reaction (PCR). The sequences of primers are listed in Table 1. The annealing temperatures for amplification of b-actin (57 ), glucokinase (57 ), insulin (52 ), and inducible nitric oxide synthase (inos) (55 ) were employed to generate respective sequences of 349 bp, 130 bp, 187 bp, and 441 bp. Once the reaction was complete, PCR products were separated by gel electrophoresis, visualized, photographed using a digital camera, and quantified with Genetools 3.06 (Syngene, Cambridge, United Kingdom). When inos mrna was determined by real-time reverse transcription-pcr, 1 μg of total RNA was reverse transcribed into cdna using the RevertAid First Strand cdna Synthesis Kit (Fermentas, Maryland, United States) before being applied to the StepOnePlus Real-Time PCR System (Applied Biosystems, Warrington, United Kingdom). Each reaction was carried out based on the description of the TaqMan Gene Expression Assay kit with pre-designed probes for inos (Rn _m1) and β-actin (Rn _m1). Data were ct values (i.e., cycle number where logarithmic PCR plots cross a calculated threshold line). For comparative quantification, ct values were firstly calculated using the formula: ct = (ct of inos) - (ct of β-actin). Then the ct value was determined using the following formula: ct = ( ct of each sample) - (average ct of control samples). Fold increase in inos level under each condition was presented as 2 - ct. Acute insulin-secretion test The acute insulin-secretion test was as previously described [14]. In brief, the culture medium was discarded and the cells were washed twice with 1 ml Krebs-Ringer bicarbonate (KRB) buffer. The cells were then pre-incubated in KRB for 40 min at 37. Pre-incubation buffer was then poured off followed by the addition of 1 ml of test medium per well at a constant rate. After 20 min, the test medium was collected at a constant rate and stored at -20 prior to insulin determination by a rat insulin enzyme-linked immunosorbent assay (ELISA) kit (Linco Research, St. Charles, Missouri, United States). The limit of sensitivity of the assay was 0.2 ng/ml (35 pmol/l) insulin using a 10 μl sample. The appropriate range of the insulin assay was 0.2 ng/ml to 10 ng/ml insulin (10 μl sample size). Samples with results greater than this range were diluted with sample buffer in order to fit the range of the standard curve. Determination of nitrite production Test samples at 100 μl were mixed with 1.32% sulfanilamide (50 μl) in 60% acetic acid and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride HCl (50 μl) in distilled water. After 10 min incubation, the absorbance was read on a spectrophotometer at 540 nm. Actual concentrations were calculated from an absorbance vs nitrite (μmol/l) standard curve. Statistical analysis The significance of various treatments was determined by the Student s unpaired t-test under non-parametric statistical conditions. Results are expressed as the mean ± SE. Differences were considered significant at P < RESULTS As shown in Figure 1, HPLC analysis of the ethanolic extract of SA-Et was carried out, and seven major peaks were obtained and identified as schiarisanrin B, taiwanschirin A, schiarisanrin E, schiarisanrin C, schiarisanrin A, macelignan, and schizanrin A, respectively, by matching them to authentic samples. In addition, the model of cytokine-mediated b cell destruction was established using BRIN-BD11 cells. As shown in Figure 2A, a synergistic effect on cytotoxicity was only observed when IL-1β was mixed with IFN-γ. Maximum cell death occurred at 48 h when cells were treated with IL-1β (2 ng/ml) and IFN-γ (100 ng/ml). As shown in Figure 2B, the pres December 14, 2012 Volume 18 Issue 46

133 Hsu YS et al. b-cell protective effects of SA-Et Peak 1 Peak 5 Peak 6 Peak 1: 17.6 min schiarisanrin B Peak 2: 24.1 min taiwanschirin A Peak 3: 32.8 min schiarisanrin E Peak 4: 34.3 min schiarisanrin C Peak 5: 39.5 min schiarisanrin A Peak 6: 52.9 min macelignan Peak 7: 52.9 min schizarin A 0.06 Peak Peak 2 Peak 3 Peak t /min Peak 1 Peak 2 Peak 3 Peak 4 O O O O CH3 O O O O CH3 CH3 O O H3C O O CH3 O O O H3OC CH3 CH3 O O OCH3 O O OCH3 CH3 CH3 O O H3CO CH3 CH3 O O O H3OC O CH3 O CH3 OCH3 OCH3 OCH3 Schiarisanrin B Taiwanschirin A Schiarisanrin E Schiarisanrin C Peak 5 Peak 6 Peak 7 O O H3C O CH3 O O O O O O O CH3 CH3 O CH3 OH O CH3 CH3 H3CO CH3 H3OC OH H3CO OCH3 OCH3 OCH3 Schiarisanrin A Macelignan Schizarin A Figure 1 Chemical fingerprinting of the ethanolic extract of Schizandra arisanensis. The ethanolic extract of Schizandra arisanensis (SA-Et) (1.0 mg) was subjected to a high-performance liquid chromatography system to obtain chemical fingerprints. According to the retention times, the chemical identities and structures of seven major peaks are listed. ence of IFN-γ alone resulted in significant G1 arrest (P < 0.05). In contrast, the presence of IL-1β alone caused significant inhibition at S phase. However, when IFN-γ was combined with IL-1β, the accumulated G1 arrested cells appeared to have progressed into the subg1 phase (P < 0.01) at the end of cytokine treatment. By affecting the mechanisms of IL-1β + IFN-γ using various inhibitors, our results showed that IL-1β + IFN-γ-mediated cytotoxicity could be attenuated by SP and SB On the other hand, the presence of U0126 and L-NAME had no beneficial effects on cell viability. Moreover, the viability of cytokine-treated cells was worse in the presence of wortmannin (Figure 2C). By employing our cell model, as shown in Figure 3A, the SA-Et at 20 μg/ml provided b cell protective activity as shown by an increase of approximately 1.3-fold (P < 0.01) in the viability in the cytokine treated condition. Consistently, induction of the subg1 phase in the presence of IL-1β + IFN-γ was attenuated by the SA- Et (Figure 3B). Finally, full-length caspase-3 degradation in the presence of IL-1β + IFN-γ was observed and was parallel to the increase in cleaved caspase-3 (active form) of about 1.2-fold at 24 h post-treatment with IL-1β + IFN-γ (Figure 3C). However, while SA-Et alone reduced the cleaved form of caspase-3 by 30%, the proteasemediated caspase-3 activation by IL-1β + IFN-γ was also ameliorated by SA-Et. Following determination of the protective effect of 6812 December 14, 2012 Volume 18 Issue 46

134 Hsu YS et al. b-cell protective effects of SA-Et A Viability (control = 100%) b a a b b b b b b b b b b b b 24 h 48 h 72 h 20 B IL-1b + IFN-g (ng/ml) Control IL IFN IL-1 + IFN Counts M4 M1 M2 M3 Counts M4 M1 M2 M3 Counts M4 M1 M2 M3 Counts M4 M1 M2 M Ratio (% of total events) FL2-A a FL2-A b b b b FL2-A b Control IL-1b (5 ng/ml) IFN-g (100 ng/ml) IL-1b + IFN-g b FL2-A C Viability (% of control cells) G1 (M1) S (M2) M (M3) SubG1/Apop (M4) b bd Control Vehicle SP (10 mmol/l) Cell cycle bd SB (20 mmol/l) IL-1b + IFN-g b U0126 (10 mmol/l) bd Wortmannin (100 nmol/l) b L-NAME (2.5 mmol/l) Figure 2 Interleukin-1b and interferon-g-mediated BRIN-BD11 cell apoptosis is attenuated by p38mapk and stress-activated protein kinase/c-jun NH2-terminal kinase inhibitors. A: The viability of interferon (IFN)-γ (100 ng/ml)-treated cells in the presence of various concentrations of interleukin (IL)-1β for 24 h, 48 h and 72 h was measured. Data are presented as the mean ± SE, n = 4. a P < 0.05, b P < 0.01 vs the viability of untreated cells; B: Cell cycle analysis was carried out at 48 h posttreatment with IL-1β (5 ng/ml), IFN-γ (100 ng/ml), or a mixture of the two. Data are presented as the mean ± SE, n = 4. a P < 0.05, b P < 0.01 vs the control; C: The viability of cytokine-treated BRIN-BD11 cells in the presence of inhibitors was measured. Data are presented as the mean ± SE, n = 4. b P < 0.01 vs the viability of untreated cells; d P < 0.01 vs the viability of cells with the cytokine mix December 14, 2012 Volume 18 Issue 46

135 Hsu YS et al. b-cell protective effects of SA-Et A Viability (% of control) B Ratio (% of total events) C Caspase-3 (full) Caspase-3 (cleaved) PKB IL-1b (5 ng/ml) + IFN-g (50 ng/ml) Control SA-Et (mg/ml) b b b b G1 (M1) S (M2) M (M3) SubG1/Apop (M4) Cell cycle None SA-Et (20 mg/ml) Control SA-Et IL-1b + IFN-g IL-1b + IFN-g + SA-Et SA-Et, we further examined the impact of SA-Et on signal transduction of IL-1β + IFN-γ. As shown in Figure 4A, stress-activated protein kinase/c-jun NH2-terminal kinase (SAPK/JNK), p38mapk, and STAT-1α activation in cytokine-treated cells was evident, while ERK1/2 was not activated. In the presence of SA-Et, the phosphorylation of SAPK/JNK (at T183/Y182) and its substrate c-jun (at S63) was attenuated by this treatment, while other pathways appeared to be unaffected by SA-Et. In addition, cytokine treatment also promoted IκBα degradation which was unaffected by the addition of SA- b a IL-1b + IFN-g IL-1b + IFN-g + SA-Et Figure 3 The anti-apoptotic effect of the ethanolic extract of Schizandra arisanensis. A: The viability of BRIN BD11 cells was determined after 48 h of cytokine treatment in the presence or absence of the ethanolic extract of Schizandra arisanensis (SA-Et); B: Cell cycle of BRIN BD11 cells was determined after 48 h of cytokine treatment in the presence or absence of the SA-Et. Data are presented as the mean ± SE, n = 4. a P < 0.05, b P < 0.01 vs the control; C: Both full and cleaved forms of caspase-3 protein were analyzed by Western blot after 24 h of cytokine treatment in the presence or absence of the SA-Et. A representative from three experiments is shown. IL-1b: Interleukin 1b; IFN-g: Interferon-g. b Et (Figure 4B). Consistently, the presence of SA-Et (20 μg/ml) was unable to inhibit cytokine-mediated inos mrna through cytokine induction (Figure 4C and D). Interestingly, SA-Et administration up to 20 μg/ml provided minor, but significant inhibition of cytokine-mediated NO formation. However, such inhibition did not reach the level of the known inos inhibitor, L-NAME, which provided > 50% of the inhibition of cytokinemediated NO production (Figure 4E). In terms of SA-Et treatment on b-cell function with or without cytokine challenge (Figure 5A), SA-Et treatment enhanced the insulinotropic effects of glucose and extracellular Ca 2+, and modestly but significantly affected the insulinotropic effect of extracellular K +. As shown in Figure 5B, glucose responsiveness of BRIN-BD11 cells was unaffected by SA-Et alone. On the other hand, a detectable amount of secreted insulin under a basal or high concentration of glucose was not detected in surviving BRIN-BD11 cells following cytokine treatment. In contrast, we detected partial secretion of insulin from cytokine-treated BRIN-BD11 cells in the presence of SA-Et (P < 0.01). When determining the insulin level in BRIN-BD11 cells under the indicated conditions, there was a modest reduction in insulin mrna expression in the presence of SA-Et or the cytokine mix 48 h post-treatment (Figure 5C). In addition, insulin protein expression and the intracellular insulin content of BRIN-BD11 cells were consistent with each other (Figure 5D). Cellular insulin protein level appeared to be similar regardless of the addition of SA-Et. In contrast, cellular insulin contents in cytokinetreated cells were significantly elevated (P < 0.001). Interestingly, insulin content in cytokine-treated BRIN-BD11 cells with SA-Et treatment was significantly reduced (P < 0.05) compared with that in untreated cells. Finally, we compared the biological activities of four major peaks as shown in Figure 6. Epigallocatechin gallate (EGCG) was employed as a reference drug which has been shown to protect against cytokine-mediated b cell death [16]. Our results indicated that both peak 1 (schiarisanrin B) and peak 5 (schiarisanrin A) provided b cell protective bioactivity similar to EGCG (20 μg/ml). The protective potency of schiarisanrin B reached its maximum at a concentration of 5 μg/ml, and higher concentrations led to a deleterious outcome. On the other hand, schiarisanrin A provided a dose-dependent protective effect starting at a concentration of 5 μg/ml (P < 0.01). Peak 4 (schiarisanrin C) led to cell death in a dose-dependent manner. Different from C19 homolignans, the C18 lignan, peak 6 (macelignan), played no role in cytokine-mediated cytotoxicity at any concentration. DISCUSSION Conventional therapy in patients with type 1 diabetes does not allow minute-to-minute control of blood glucose and does not prevent complications associated with the disease. Whole pancreas or islet transplantation facilitates glucose control on a real-time basis but the lack of 6814 December 14, 2012 Volume 18 Issue 46

136 Hsu YS et al. b-cell protective effects of SA-Et A None IL-1b + IFN-g SA-Et (20 mg/ml) None IL-1b + IFN-g C IL-1b + IFN-g time course (h) SAPK/JNK Phos (T183/Y182) None inos c-jun Phos (S63) Actin p38 Phos (T180/Y182) ERK1/2 Phos (T202/Y204) STAT1 Phos (Y701) SA-Et (20 mg/ml) inos Actin STAT1 Actin B IkBa Actin D 25 None IL-1b + IFN-g SA-Et (20 mg/ml) None IL-1b + IFN-g IL-1b + IFN-g b Relative inos expression (4 h IL-1 + IFN = 100%) E IL-1b + IFN-g IL-1b + IFN-g + SA-Et Incubation t /h inos mrna level (qpcr) b Nitrite (mmol/l) 4 2 IL-1b + IFN-g b b b 0 None SA-Et None Conditions SA-Et 0 Control L-NAME SA-Et (mg/ml) Figure 4 Characterizing the impact of ethanolic extract of Schizandra arisanensis on cytokine signal transduction. A: BRIN-BD11 cells stimulated with the cytokine mix in the presence or absence of ethanolic extract of Schizandra arisanensis (SA-Et) (20 μg/ml) were harvested after 30 min treatment for Western blot with the indicated antibodies. A representative Western blot from three experiments is shown; B: BRIN-BD11 cells stimulated with the cytokine mix in the presence or absence of SA-Et were harvested after 60 min treatment to measure IkBα protein level. A representative Western blot from three experiments is shown; C: Cytokine-induced inducible nitric oxide synthase (inos) mrna levels in BRIN-BD11 cells during 24 h were determined by an reverse transcription-polymerase chain reaction. Relative inos gene expression was calculated by employing endogenous b-actin mrna level as an internal control. The maximum inos gene expression at 4 h post-treatment with the cytokines alone was set to 100%. Data are presented as the mean ± SE, n = 4; D: Changes in cytokine-induced inos mrna levels in BRIN-BD11 cells at 4 h in the presence or absence of the SA-Et (20 μg/ml) were also determined by real-time reverse transcription-polymerase chain reaction. b-actin was used as an internal control. The relative quantification of inos mrna was presented as 2 - ct. Data are presented as the mean ± SE, n = 3. b P < 0.01 vs cells under control conditions; E: Nitric oxide production in cytokine-treated BRIN-BD11 cells in the presence or absence of the SA-Et (0-20 μg/ml) or nitro-l-arginine methyl ester (L-NAME) (0.5 mmol/l) was determined. Data are presented as the mean ± SE, n = 4. b P < 0.01 vs cells treated with the cytokine mixture alone. IL-1b: Interleukin 1b; IFN-g: Interferon-g. sufficient organs for transplantation is a major obstacle. Employing toxin-resistant cells for type 1 diabetes therapy is another developing approach in cell-based therapies. However, disassociation between toxin resistance and insulin-secretory functions may occur during the induction of toxin resistance [17]. Moreover, to our knowledge, no master toxin-resistant cells have been created so far [17-19]. As a result, supplementation with b cell protective phytochemicals to enhance the survival rate and secretory functions of cell-based therapeutics for curing type 1 diabetes is an interesting and flexible approach [20,21]. To pursue this objective, we created a simple platform by employing BRIN-BD11 cells. The use of a cytokine mix successfully promoted b cell death and abolished glucose-stimulated insulin secretion after 48 h of treatment. Through cell cycle analysis, it was apparent that only the combination of IL-1β and IFN-γ elicited apoptosis as judged by caspase 3 cleavage and a significant increment of the subg1 population in BRIN-BD11 cells under such conditions. In the present investigation, the anti-apoptotic effect of SA-Et against cytokine-mediated b cell death was clearly demonstrated. Consistent with the restoration 6815 December 14, 2012 Volume 18 Issue 46

137 Hsu YS et al. b-cell protective effects of SA-Et A Insulin secretion (ng/10 6 cells/20 min) d a bd b bd b bd B Insulin secretion (ng/10 6 cells/20 min) b b IL-1b + IFN-g d 1.1 G 5.6 G 16.7 G d G 16.7 G 16.7 G + KCl 16.7 G + Ca ND ND SA-Et (20 mg/ml) Vehicle SA-Et (20 mg/ml) C None SA-Et IL-1b + IFN-g IL-1b + IFN-g + SA-Et D None SA-Et IL-1b + IFN-g IL-1b + IFN-g + SA-Et Insulin IB: Insulin Actin IB: Actin b Insulin/actin a a Cellular insulin content (ng insulin/mg total protein) a 0 None SA-Et IL-1b + IFN-g IL-1b + IFN-g + SA-Et 0.0 None SA-Et IL-1b + IFN-g IL-1b + IFN-g + SA-Et Figure 5 Ethanolic extract of Schizandra arisanensis treatment partially rescued the abolished insulin secretion in cytokine-treated BRIN-BD11 cells. A: Acute insulin release in response to glucose, KCl and Ca 2+ in the presence or absence of the ethanolic extract of Schizandra arisanensis (SA-Et) was evaluated. Data are presented as the mean ± SE, n = 4. a P < 0.05, b P < 0.01 vs insulin released from cells treated with 1.1 mmol/l glucose; d P < 0.01 vs control cells under the same conditions; B: Cultured BRIN-BD11 cells were treated with the cytokine mixture in the presence or absence of the SA-Et. At 48 h post-treatment, treated cells were evaluated for glucose responsiveness. Data are presented as the mean ± SE, n = 4. b P < 0.01 vs insulin released from cells treated with 1.1 mmol/l glucose; d P < 0.01 vs SA-Et-treated cells under the same glucose conditions. C: In addition, a portion of the treated cells was harvested to measure insulin mrna level using reverse transcription-polymerase chain reaction. Relative insulin gene expression was calculated by employing endogenous b-actin mrna level as an internal control. a P < 0.05 vs control conditions (none); D: Protein level and cellular insulin content were independently measured by Western blot and enzyme-linked immunosorbent assay. Data are presented as the mean ± SE, n = 4. a P < 0.05, b P < 0.01 vs control conditions (none). IL-1b: Interleukin 1b; IFN-g: Interferon-g; ND: Not determined. of cell viability, the ratio of the subg1 phase and protein level of cleaved caspase-3 in the cytokine-treated condition was blocked or reduced by SA-Et. According to previous research, NFκB, p38mapk, and SAPK/JNK pathways all play a role in cytokinemediated cytotoxicity in b cells. Interestingly, unlike other cell lines, NFκB downstream inos/no induction is not responsible for apoptosis of BRIN-BD11 cells. Instead, insulin secretion in response to glucose was significantly affected by NO production in BRIN-BD11 cells [22]. Our results are consistent with previous findings and further demonstrated that p38mapk and JNK/SAPK play important roles in cytokine-mediated BRIN-BD11 cell death. Using this cell model, we screened several antidiabetic herbal extracts and discovered that SA-Et has protective bioactivity. According to the HPLC profile of the SA-Et, seven major peaks were identified: five C19 homolignans (schiarisanrins) and two C18 lignans (schizanrin A and macelignan). Through bioactivity screening, it was evident that some C19 homolignans could account for the b cell protective effects of the SA-Et against the cytokine challenge. To our knowledge, this is the first report to reveal such bioactivity in C19 homolignans. The results also implied that C19 homolignans which possess an acetoxyl group at C-5 exhibited more-potent bioactivity than did those with the benzonic acid ester at C-5, regardless of the different C19 homolignan skeletons as shown in Figure 1. Notably, schiarisanrin C exhibited dose-dependent negative protection activity, which was consistent with a previous report which found that schiarisanrin C exhibited cytotoxicity against other cell lines [8]. The structure December 14, 2012 Volume 18 Issue 46

138 Hsu YS et al. b-cell protective effects of SA-Et Viability (% of control) IL-1b + IFN-g b b b b b b b b b b b b Peak1 (Schiarisanrin B) Peak4 (Schiarisanrin C) Peak5 (Schiarisanrin A) Peak6 (Macelignan) Reference drug (EGCG) Control Veilcle Test compounds (mg/ml) Figure 6 Identification of bio-active compounds isolated from Schizandra arisanensis. After major peak compounds were collected, BRIN-BD11 cells were treated with cytokine mix in the presence of each peak compounds for 48 h. The viability of cells was then measured by neutral red assay. Epigallocatechin gallate (EGCG) is used as reference drug. Data are presented as the mean ± SE, n = 8. b P < 0.01 vs the viability of cells with no treatment. activity relationship will likely be more obvious when more types of C19 homolignan derivatives are compared. To further elucidate the potential drug target of SA- Et, we evaluated the effects of SA-Et on key players in each pathway involved in cytokine-mediated b cell death. The results indicated that SA-Et appeared to impact on JNK/SAPK kinase activities and downstream substrate, i.e., c-jun, to facilitate its protective activity against the cytokine mix. Recent research discovered that the JNK/ c-jun cascade could transactivate the expression of Bcl-2 homology 3 (BH3)-only member death protein 5 (DP5)/ harakiri (Hrk) leading to b cell apoptosis [23] ; therefore, our results suggest that SA-Et treatment might intervene in such a cytokine pathway to facilitate b cell protection. Interestingly, our results also demonstrated that SA-Et treatment had no inhibitory effect on cytokine-mediated NF-kB release judged by a similar pattern of cytokinemediated IkB degradation and inos mrna expression in the presence of SA-Et. NF-kB is an important player in abolishing glucose-stimulated insulin secretion (GSIS) by affecting the glucose-induced influx of Ca 2+[24,25]. In addition, IL-1β-mediated NO production also plays a negative role in insulin secretion in BRIN-BD11 cells [22]. Therefore, whether SA-Et can not only preserve the viability of cytokine-treated BRIN-BD11 cells, but also rescue the impaired insulin secretion of the cytokine-treated cells was our next question. In terms of the insulinotropic action of SA-Et, our results indicate that SA-Et acutely enhanced both glucose- and calcium-stimulated insulin secretion, although K + -stimulated insulin secretion appeared to be slightly reduced by SA-Et. As a result, we speculated that the insulinotropic effect of SA-Et might be associated with calcium-mediated insulin exocytosis, an overall proximal step in insulin secretion. In contrast, under cytokine treatment, insulin exocytosis rather than insulin mrna expression appeared to be affected in BRIN-BD11 cells. This was based on the observations that insulin secreted by cytokine-treated BRIN-BD11 cells was undetectable using the insulin ELISA kit; however, the insulin mrna and intracellular insulin content (protein level) of BRIN- BD11 cells remained intact. When SA-Et was present, the abolished insulin exocytosis by the cytokine mix might be counteracted; therefore, resulting in partial recovery of glucose-stimulated insulin secretion and relief of accumulated insulin content in cytokine treated BRIN-BD11 cells. In conclusion, the current investigation revealed, for the first time, that C19 homolignans isolated from SA-Et possessed protective bioactivity against cytokine-induced b cell death. The cytokine-impaired insulin secretory function was also partially restored. However, inos/no inhibition appeared to be an important factor if intact b cell insulin secretory function is to be preserved after cytokine challenge. In the future, continued research on potent b cell-protective phytochemicals should substantially contribute to the development of islet/stem cell transplantation or cell-based therapies for type 1 diabetes. COMMENTS Background Conventional therapy (i.e., insulin injection) in patients with type 1 diabetes does not allow minute-to-minute control of blood glucose and does not prevent complications associated with the disease. However, the employment of whole pancreas or islet transplantation suffers from a lack of sufficient organs for transplantation. Therefore, the application of cell-based therapeutics for curing type 1 diabetes is an interesting and hot topic in the field. Research frontiers Employing toxin-resistant cells for type 1 diabetes therapy is a developing cellbased approach for curing type 1 diabetes. At least 5 toxin resistant β cell lines have been generated and investigated for defensive mechanisms. However, disassociation between toxin resistance and insulin-secretory functions may occur during the induction of toxin resistance. To our knowledge, no master toxin-resistant cells have been created so far. As a result, employing β-cell protective agent(s) including phytochemicals from traditional Chinese medicine to protect cell-based transplants has become an alternative and practical approach in the field. Innovations and breakthroughs By employing the glucose-responsive, insulin-secreting cell line, BRIN-BD11 cells, the authors successfully established a platform mimicking hormonal part of immune-attack in type 1 diabetes. In addition, the authors noted dissociation not only between cytokine-mediated nitric oxide and cell death, but also 6817 December 14, 2012 Volume 18 Issue 46

139 Hsu YS et al. b-cell protective effects of SA-Et between cell survival rate and secretory function. Moreover, the NFkB-induced inducible nitric oxide synthase-nitric oxide pathway is not necessarily a maser regulator for cell viability and function. Schizandra arisanensis (SA-Et) is one of the schizandraceous plants from Taiwan. The indications for this herb in traditional Chinese medicine include diabetes, hepatitis, immunomodulation and cancer. The current investigation is likely to be the first report on C19 homolignans from SA-Et which possess β-cell protective effects. Different to previous β cell protective phytochemicals, such as epigallocatechin gallate and silymarin, the action of C19 homolignans is not due to the blockage of NFkB-inducible nitric oxide synthase-nitric oxide. Applications By employing this glucose-responsive, insulin-secreting cell line to generate several platforms which represent key steps in autoimmune mediated β-cell destruction. The authors will be able to design a formulation which can be used to enhance the survival rate and maintain secretory functions in cell-based therapeutics to achieve needle-free control of type 1 diabetes in the future. Terminology Type 1 diabetes (T1D) is an autoimmune disease which occurs when approximately 60%-90% of insulin secreting cells are lost and/or are dysfunctional due to β-cell directed autoimmunity. During the progression of T1D, heavy infiltrations of mononuclear cells lead to substantial damage to β cells by generating locally high concentrations of pro-inflammatory cytokines, perforin, and FasL- Fas interactions within the micro-environment of islets. As a result, deleterious outcomes including excessive pro-insulin secretion, the abolition of glucoseinduced insulin secretion, and ultimately β cell death are observed. Peer review The article is very interesting and shows novel findings in insulin secreting cells. The article is well written. Furthermore, it correctly cites the most important articles in the field. REFERENCES 1 van Belle TL, Coppieters KT, von Herrath MG. Type 1 diabetes: etiology, immunology, and therapeutic strategies. Physiol Rev 2011; 91: Bergholdt R, Heding P, Nielsen K, Nolsøe R, Sparre T, Størling J, Nerup J, Pociot F, Mandrup-Poulsen T. Type 1 database mellitus: an inflammatory disease of the islet. Adv Exp Med Biol 2004; 552: Donath MY, Størling J, Berchtold LA, Billestrup N, Mandrup-Poulsen T. Cytokines and beta-cell biology: from concept to clinical translation. Endocr Rev 2008; 29: Cnop M, Welsh N, Jonas JC, Jörns A, Lenzen S, Eizirik DL. Mechanisms of pancreatic beta-cell death in type 1 and type 2 diabetes: many differences, few similarities. Diabetes 2005; 54 Suppl 2: S Kutlu B, Cardozo AK, Darville MI, Kruhøffer M, Magnusson N, Ørntoft T, Eizirik DL. Discovery of gene networks regulating cytokine-induced dysfunction and apoptosis in insulin-producing INS-1 cells. 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Endocrine 2007; 32: Zhang XZ, Li S, Wu XZ. [Effects of Tripterygiitotorum in the treatment of insulin dependent diabetes mellitus with islet transplantation]. Zhongguo Zhongxiyi Jiehe Zazhi 1994; 14: Onaca N, Naziruddin B, Matsumoto S, Noguchi H, Klintmalm GB, Levy MF. Pancreatic islet cell transplantation: update and new developments. Nutr Clin Pract 2007; 22: Stickings P, Cunningham JM. Interleukin-1beta-induced nitric oxide production and inhibition of insulin secretion in rat islets of langerhans is dependent upon the nitric oxide synthase cofactor tetrahidrobiopterin. Cytokine 2002; 18: Gurzov EN, Ortis F, Cunha DA, Gosset G, Li M, Cardozo AK, Eizirik DL. Signaling by IL-1beta+IFN-gamma and ER stress converge on DP5/Hrk activation: a novel mechanism for pancreatic beta-cell apoptosis. Cell Death Differ 2009; 16: Kim HE, Choi SE, Lee SJ, Lee JH, Lee YJ, Kang SS, Chun J, Kang Y. Tumour necrosis factor-alpha-induced glucosestimulated insulin secretion inhibition in INS-1 cells is ascribed to a reduction of the glucose-stimulated Ca2+ influx. J Endocrinol 2008; 198: Heitmeier MR, Kelly CB, Ensor NJ, Gibson KA, Mullis KG, Corbett JA, Maziasz TJ. Role of cyclooxygenase-2 in cytokine-induced beta-cell dysfunction and damage by isolated rat and human islets. J Biol Chem 2004; 279: S- Editor Gou SX L- Editor Kerr C E- Editor Lu YJ 6818 December 14, 2012 Volume 18 Issue 46

140 Online Submissions: doi: /wjg.v18.i World J Gastroenterol 2012 December 14; 18(46): ISSN (print) ISSN (online) 2012 Baishideng. All rights reserved. ORIGINAL ARTICLE Moxibustion inhibits interleukin-12 and tumor necrosis factor alpha and modulates intestinal flora in rat with ulcerative colitis Xiao-Mei Wang, Yuan Lu, Lu-Yi Wu, Shu-Guang Yu, Bai-Xiao Zhao, Hong-Yi Hu, Huan-Gan Wu, Chun-Hui Bao, Hui-Rong Liu, Jin-Hai Wang, Yi Yao, Xue-Gui Hua, Hui-Ying Guo, Li-Rong Shen Xiao-Mei Wang, Huan-Gan Wu, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai , China Yuan Lu, Lu-Yi Wu, Chun-Hui Bao, Hui-Rong Liu, Jin-Hai Wang, Yi Yao, Xue-Gui Hua, Hui-Ying Guo, Li-Rong Shen, Key Laboratory of Acupuncture- Moxibustion and Immunological Effects, Shanghai University of Traditional Chinese Medicine, Shanghai , China Shu-Guang Yu, Laboratory of Experimental Acupuncture of College of Acumox and Tuina, Chengdu University of Traditional Chinese Medicine, Chengdu , Sichuan Province, China Bai-Xiao Zhao, School of Acupuncture-Moxibustion and Tuina, Beijing University of Chinese Medicine, Beijing , China Hong-Yi Hu, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai , China Author contributions: Wang XM and Wu HG designed the research; Lu Y, Wu LY, Bao CH, Wang JH and Wang XM performed experiments; Yu SG, Wang JH, Yao Y and Wu LY analyzed the data; Wang XM, Liu HR, Hu HY, Hua XG, Guo HY and Shen LR wrote the manuscript; Hu HY, Yu SG and Zhao BX revised the manuscript; Wu HG supervised the research and edited the manuscript. Supported by National Natural Science Foundation of China, No ; National Basic Research Program of China (973 program), No. 2009CB522900; Shanghai Health System of Outstanding Young Talent Cultivation Program, No. XYQ ; Shanghai Rising-Star Program, No. 10QA Correspondence to: Huan-Gan Wu, Professor, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, 650 South Wanping Road, Xu Hui District, Shanghai , China. [email protected] Telephone: Fax: Received: September 14, 2012 Revised: November 6, 2012 Accepted: November 11, 2012 Published online: December 14, 2012 Abstract AIM: To investigate the effect of moxibustion on intestinal flora and release of interleukin-12 (IL-12) and tumor necrosis factor-α (TNF-α) from the colon in rat with ulcerative colitis (UC). METHODS: A rat model of UC was established by local stimulation of the intestine with supernatant from colonic contents harvested from human UC patients. A total of 40 male Sprague-Dawley rats were randomly divided into the following groups: normal (sham), model (UC), herb-partition moxibustion (HPM-treated), and positive control sulfasalazine (SA-treated). Rats treated with HPM received HPM at acupuncture points ST25 and RN6, once a day for 15 min, for a total of 8 d. Rats in the SA group were perfused with SA twice a day for 8 d. The colonic histopathology was observed by hematoxylin-eosin. The levels of intestinal flora, including Bifidobacterium, Lactobacillus, Escherichia coli (E. coli ), and Bacteroides fragilis (B. fragilis ), were tested by real-time quantitative polymerase chain reaction to detect bacterial 16S rrna/dna in order to determine DNA copy numbers of each specific species. Immunohistochemical assays were used to observe the expression of TNF-α and IL-12 in the rat colons. RESULTS: HPM treatment inhibited immunopathology in colonic tissues of UC rats; the general morphological score and the immunopathological score were significantly decreased in the HPM and SA groups compared with the model group [3.5 ( ), 3.0 ( ) vs 6.0 ( ), P < 0.05 for the general morphological score, and 3.00 ( ), 3.00 ( ) vs 5.00 ( ), P < 0.01 for the immunopathological score]. As measured by DNA copy number, we found that Bifidobacterium and Lactobacillus, which are associated with a healthy colon, were significantly higher in the HPM and SA groups than in the model group (1.395 ± 1.339, ± vs ± 0.036, P < 0.01 for Bifidobacterium, and ± 0.325, ± vs ± , P < 0.01 for Lactobacillus ). On the 6819 December 14, 2012 Volume 18 Issue 46

141 Wang XM et al. Moxibustion modulates intestinal flora other hand, E. coli and B. fragilis, which are associated with an inflamed colon, were significantly lower in the HPM and SA groups than in the model group (0.244 ± 0.107, ± vs ± 0.683, P < 0.01 for E. coli, and ± 0.181, ± vs ± 1.039, P < 0.01 for B. fragilis ). The expression of TNF-α and IL-12 was decreased after HPM and SA treatment as compared to UC model alone ( ± , ± vs ± , P < 0.01 for TNF-α, and ± , ± vs ± , P < 0.01 for IL-12). CONCLUSION: HPM treatment can regulate intestinal flora and inhibit the expression of TNF-α and IL-12 in the colon tissues of UC rats, indicating that HPM can improve colonic immune response Baishideng. All rights reserved. Key words: Ulcerative colitis; Herb-partition moxibustion; Intestinal flora; Immune regulation Peer reviewer: Mitsunori Yamakawa, Professor, Department of Pathological Diagnostics, Yamagata University, Faculty of Medicine, Iida-Nishi, Yamagata , Japan Wang XM, Lu Y, Wu LY, Yu SG, Zhao BX, Hu HY, Wu HG, Bao CH, Liu HR, Wang JH, Yao Y, Hua XG, Guo HY, Shen LR. Moxibustion inhibits interleukin-12 and tumor necrosis factor alpha and modulates intestinal flora in rat with ulcerative colitis. World J Gastroenterol 2012; 18(46): Available from: URL: i46/6819.htm DOI: INTRODUCTION Ulcerative colitis (UC) is a chronic inflammatory disease of the gastrointestinal tract characterized by increased stool frequency, bleeding and abdominal pain, and ulceration limited to the colon mucosa. The role of immunological pathogenesis in UC has been widely recognized [1,2]. In recent years, the involvement of pathogenmediated immune dysfunction has received extensive attention. Studies have shown that UC patients have altered intestinal flora species. For example, Bifidobacterium, Lactobacillus and fusobacterium Escherichia coli (E. coli) desulfovibrio were significantly decreased, while Bacteroides fragilis (B. fragilis), were significantly increased [3-6]. In hosts with altered intestinal flora, the immune system recognizes intestinal flora and its metabolites as pathogenic antigens that can cause an abnormal immune response and stimulate bowel epithelial cells in UC patients with genetic susceptibility. This activity then destroys the structure and function of colonic mucosa, leading to the ongoing pathology associated with UC [7]. Alteration of the intestinal flora has been considered to be the initiating and continuous factor in the onset of UC [7-10]. Therefore, the modulation of intestinal flora has been suggested as a potential approach to treat UC. Previous studies have shown that herb-partition moxibustion (HPM) can be anti-inflammatory [11]. It can also improve symptoms in mild-to-moderate UC patients, such as relieving abdominal pain and decreasing bloody diarrhea [12-14]. Experiments have also demonstrated that HPM can down-regulate the protein and mrna expression of interleukin (IL)-8 and intercellular adhesion molecule-1 in colon tissues of UC patients, indicating that HPM can inhibit chemotaxis and cell migration into inflamed tissues [14]. HPM can also promote neutrophil apoptosis and down-regulate cytokines, such as IL-1 beta, IL-6, and tumor necrosis factor-alpha (TNF-α), further suggesting an anti-inflammatory role for HPM treatment [15]. Moreover, a recent report shows that intestinal injury was decreased and TNF-α was also found to be decreased after HPM treatment in a Crohn s disease model [16]. Taken together, these results indicate that HPM can regulate immune function in UC. However, whether HPM can also alter the intestinal flora composition is still unknown. A large number of bacteria comprise the human intestinal flora, outnumbering host cells by a ratio of 10:1. Various types of bacteria constitute the gut flora, including symbiotic and pathogenic bacteria whose pathogenicity is hidden from the immune system from various barrier and immune-mediated mechanisms. For this study, we observed the relative proportions of both the symbiotic - and most prevalent - bacteria, such as bifidobacteria and lactobacilli, and the conditioned pathogenic flora, such as E. coli and B. fragilis, as readouts to explore the effects of HPM on modulating the intestinal flora of rats in a model of UC. We also investigated whether HPM regulates the expression of pro-inflammatory cytokines, such as TNF-α and IL-12, in colon tissues of UC rats. This study would aid in understanding the mechanisms of how HPM can alleviate symptoms of UC. MATERIALS AND METHODS Animals Sprague-Dawley rats were obtained from the Department of Experimental Animal Science of Shanghai Medical College at Fudan University [No. SCXK(SH) ]. All protocols were performed in strict accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of the People s Republic of China [17]. This study received permission from the Ethics Committee in Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, which is affiliated with Shanghai University of Traditional Chinese Medicine, China. Establishment of the UC rat model Forty neonatal, male Sprague Dawley rats weighing 183 ± 10 g were randomly divided into the following experimental groups: normal, model, HPM, and sulfasalazine (SA). The UC rat model was established according to an 6820 December 14, 2012 Volume 18 Issue 46

142 Wang XM et al. Moxibustion modulates intestinal flora Table 1 General morphological scoring General morphological manifestation Score Colon adhesion No adhesion 0 Mild adhesion 1 Severe adhesion 2 Ulcer and No ulcer and no inflammation 0 inflammation Local congestion, no ulcer 1 1 ulcer without congestion or bowel wall 2 thickening 1 ulcer with inflammation 3 > 2 ulcer and inflammation 4 > 2 ulcer and/or inflammation area > 1 cm 5 > 2 ulcer and/or inflammation area > 2 cm, one more damage, plus Table 2 Immunopathological scoring Immunopathological manifestation Score Ulcer No ulcer 0 Ulcer area < 3 cm 1 Ulcer area > 3 cm 2 Inflammation No inflammation 0 Mild inflammation 1 Severe inflammation 2 Granuloma No granuloma 0 Granuloma 1 Lesion depth No lesion 0 Submucosa 1 Muscular layer 2 Serosa layer 3 Fibrosis No fibrosis 0 Mild fibrosis 1 Severe fibrosis 2 immunological method and local stimulation [15,18-20]. In brief, fresh human colonic mucosa was obtained from surgical colonic specimens, homogenized in normal saline and centrifuged for 30 min at 3000 rpm in order to remove cells and bacteria. The supernatant, containing antigens released from UC colon patients, was diluted into an appropriate protein concentration and mixed with Freund s adjuvant (Shanghai Chemical Reagent Company, Shanghai, China). One ml of the antigen plus adjuvant mixture, containing a total of 6 mg protein, was injected into the front footpad at day 0. Following this initial dose, the same mixture was then injected into the back footpad, dorsa, inguina, and abdominal cavities on days 10, 17, 24 and 31, respectively. On day 38, rats were anesthetized intraperitoneally with 2% pentobarbital sodium (30 mg/kg) and 3 ml enema of 3% formalin were administered to the rats - in order to stimulate the colon immune response - for 1 h following a saline wash after a 2 ml enema of antigen fluid (without Freund s adjuvant) for 2 h following a saline wash. Treatment After the UC model was established in the rats, HPM was performed during days of UC model in the treatment groups. Moxa cones (0.5 cm in diameter and 0.6 cm high) (Nanyang Hanyi Moxa Co., Ltd. China) were placed vertically on a medicinal formula composed of radix aconite, cortex, radix, carthami and salviae miltiorrhizae. The medicinal formula was then placed on acupoints ST25 (located on a horizontal line 2 cun laterally to the midline and 5 cun above the symphysis pubis, a point of hand Yangming, the mu point of the large intestine meridian, and it regulates the function of the intestine and stomach) [21,22] and RN6 (located on ventral midline 3.5 cun above the symphysis pubis and 1.5 cun below the navel, a point of the conception meridian, and it strengthens original qi and improves immune function); the moxa cones were then ignited and each acupoint was treated twice in 15 min increments. This treatment was repeated once daily for a total of 8 d [23]. As a positive control for inhibition of UC symptoms, the immunosuppressant SA was used. For the SA treatment group, salicylazosulfapyridine solution (0.25 g/tablet) was intragastrically administered to rats (Sanwei Pharmaceutical, Shanghai, China; Batch No C30) twice daily for a total of 8 d. The salicylazosulfapyridine solution concentration was 20 mg/ml with a daily dose of 100 mg/kg, which is equivalent to 0.1 g/kg in a human patient [24]. Morphological observation of fixed colon samples Following treatment and sacrifice by cervical dislocation, samples were collected from the descending colon (5 cm above the anus), cleaned with normal saline, and general morphology was then scored (Table 1). The samples were fixed in 10% formalin, dehydrated, embedded in paraffin, and sectioned into 4 μm thick slices. These sections were then stained by hematoxylin-eosin for pathological observation and the histological grade was scored with the bland method [25,26] (Table 2). TNF-α and IL-12 immunohistochemistry Paraformaldehyde-fixed and paraffin-embedded samples were sliced into 4 μm slices. Paraffin slides were deparaffinized in xylene Ⅰ, xylene Ⅱ, and xylene Ⅲ; dehydrated in 95%, 90% and 70% ethanol; and then incubated with primary rabbit antibodies at 4 for 18 h (anti-tnf-α was diluted to 1:150; IL-12 was diluted to 1:200) (Abcam Co., United Kingdom). The tissues were then visualized with 0.5 g/l diaminobenzidine and 0.3 g/l H2O2 in distilled water, and rinsed in phosphate buffered saline for 10 min. A known positive sample was used as a positive control for all slices, and phosphate buffered saline was substituted for the primary antibody and isotype controls as negative controls. All the samples were analyzed by a Motic Med 6.0 image analysis system (Motic Group Co., Ltd.). Three fields were randomly selected under an optical microscope (Olympus Co., Ltd.) at 400 magnification to calculate the positive target value of the integral optical density. 16S rrna real-time quantitative polymerase chain reaction Rat feces taken directly from inoculated rats was placed 6821 December 14, 2012 Volume 18 Issue 46

143 Wang XM et al. Moxibustion modulates intestinal flora Table 3 Oligonucleotide primers and TaqMan fluorescent probe sequences used for real-time polymerase chain reaction assays Primer or probe name Primer sense Sequence (5 3 ) Amplification product (bp) Bifidobacterium Forward 5'-ACTGGAATTCCCGGTGTAAC-3' 85 Reverse 5'-GTCAGTAACGGCCCAGAGAC-3' Lactococcus lactis Forward 5'-CAACATTTGGAAACGAATGC-3' 134 Reverse 5'-CCTTGGTGAGCCTTTACCTC-3' Bacteroides Forward 5'-ATTGCAGTGGAATGATGTGG-3' 106 Reverse 5'-TATGGCACTTAAGCCGACAC-3' Bacillus vallismortis Forward 5'-ACCGCATGGTTCAGACATAA-3' 88 Reverse 5'-AGCCGTTACCTCACCAACT-3' General morphological score P = P = 0.01 P = NC MC HPM SA Groups P25 - Maximum - Minimum +P50 P75 Figure 1 Herb-partition moxibustion inhibits tissue damage in colonic tissues of rats with ulcerative colitis. The general morphological score of the colonic tissue in the model control (MC) group was significantly higher than the normal control (NC) group (P = 0.000). After treatment, the scores were lower in both the herb-partition moxibustion (HPM) group (P = 0.01) and the control sulfasalazine (SA) group (P = 0.002). into 5-10 ml of nutrient broth and was shaken and cultivated overnight at 37. To extract DNA, 50 μl of the bacterial culture fluid was placed in a 1.5 ml sterile centrifuge tube, and 50 μl of DNA extract was added. This was mixed well at a constant temperature of 100 C for 10 min. It was then centrifuged at rpm for 5 min and saved for later use. For the quantitative reverse transcription-polymerase chain reaction, the 7500 Sequence Detection System was used (ABI Co., USA). The sequence of the forward primer (F) and reverse primer (R) are shown in Table 3. DNA was prepared for PCR amplification in the following way: 10 μl 5 PCR buffer, 0.5 μl forward primer F, 0.5 μl reverse primer R, 0.5 μl dntps, 0.5 μl TaqMan fluorescent probe, 1 μl Taq enzyme, 32 μl dho2 water, and 5 μl cdna template. We used the following amplification conditions: (1) 50 2 min, (2) 95 5min, (3) s, and (4) s, for 40 cycles. Statistical analysis All data were analyzed using SPSS 10.0 statistical software (SPSS Inc., United States), and all data were expressed as mean ± SD for normally distributed continuous variables and as median (QL-QU) for abnormal variables. A one-way analysis of variance was used for normal distributions, LSD/SNK-q for homogeneity of variance, and Dunnett s T3 for heterogeneity of variance. A non-parametric test was used for when the data did not follow Gaussian distribution, and the Spearman method was used for correlation analysis. A value of P < 0.05 was considered statistically significant. RESULTS HPM inhibits tissue damage in colonic tissues of UC rats When the colonic tissues were observed for morphological changes upon treatment, we observed that our UC model group exhibited edematous colonic mucosa and there was serious congestion, erosion, and ulcer formation as compared to the normal group, which exhibited a smooth colonic mucosa surface, clear vascular texture, and no erosion or ulcers. Interestingly, HPM treatment was able to decrease the observed changes in UC treated rats and return the colon to a more normal state, as the colonic mucosa surface of these rats was smooth, there was accidental edema, the vascular texture was clear, and congestion, edema and erosion were significantly reduced compared to the model group. As shown in Figure 1, the general morphological score of the colonic tissue in the model group was significantly higher than the normal group [6.0 ( ) vs 0.0 ( ), P = 0.000]. After treatment, the scores were lower in both the HPM group [3.5 ( ) vs 6.0 ( ), P = 0.01] and the control SA group [3.0 ( ) vs 6.0 ( ), P = 0.002]. This data suggests that HPM is able to dampen the tissue-damaging effects of UC. HPM inhibits immunopathology in colonic tissues of UC rats To evaluate whether HPM was also able to regulate the inflammatory cell infiltration that causes tissue damage in UC, we evaluated the extent of immunopathology by looking for inflammatory cell tissue infiltration by HE staining. As shown in Figure 2A-D, the colonic mucosa and mucosa villi were damaged or missing in the UC model group, and large mononuclear cell and macrophage infiltration appeared in the mucosa or submucosa with congestion, edema, and ulceration as compared to the normal group, where the colonic mucosa epithelium was complete and the colonic gland was regularly arranged with inconspicuous inflammatory cell infiltration. However, HPM treatment decreased the inflammatory 6822 December 14, 2012 Volume 18 Issue 46

144 Wang XM et al. Moxibustion modulates intestinal flora A B C D E P = P25 P = P = Maximum - Minimum 8 7 +P50 P75 Histopathological score NC MC HPM SA Groups Figure 2 Herb-partition moxibustion inhibits immunopathology and decreases the histopathological scores in colonic tissues of rats with ulcerative colitis. A: Normal; B: Ulcerative colitis (UC); C: Herb-partition moxibustion (HPM); D: Sulfasalazine (SA); E: The histopathological scores for the colonic tissue in the UC model control (MC) group were significantly higher than the normal control (NC) group (P = 0.000). After treatment, the scores were lower in both the HPM group (P = 0.007) and SA group (P = 0.015). The colonic mucosa was damaged in the UC model group with cell infiltration, congestion, edema, and ulceration. After HPM treatment, there are only slight submucosal edema and inflammatory cell infiltration, and the colonic mucosa epithelium and the colonic gland were more regularly arranged than in the model group, new epithelial cells were observed to be covering the ulcers. Positive control SA treatment showed similar recovery of the UC model as the HPM group. cell infiltration and the ensuing immunopathology, as we observed only slight submucosal edema and inflammatory cell infiltration, and the colonic mucosa epithelium and the colonic gland were more regularly arranged than in the model group. Additionally, new epithelial cells were observed to be covering the ulcers that developed under UC conditions, indicating that HPM treatment could induce recovery of these ulcers. Positive control SA treatment showed similar recovery of the UC model as the HPM group. As shown in Figure 2E, the histopathological scores for the colonic tissue in the model group were significantly higher than the normal group [5.00 ( ) vs 0.00 ( ), P = 0.000]. After treatment, the scores were lower in both the HPM group [3.00 ( ) vs 5.00 ( ), P = 0.007] and SA group [3.00 ( ) vs 5.00 ( ), P = 0.015]. Taken together, these data indicate that HPM treatment can inhibit inflammatory cell infiltration under UC conditions and therefore limit the resulting immunopathology in the colon tissue. HPM treatment rebalances the colonic intestinal flora of UC rats Because the deregulation of intestinal flora is now understood to be a major component of UC pathogenesis, we tested whether HPM could restore the balance of 6823 December 14, 2012 Volume 18 Issue 46

145 Wang XM et al. Moxibustion modulates intestinal flora 90 P = P = P = Normal UC HPM SA 25 P = P = P = P = P = P = Normal UC HPM SA DNA copy P = P = P = DNA copy P = Bifidobacterium Lactobacillus 0 Bacteroides fragilis Colibacillus Figure 3 Herb-partition moxibustion treatment increases the colonic Bifidobacterium and Lactobacillus of rats with ulcerative colitis. The DNA copies of the symbiotic groups Bifidobacterium and Lactobacillus were both significantly decreased in the ulcerative colitis (UC) group compared to the normal control (NC) group (P = 0.000). After herb-partition moxibustion (HPM) treatment, the DNA copies of Bifidobacterium and Lactobacillus were both significantly increased in the HPM group (P = and P = 0.000) and sulfasalazine (SA) group (P = and P = 0.000) compared with the UC model group. Figure 4 Herb-partition moxibustion treatment decreases the colonic pathogenic bacteria Bacteroides fragilis and Escherichia coli of rats with ulcerative colitis. The DNA copies of the pathogenic bacteria Bacteroides fragilis (B. fragilis), and Escherichia coli (E. coli) were both significantly increased in the ulcerative colitis (UC) rats compared to the normal control (NC) rats (P = 0.000). After herb-partition moxibustion (HPM) treatment, the DNA copies of B. fragilis and E. coli were both significantly decreased in the HPM group (P = and P = 0.000) and sulfasalazine (SA) group (P = and P = 0.003) compared with the UC model group. the bacteria species back to normal by using the readout of the levels of symbiotic and pathogenic bacteria. To do this, we tested the levels of species-specific bacterial DNA by detecting 16S RNA from rat feces among the different groups. As shown in Figure 3, DNA copies of the symbiotic groups Bifidobacterium and Lactobacillus were both significantly decreased in the UC group compared to the normal group (0.045 ± vs ± for Bifidobacterium, ± vs ± for Lactobacillus, P = 0.000). After HPM treatment, the levels were restored, because DNA copies of Bifidobacterium and Lactobacillus were both significantly increased in the HPM group and SA group compared with the model group (1.395 ± 1.339, ± vs ± , P = in HPM group and P = in SA group for Bifidobacterium, and ± 0.325, ± vs ± , P = for Lactobacillus). As shown in Figure 4, the DNA copies of the pathogenic bacteria B. fragilis and E. coli were both significantly increased in the UC rats compared to the normal rats (1.285 ± vs ± for B. fragilis, ± vs ± for E. coli, P = 0.000). After HPM treatment, the DNA copies of B. fragilis and E. coli were both significantly decreased in the HPM group and SA group compared with the UC model group (0.351 ± , ± vs ± , P = for B. fragilis and ± , ± vs ± P = in HPM group and P = in SA group for E. coli). These data indicate that HPM treatment can rebalance the intestinal flora toward more normal levels after alteration under UC conditions. HPM inhibits the secretion of the pro-inflammatory mediators TNF-α and IL-12 in the colon tissue of UC rats To determine whether the HPM-induced reduction of inflammation was due to an effect of HPM on the secretion of pro-inflammatory cytokines, we measured TNF-α and IL-12 levels in colon tissue by immunohistochemistry. As shown in Figure 5A1-E1, TNF-α expression was significantly increased in the UC model group compared to the normal group ( ± vs ± , P = 0.000). After treatment, TNF-α expression was significantly decreased in both the HPM group and SA group compared with the UC model group ( ± , ± vs ± , P = 0.000). There was a difference in TNF-α expression between the HPM group and positive control SA group ( ± vs ± , P = 0.034). As shown in Figure 5A2-E2, IL-12 expression was significantly increased in the model group compared to the normal group ( ± vs ± , P = 0.000). HPM treatment inhibits IL-12 expression because it was significantly decreased in both the HPM group and positive control SA group compared with the model group ( ± , ± vs ± , P = in the HPM group and P = in the SA group). There was a difference in IL-12 expression between the HPM group and the SA group ( ± vs ± , P = 0.033). This data indicates that HPM treatment exerts its anti-inflammatory effects through the inhibition of pro-inflammatory cytokine secretion, such as TNF-α and IL December 14, 2012 Volume 18 Issue 46

146 Wang XM et al. Moxibustion modulates intestinal flora A1 B1 E1 IOD of TNF-a P = P = P = P = Normal UC HPM SA Groups C1 D1 A2 B2 E2 IOD of IL P = P = P = P = Normal UC HPM SA Groups C2 D2 Figure 5 Herb-partition moxibustion inhibits the secretion of the pro-inflammatory mediators tumor necrosis factor-α and interleukin-12 in the colon tissue of rats with ulcerative colitis ( 400). A1, A2: Normal control; B1, B2: Ulcerative colitis (UC); C1, C2: Herb-partition moxibustion (HPM); D1, D2: Sulfasalazine (SA). A1-E1: Tumor necrosis factor-α (TNF-α) expression was significantly increased in the UC model group compared to the normal group (P = 0.000). After treatment, TNF-α expression was significantly decreased in both the HPM group (P = 0.000) and SA group (P = 0.000) compared with the UC model group. There was a difference in TNF-α expression between the HPM group and positive control SA group (P = 0.034); A2-E2: Interleukin-12 (IL-12) expression was significantly increased in the model group compared to the normal group (P = 0.000). HPM treatment inhibits IL-12 expression because it was significantly decreased in both the HPM group (P = 0.001) and positive control SA group (P = 0.012) compared with the model group. There was a difference in IL-12 expression between the HPM group and SA group (P = 0.033). IOD: Integrated optical density per dye December 14, 2012 Volume 18 Issue 46

147 Wang XM et al. Moxibustion modulates intestinal flora DISCUSSION In normal physiological conditions, the overgrowth of pathogenic microorganisms is kept in check by the balance of gut flora species as well as the interaction between microbes and the host s immune system. These mechanisms limit the pathogenic microorganisms from sticking to the intestinal mucosa and act as a biological barrier to the host [7,27,28]. Altered bacterial composition and function of the gut flora in UC results in increased immune stimulation, epithelial dysfunction, and enhanced mucosal permeability [7]. Moreover, this barrier dysfunction leads to increased recognition of pathogens and their metabolites by the immune system and cause an abnormal intestinal immune response [7] that constantly stimulates intestinal epithelium cells, among other effects. This immune response leads to the production of a large amount of cytokines, such as TNF-α, IL-12 and IL-6, that further aggravate the local immune response of the intestinal mucosa and cause continual damage of the intestinal mucosa [29,30]. In recent years, studies have widely focused on the relationship between intestinal bacteria and UC. Changes in gut flora have been considered as key in the initiation and maintenance stages of the inflammatory processes in UC [7,10,31-35]. There is evidence to support that UC patients undergo alterations in intestinal flora: the amount of Enterobacter, Enterococcus, and small-bowel Clostridium increased significantly, while Bifidobacteria and Lactobacilli decreased significantly in acute UC. In patients undergoing remission from UC, bacteroid and Bifidobacteria were increased markedly and small-bowel Clostridium was decreased significantly [36,37]. In some studies, bacteroids were significantly increased [38] and Bifidobacteria and Lactobacilli in the gut were both decreased in acute and remission UC [39]. Other studies have indicated that Bifidobacteria, Lactobacilli, and Fusobacterium in the gut of UC patients decreased significantly, while Bacteroides, E. coli, and Desulfovibrio increased remarkably [3-6]. The results of the present study showed that Bifidobacteria and Lactobacilli decreased significantly, while E. coli and B. fragilis significantly increased by testing the bacterial DNA copy numbers in the stool of UC rats as compared to normal rats. These results indicate that gut flora alterations also occur in the rat model of UC used in these studies and provide further evidence that alterations in gut flora play an important role in the pathogenesis of UC. In recent years, modulation of gut flora has been suggested as an approach to treat UC. The balance between beneficial and detrimental bacterial species determines homeostasis vs inflammation. This balance can be manipulated by antibiotics, probiotics, and prebiotics to treat and prevent relapses of UC [40]. Additionally, alternative treatments or complementary therapy, such as the use of probiotics, have been considered good candidates for treatment of UC because there are less problems with resistance, it means less risk for the development of bacterial resistance to antibiotic treatment, and there are less potential side effects and fewer ecological concerns than using antibiotic drugs [41]. Our previous studies in humans have indicated that HPM treatment is safe and has a therapeutic effect on UC patients [12-14]. To determine the mechanism of how HPM might control UC, the aim of this study was to analyze whether HPM can modulate the gut flora in a rat model of UC. The results shown here demonstrate that HPM treatment does indeed alter the intestinal flora bacterial composition, as Bifidobacteria and Lactobacilli increased significantly, while E. coli and B. fragilis decreased remarkably in the stool of HPM-treated UC rats as compared to the stool of UC rats without treatment. Damage to the colonic mucosa was also remarkably improved after HPM treatment of UC rats, which indicated that the alteration of the gut flora back towards normal rat flora correlated with increased recovery of gut pathology in the UC rats treated with HPM. Therefore, HPM could modulate the alteration of gut flora in the UC rat model, recover intestinal micro-ecological balance, and inhibit the development and/or maintenance of gut pathology. The resultant modulation of gut flora by HPM is similar to that of probiotic supplements [29], and therefore deserves further study as an alternative treatment for UC symptoms. IL-12 and TNF-α are pre-inflammation factors and play an important role in the pathology of intestinal bowel disease (IBD). IL-12 from dendritic cells induces the differentiation of CD4 + T cells to Th1 cells. IL-12 also stimulates natural killer cells and T cells to secrete γ-interferon and TNF-α cytokines to mediate the inflammation. TNF-α cooperates with γ-interferon and can change the function of the intestinal barrier; it can also enhance the permeability of the mucous membrane or induce apoptosis of bowel epithelial cells. This tissue damage is the key process in the colonic inflammatory response in UC [3,39] and is closely related to the pathogenesis of UC [4,5]. Studies have revealed that IL-12 protein and mrna expression is increased and correlates with the active index score of UC [5,6]. IL-12 and TNF-α are significantly increased in the peripheral blood of patients with IBD [40-43]. These previous studies indicate the relevance of IL-12 and TNF-α cytokines in the initiation and maintenance of UC pathogenesis. Previous studies showed that HPM could rectify abnormal mucosal immune responses [43], regulate the expression of IL-1 and IL-1β [44,45], and improve intestinal mucus damage by down-regulating the expression of TGF-β1 and IGF-1 in the inflamed tissue of UC [46,47]. The results of the present study indicate that TNF-α and IL-12 are expressed at low levels in the colonic mucosa and the submucosa of normal rats. However, in UC rats, TNF-α and IL-12 were expressed at high levels that correlated with the observed higher morphological and histopathological scores. After HPM treatment, however, the expression of TNF-α and IL-12 decreased and the general morphological and histopathological scores were lower than those of the UC group, indicating that HPM treatment had an anti-inflammatory effect and improved the UC-mediated pathology. These results further support the previous reports that suggest that HPM can 6826 December 14, 2012 Volume 18 Issue 46

148 Wang XM et al. Moxibustion modulates intestinal flora improve the inflammatory response induced by UC, and that the mechanism may be regulation of the expression of inflammatory cytokines IL-6, IL-1β, TNF-α, and IL-12 in the colon mucosa of UC rats. Additionally, the intestinal flora and their products have been found to trigger cytokine expression, such as inducing TNF-α in macrophage and epithelial cell systems of inflammatory bowel disease [48]. In addition to restoring beneficial intestinal flora, probiotics may enhance host protective immunity such as down-regulation of pro-inflammatory cytokines, IL-12 and TNF-α in colitis [49,50]. Rifaximin administration decreased the protein and mrna levels of IL-12 and TNF-α, and caused a significant reduction of colon bacterial translocation towards mesenteric lymph nodes, in colon of 2, 4, 6-trinitrobenzene sulfonic acid-induced colitis in mice [51]. The results of this study showed that HPM can modulate intestinal flora towards a more normal flora and inhibit the expression of TNF-α and IL-12 in UC rats. These indicate that there may be a relationship between the release of IL-12 /TNF-α and the modulation of bacterial flora. In conclusion, we suggest that HPM can modulate intestinal flora towards a more normal flora and inhibit the expression of the pro-inflammatory mediators TNF-α and IL-12 in UC rats. Therefore, whether HPM can improve the intestinal inflammation response of UC by modulating intestinal flora deserves further study as a treatment for patients suffering with UC. COMMENTS Background Intestinal flora plays an important role in human health. Previous studies indicated that herb-partition moxibustion (HPM) has a beneficial effect on ulcerative colitis (UC), not just to relieve the symptoms, but also to improve the stool property, but whether the effect of HPM is related to intestinal flora remains unknown. Research frontiers More and more data show that the key role of bacteria in the pathogenesis of inflammatory bowel disease, which has become a hot spot of study. Innovations and breakthroughs This study is the first to report on the effects of HPM on intestinal flora of UC. HPM treatment can regulate intestinal flora of the UC model rat. Applications The experimental data can be used in the further study of moxibustion therapy in the treatment of inflammatory bowel disease. Peer review This is a good original study in which authors observed the relative proportions of both the symbiotic and most prevalent bacteria, and the conditioned pathogenic flora to explore the effects of HPM on modulating the intestinal flora of rats in a model of UC. They also investigated whether HPM regulates the expression of pro-inflammatory cytokines in colon tissues of UC rats. 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Nestle Nutr Workshop Ser Pediatr Program 2009; 64: ; discussion , Tursi A, Brandimarte G, Papa A, Giglio A, Elisei W, Giorgetti GM, Forti G, Morini S, Hassan C, Pistoia MA, Modeo ME, Rodino S, D Amico T, Sebkova L, Sacca N, Di Giulio E, Luzza F, Imeneo M, Larussa T, Di Rosa S, Annese V, Danese S, Gasbarrini A. Treatment of relapsing mild-to-moderate ulcerative colitis with the probiotic VSL#3 as adjunctive to a standard pharmaceutical treatment: a double-blind, randomized, placebo-controlled study. Am J Gastroenterol 2010; 105: Brauchle M, Angermeyer K, Hübner G, Werner S. Large induction of keratinocyte growth factor expression by serum growth factors and pro-inflammatory cytokines in cultured fibroblasts. Oncogene 1994; 9: D Haens G, Daperno M. Advances in biologic therapy for ulcerative colitis and Crohn s disease. Curr Gastroenterol Rep 2006; 8: Verri WA, Souto FO, Vieira SM, Almeida SC, Fukada SY, Xu D, Alves-Filho JC, Cunha TM, Guerrero AT, Mattos- Guimaraes RB, Oliveira FR, Teixeira MM, Silva JS, McInnes IB, Ferreira SH, Louzada-Junior P, Liew FY, Cunha FQ. IL-33 induces neutrophil migration in rheumatoid arthritis and is a target of anti-tnf therapy. Ann Rheum Dis 2010; 69: Pang YH, Zheng CQ, Yang XZ, Zhang WJ. Increased expression and activation of IL-12-induced Stat4 signaling in the mucosa of ulcerative colitis patients. Cell Immunol 2007; 248: Nielsen OH, Kirman I, Rüdiger N, Hendel J, Vainer B. Upregulation of interleukin-12 and -17 in active inflammatory bowel disease. Scand J Gastroenterol 2003; 38: Tessner TG, Cohn SM, Schloemann S, Stenson WF. Prostaglandins prevent decreased epithelial cell proliferation associated with dextran sodium sulfate injury in mice. Gastroenterology 1998; 115: Klapproth JM, Sasaki M. Bacterial induction of proinflammatory cytokines in inflammatory bowel disease. Inflamm Bowel Dis 2010; 16: Chen CC, Kong MS, Lai MW, Chao HC, Chang KW, Chen SY, Huang YC, Chiu CH, Li WC, Lin PY, Chen CJ, Li TY. Probiotics have clinical, microbiologic, and immunologic efficacy in acute infectious diarrhea. Pediatr Infect Dis J 2010; 29: Nanda Kumar NS, Balamurugan R, Jayakanthan K, Pulimood A, Pugazhendhi S, Ramakrishna BS. Probiotic administration alters the gut flora and attenuates colitis in mice administered dextran sodium sulfate. J Gastroenterol Hepatol 2008; 23: Fiorucci S, Distrutti E, Mencarelli A, Barbanti M, Palazzini E, Morelli A. Inhibition of intestinal bacterial translocation with rifaximin modulates lamina propria monocytic cells reactivity and protects against inflammation in a rodent model of colitis. Digestion 2002; 66: S- Editor Gou SX L- Editor O Neill M E- Editor Xiong L 6828 December 14, 2012 Volume 18 Issue 46

150 Online Submissions: doi: /wjg.v18.i World J Gastroenterol 2012 December 14; 18(46): ISSN (print) ISSN (online) 2012 Baishideng. All rights reserved. BRIEF ARTICLE Minimally invasive treatment of pancreatic necrosis Brian Bello, Jeffrey B Matthews Brian Bello, Jeffrey B Matthews, Department of Surgery, School of Medicine, University of Chicago, Chicago, IL 60637, United States Author contributions: Bello B and Matthews JB wrote the paper. Correspondence to: Jeffrey B Matthews, MD, FACS, Professor, Chairman, Department of Surgery, University of Chicago, 5841 S Maryland Ave, MC5029, Chicago, IL 60637, United States. [email protected] Telephone: Fax: Received: April 18, 2012 Revised: September 3, 2012 Accepted: September 6, 2012 Published online: December 14, 2012 Abstract AIM: To systematically review these minimally invasive approaches to infected pancreatic necrosis. METHODS: We used the MEDLINE database to investigate studies between 1996 and 2010 with greater than 10 patients that examined these techniques. Using a combination of Boolean operators, reports were retrieved addressing percutaneous therapy (341 studies), endoscopic necrosectomy (574 studies), laparoscopic necrosectomy via a transperitoneal approach (148 studies), and retroperitoneal necrosectomy (194 studies). Only cohorts with at least 10 or more patients were included. Non-English papers, letters, animal studies, duplicate series and reviews without original data were excluded, leaving a total of 27 studies for analysis. RESULTS: Twenty-seven studies with 947 patients total were examined (eight studies on percutaneous approach; ten studies on endoscopic necrosectomy; two studies on laparoscopic necrosectomy via a transperitoneal approach; five studies on retroperitoneal necrosectomy; and two studies on a combined percutaneous-retroperitoneal approach). Success rate, complications, mortality, and number of procedures were outcomes that were included in the review. We found that most published reports were retrospective in nature, and thus, susceptible to selection and publication bias. Few reports examined these techniques in a comparative, prospective manner. CONCLUSION: Each minimally invasive approach though was found to be safe and feasible in multiple reports. With these new techniques, treatment of infected pancreatic necrosis remains a challenge. We advocate a multidisciplinary approach to this complex problem with treatment individualized to each patient Baishideng. All rights reserved. Key words: Acute pancreatitis; Pancreatic abscess; Pancreatic necrosis; Necrosectomy; Laparoscopic necrosectomy Peer reviewer: Edward L Bradley Ⅲ, MD, Professor of Surgery, Department of Clinical Science, Florida State University College of Medicine, 1600 Baywood Way, Sarasota, FL 34231, United States Bello B, Matthews JB. Minimally invasive treatment of pancreatic necrosis. World J Gastroenterol 2012; 18(46): Available from: URL: v18/i46/6829.htm DOI: i INTRODUCTION Most cases of acute pancreatitis are self-limited and resolve without serious complications. However, severe acute pancreatitis is associated with the development of potentially life-threatening complications including pancreatic necrosis and pancreatic abscess. The 1992 international consensus conference held in Atlanta established uniform terminology for acute pancreatitis and its complications. According to the Atlanta Classification, pancreatic necrosis refers to diffuse or focal areas of nonviable pancreatic parenchyma, typically associated with peripancreatic fat necrosis, whereas pancreatic abscess is defined as a circumscribed intra-abdominal col December 14, 2012 Volume 18 Issue 46

151 Bello B et al. Minimally invasive treatment of pancreatic necrosis lection of pus, usually in proximity to the pancreas arising as a consequence of acute pancreatitis or pancreatic trauma [1]. Treatment for pancreatic necrosis has evolved considerably over the past decade with respect to both the timing of intervention and the development of alternatives to traditional open necrosectomy. Pancreatic necrosis may be sterile or infected. The prognosis (with or without intervention) is much worse for infected than sterile necrosis. Historically, early surgical intervention was considered mandatory for cases of suspected infection. The 2002 International Acute Pancreatitis (IAP) guidelines recommended early fineneedle aspiration to discriminate between sterile and infected pancreatic necrosis, with continued non-operative management for stable or improving patients with sterile necrosis but surgical intervention for those with documented infection [2]. The traditional surgical approach to infected necrosis was open necrosectomy with the goals of wide drainage of all infected compartments and complete removal of all necrotic tissue with the placement of drains for continuous postoperative closed lavage. Frequently, repeat laparotomy was needed to ensure complete debridement [2]. Historically, open necrosectomy was associated with substantial morbidity and rates of perioperative mortality that exceeded 50% in some reports [3,4], although mortality in some contemporary series has been 11% or lower [5,6]. Because various studies showed high mortality with early operation for severe necrotizing pancreatitis, the IAP recommended avoidance of surgical intervention within the first 14 d after onset unless there was progressive multiple organ failure and clinical deterioration. Subsequent studies suggested that morbidity and mortality can be further reduced if operation is delayed beyond d [7], presumably because the extended interval allows sufficient demarcation between normal and necrotic tissue, thereby reducing the risk of inciting overwhelming postoperative septic and systemic inflammatory responses, as well as the risk of intraoperative injury to surrounding organs and hemorrhage. In addition to the open approach for pancreatic necrosis and abscess, the last two decades have brought about alternative minimally-invasive techniques. Percutaneous drainage of infected pancreatic necrosis has been shown to be safe and effective in highly-selected patients [8,9], but multiple procedures are often needed, and adjunctive treatment is often required. Gagner [10] first described minimally invasive surgical treatment of necrotizing pancreatitis in 1996, including laparoscopic retrocolic, retroperitoneoscopic, and transgastric procedures. Over the past 15 years, a number of other minimally invasive surgical, endoscopic, and radiological approaches to drain and debride pancreatic necrosis have been described. These alternatives appear to be feasible and safe, although comparisons among approaches have been difficult due to small numbers, lack of uniform reporting criteria, and varying degrees of operator dependence. The advent of some of these alternatives has led to reconsideration of some of the fundamental tenets of open surgical necrosectomy, particularly with respect to the timing and completeness of debridement. In this report, we review current techniques of minimally invasive pancreatic necrosectomy. MATERIALS AND METHODS A literature search was performed of the MEDLINE database from April 1996 to November 2010 for each of four techniques well described for minimally invasive necrosectomy: percutaneous therapy (341 studies), endoscopic necrosectomy (574 studies), laparoscopic necrosectomy via a transperitoneal approach (148 studies), and retroperitoneal necrosectomy (194 studies). Only cohorts with at least 10 or more patients were included. Non- English papers, letters, animal studies, duplicate series and reviews without original data were also excluded. For percutaneous drainage, a search was conducted using subject headings: percutaneous, drainage, pancreatic necrosis, and necrotizing pancreatitis with the aid of Boolean operators. There were 341 initial hits in MEDLINE. After exclusion using the abovementioned criteria, eight studies were included in this review. For endoscopic necrosectomy, a search was conducted using subject headings: endoscopic, endotherapy, drainage, pancreatic necrosis, and necrotizing pancreatitis with the aid of Boolean operators. There were 574 initial hits in MEDLINE. After exclusion using the above-mentioned criteria, ten studies were included in this review. For laparoscopic necrosectomy via a transperitoneal approach, a search was conducted using subject headings: laparoscopic, minimally invasive, necrosectomy, pancreatic necrosis, and necrotizing pancreatitis with the aid of Boolean operators. There were 148 initial hits in MEDLINE. After exclusion using the above-mentioned criteria, two studies were included in this review. For retroperitoneal necrosectomy, a search was conducting using subject headings: laparoscopic, minimally invasive, retroperitoneal, necrosectomy, pancreatic necrosis and necrotizing pancreatitis with the aid of Boolean operators. There were 194 initial hits in MEDLINE. After exclusion using the above-mentioned criteria, five studies were included in this review. RESULTS Percutaneous therapy The results of the literature search for percutaneous therapy for pancreatic necrosis are summarized in Table 1. Technique was similar throughout the studies, access to the area of necrosis was obtained utilizing ultrasound and/or computed tomography to place percutaneous drains ranging in size from 10 to 28 French drains. Afterward, saline flushes were often used every 8 h December 14, 2012 Volume 18 Issue 46

152 Bello B et al. Minimally invasive treatment of pancreatic necrosis Table 1 Percutaneous drainage, endoscopic, laparoscopic and retroperitoneal necrosectomy and combined approach Study n Median delay to drainage, d (range) Median procedures per patient (range) Success (%) Median follow-up, mo (range) Complications (%) Overall mortality (%) Percutaneous drainage Freeny et al [8] 34 9 d (1-48 d) 4 (1-12) 16 (47) NA 24 (71) 4 (12) Van Santvoort et al [11] 43 NA (35) 6 17 (40) 8 (19) Rocha et al [12] 28 NA NA 5 (18) NA 3 (11) 8 (29) Mortelé et al [13] d (2-33 d) 3 (1-7) 17 (49) NA 4 (11) 6 (17) Bruennler et al [14] d (1-40 d) 2 (1-9) 42 (53) NA 23 (29) 27 (34) Lee et al [15] 18 NA NA 14 (78) (11) 1 (6) Baril et al [16] 38 NA 2.4 (2-4) 30 (79) NA 1 (3) 2 (5) Gambiez et al [17] d (10-25 d) NA 3 (30) NA 6 (60) 2 (20) Endoscopic necrosectomy Seifert et al [18] d (4-158 d) 6.2 (1-35) 63 (68) (26) 13 (19) Coelho et al [19] 56 5 wk (4-10 wk) 4 (2-8) 49 (87) (20) 2 (3.5) Escourrou et al [20] d (23-32 d) 1.8 (1-3) 13 (100) (46) 0 (0) Lopes et al [21] 26 NA NA 24 (94) 9 2 (7.7) 0 (0) Voermans et al [22] d ( d) NA 23 (95) (40) 0 (0) Papachristou et al [23] d ( d) 3 (1-12) 41 (77) (49) 3 (6) Hookey et al [24] d (10-45 d) 2 (1-2) 10 (59) NA 2 (12) NA Charnley et al [25] d (3-180 d) 4 (1-10) 11 (85) 16 0 (0) 2 (15) Seewald et al [26] 13 NA NA 9 (69) (5.6) 0 (0) Baron et al [27] 43 NA 2 (1-6) 31 (72) (37) NA Laparoscopic necrosectomy Parekh [28] d ( d) 1 (1-3) 14 (74) NA 11 (58) 2 (10.5) Zhu et al [29] wk (NA) NA 9 (90) NA NA 1 (10) Retroperitoneal necrosectomy Gambiez et a l[17] d (NA) 5 (1-9) 17 (85) NA 6 (30) 2 (10) Raraty et al [30] d (1-181 d) 3 (1-9) 120 (86) NA 75 (55) 26 (19) Chang et al [31] d (14-56 d) NA 17 (89.5) NA 4 (21) 3 (15.8) Castellanos et al [32] d (1-28 d) 5 (3-10) 11 (100) (0) 0 (0) Carter et al [33] d ( d) 3 (1-6) 8 (80) NA 5 (50) 2 (20) Combined approach Van Sanvoort et al [11] 43 4 wk (95) (40) 8 (19) Horvath et al [34] 40 4 wk 1 (1-2) 24 (60) 6 29 (72.5) 2 (5) NA: Not available. Eight studies included 286 patients ranging from 10 to 80 patients. There were six retrospective case series, one prospective case series [15], and one randomized control trial [11]. In the randomized control trial, percutaneous drainage was used as part of the minimally invasive step-up approach, in which a retroperitoneal necrosectomy was necessary in 65% of patients. This technique is further described later in the Results section under combined approach. One hundred and twenty seven of 286 (44%) patients had successful percutaneous therapy and did not subsequently require surgical necrosectomy. Mortality was reported in 58 of 286 patients (20.2%). Complications were reported in 80 of 286 patients (28%) and included multiple organ failure [8,11], colonic perforation [16], intrabdominal bleeding [8,11-13], gastrointestinal fistula [8,11,12,14,15], biliary obstruction [8], respiratory failure [8], renal failure [8], pancreatic fistula [11,14], new-onset diabetes [17], pseudocyst [17], and use of pancreatic enzymes [11]. Endoscopic necrosectomy The results of the literature search for endoscopic necrosectomy for pancreatic necrosis are summarized in Table 1. Ten studies, all retrospective, included 352 patients ranging from 13 to 93 patients. Two hundred and seventy four of 352 (78%) patients had successful endoscopic necrosectomy therapy and did not subsequently require surgical necrosectomy. Mortality was reported in 20 of 352 patients (5.6%). Complications were reported in 100 of 352 patients (28%) and included bleeding, fever [20,23,24], gallbladder puncture [23], hypotension [23], deep-vein thrombosis/ pulmonary embolism [23], peritonitis [23], Clostridium difficile colitis [23], perforation of necrosis into abdominal cavity [18,22], pneumoperitoneum [21,24,27], migration of stent into cyst [21,23], post-endoscopic retrograde cholangiopancreatography pancreatitis [24], fistula [18,23], bowel obstruction [23], and air embolism [18]. Median follow up was reported as 5.7 mo to 43 mo. The specific technique varied among the reports. Usually, a therapeutic or pediatric gastroscope or a videoduodenoscope was used. Endoscopic ultrasound was used often to locate the necroma cavity and guide initial access. Transmural drainage was then usually performed followed by dilation and irrigation of the debris. This was accomplished most often via the stomach, although the duodenum was used in approximately 10%-50% of these patients. The size of the endoscopic cystgastros December 14, 2012 Volume 18 Issue 46

153 Bello B et al. Minimally invasive treatment of pancreatic necrosis tomy was typically 1.5 cm to 2.0 cm. Endoscopic biopsy forceps and baskets was used to remove solid debris into the stomach, allowing it to be eliminated by digestion and peristalsis. Multiple pigtail stents were left in place to maintain cystgastrostomy patency, and often a nasocystic tube was left in place for post-procedure irrigation. On average, 3.2 endoscopic debridement procedures were necessary to achieve resolution of necrosis. Laparoscopic necrosectomy The results of the literature search for laparoscopic necrosectomy for pancreatic necrosis are summarized in Table 1. Two studies included 29 patients ranging from 10 to 19 patients [28,29]. Each was a retrospective study. Twenty-three of 29 (79%) patients had successful laparoscopic necrosectomy therapy and did not subsequently require open necrosectomy. Mortality was reported in 3 of 29 patients (10.3%). Complications were reported in only the study by Parekh [28] and included Clostridium difficile infection, reintubation, central line infection, delirium tremens, pseudomonas pneumonia, and minor wound complications. The study also notes 11 patients were diagnosed with a pancreatic fistula. Thus the complication rate was calculated as 11 of 19 patients (58%). Zhu et al [29] did not report complications of the procedure, only mortality. Median follow up was not available for either study. Parekh [28] described using three ports for access: a hand access device and two standard laparoscopic ports. Access to the retroperitoneum was obtained either through an infracolic approach or through the greater omentum between the stomach and colon. Gentle finger dissection was used for debridement and several drains were left for postoperative drainage. Four patients that were referred to the service were not considered for a laparoscopic approach because two patients had ileus and anasarca, and two patients had gastrointestinal perforations from prior interventions at other hospitals. Zhu et al [29] described using at least four standard ports, and going through the gastrocolic ligament to approach the pancreas. A fan retractor was used to elevate the stomach for exposure. Four to six drainage tubes were used for postoperative lavage. Selection criteria were not described. Retroperitoneal necrosectomy The results of the literature search for endoscopic necrosectomy for pancreatic necrosis are summarized in Table 1. Five studies included 197 patients ranging from 10 to 137 patients [17,30-33]. There were three retrospective studies [17,30,31] and two prospective case series [32,33]. 173 of 197 (88%) patients had successful retroperitoneal necrosectomy therapy and did not subsequently require open necrosectomy. Mortality was reported in 33 of 197 patients (17%). Complications were reported in 90 of 197 patients (46%) and included colonic fistula [17], gastric perforation [31], duodenal perforation [31, enteric fistula [30], bleeding [17,30,33], pseudocyst [17,30,33], pancreatic fistula [17], incisional hernia [17], myocardial infarct [30], cerebrovascular event [30], biliary stricture [30], pulmonary embolus [30], colonic necrosis [30], hepatic portal/superior mesenteric/ splenic vein thrombosis [30], Clostridium difficile infection [30], residual abscess [31], pneumonia [31], respiratory and liver failure [33], and gastric ileus [33]. Technique varied among the five studies. Gambiez et al [17] described using a short lumbotomy (6 cm in length) centered on the 12th rib. The spleen and descending colon (or ascending colon if on right side) were mobilized anteriorly and pancreas was accessed without violating the peritoneum. A 23 cm mediastinoscope was used for direct vision. Necrotic areas were removed with blunt dissection using a suction metal tube. A tube drain was left to facilitate later drainage. Similarly, Castellanos et al [32] described a left translumbar approach. However, the group used a flexible endoscope for access followed by flushing and aspiration under direct guidance. Chang et al [31] described using a 5 cm skin incision below the costal margin, followed by blunt dissection. A Yankauer sucker was then used for probing the abscess cavity and/or removing necrotic discharge. Raraty et al [30] described first placing a 12F pigtail catheter under CT-guidance. After moving the patient to the operating room, the catheter was exchanged over a guide wire with serial dilators to 30F size. A nephroscope was then used for access, and a metal forceps used for piecemeal removal. Two drains were placed for irrigations. Carter et al [33] used a similar technique with the nephroscope but also used a flexible endoscope for direct access. Combined approach Based on anecdotal success of percutaneous radiological and endoscopic necrosectomy used as primary therapy with avoidance of open necrosectomy, an increasing number of reports have utilized combinations of nonsurgical approaches to treat pancreatic necrosis. A prospective randomized multicenter trial called the Minimally Invasive Step Up Approach Versus Maximal Necrosectomy in Patients with Acute Necrotising Pancreatitis (PANTER) was recently performed in the Netherlands [11]. After the diagnosis of necrotizing pancreatitis or infected pancreatic necrosis was made, patients were randomly assigned to either a step-up approach or open necrosectomy. The step-up approach consisted of percutaneous drainage or endoscopic drainage, followed by a minimally invasive retroperitoneal necrosectomy if necessary. A video-assisted retroperitoneal debridement (VARD) with postoperative lavage was then performed three days after if there was no clinical improvement. Major complications or death occurred in 31 of 45 patients after open necrosectomy (69%) vs 17 of 43 patients after the stepup approach (40%) (risk ratio with the step-up approach, 0.57; 95% CI, 0.38 to 0.87; P = 0.006). About 35% of patients in the step up group were successfully able to be managed with percutaneous drainage only [11] December 14, 2012 Volume 18 Issue 46

154 Bello B et al. Minimally invasive treatment of pancreatic necrosis Similar to the PANTER Trial, there is also a recent, prospective multicenter, single-arm study based out of the University of Washington. Percutaneous drainage was used as initial treatment for infected pancreatic necrosis. If there was a 75% reduction in size based on follow-up scan 10 d later, the remainder of their treatment would be percutaneous drains alone. If patients did not have a 75% reduction they were treated with a VARD. Twenty-three percent of patients were treated with percutaneous drains only. Sixty percent of patients were treated with a minimally invasive intervention (drains with or without VARD). Mortality at 30 d was 2.5%. The results of these two studies are summarized in Table 1. DISCUSSION Infected pancreatic necrosis and pancreatic abscess are serious complications of severe acute pancreatitis. However, the last two decades have demonstrated much innovation and variety in minimally invasive techniques. These new techniques can potentially lower the significant morbidity and mortality of these complications. The less invasive approach can potentially keep an infection compartmentalized, specifically avoiding contamination of virgin spaces, such as the peritoneal cavity. It may reduce systemic inflammatory and septic response as a consequence of a major open operation and release of infected necrosis. The percutaneous approach to infected pancreatic necrosis has been shown to be safe and feasible in multiple retrospective case series. It is significant to note, 44% of patients in the studies reviewed did not need surgical therapy. Conclusions are limited, however, since the studies are retrospective and small. We limited our review to include only studies that examined 10 or greater patients. Selection criteria was also not often reported and thus, it may be difficult to predict which subset of patients would most benefit from percutaneous drainage alone. In addition, repeat procedures were often needed, ranging from 1 to 12 procedures in the above-mentioned studies. What has become increasingly popular is the combination of percutaneous technique combined with a VARD as mentioned in the PANTER trial and the Horvath study [11,34]. These studies not only confirmed that there is a subgroup of patients that can benefit from percutaneous drainage alone but also examined a combined technique in a prospective fashion with a relatively larger amount of patients. The endoscopic approach to necrosectomy has also been shown to be safe and feasible with an acceptable procedure-related morbidity. The endoscopic ultrasound has allowed this approach to evolve allowing a better definition of the fluid collection and surrounding vasculature. The approach can also be used in poor risk surgical candidates. However, this technique though is not available at all institutions and often there is a need for repeated procedures for maximal drainage. Furthermore, not all pancreatic abscesses and pancreatic necrosis are accessible via a transgastric or transduodenal approach. Like percutaneous drainage alone, often repeat procedures are needed. Laparoscopic necrosectomy may provide better access to fluid collections not amenable to endoscopic approach. This may facilitate a more thorough debridement of the cavity. The laparoscopic approach has also been demonstrated to be safe in several small case series. In the two studies reported 23 of 29 (79%) patients did not require an open necrosectomy. As previously discussed, these are highly selected patients: these were likely stable patients that were able to tolerate pneumoperitoneum. Morbidity still exists in this approach including enterocutaneous and pancreatic fistulas. Similar to the laparoscopic approach, retroperitoneal necrosectomy has been shown to be effective in select patients: 173 of 197 (88%) patients did not need an open necrosectomy. The approach has the theoretical advantage of not having transmission of intraperitoneal infection from the necrotic area compared to laparoscopy. This technique allows access to areas not accessible via endoscopy and the potential to remove all necrotic tissue from the area. Because of their relatively rare presentation, reports on these minimally invasive approaches to pancreatic necrosis and abscess have mostly small numbers of patients and are mostly retrospective in nature. Selection and publication bias is inherent in these studies and the low morbidity and mortality demonstrated in these studies have to be interpreted as such. There is also a significant amount of heterogeneity and variety of technique within each type of different approach. In addition, several studies utilize a combined technique using percutaneous drainage first followed by endoscopic, laparoscopic, or retroperitoneal necrosectomy. Percutaneous drainage likely temporizes the patient in early phases of infected necrosis. In the future, we look for more prospective studies that use definitions as laid out clearly by the Atlanta classification. Several of the above studies do not strictly adhere to those classifications. Furthermore, complications, specifically major ones including pancreatic or enteric fistula and major hemorrhage should separately be reported. Additionally, some studies do not report the amount of days from presentation to time of necrosectomy. It is well-accepted now that a delay of 28 d between onset of symptoms and intervention can potentially lower morbidity and mortality [7]. Up until recently, there has been no comparison of results between open necrosectomy and these minimally invasive techniques. The only randomized control trial to date comparing a minimally invasive approach versus an open approach is the PANTER trial recently published. The minimally invasive approach is actually a step-up approach in which the first step is either a percutaneous or endoscopic transgastric approach. A VARD procedure was then performed if those approaches were unsuccessful or if the patient did not clinically improve in 72 h. The step-up approach reduced the rate of the 6833 December 14, 2012 Volume 18 Issue 46

155 Bello B et al. Minimally invasive treatment of pancreatic necrosis composite end point of major complications or death. In addition, the step-up approach yielded less incisional hernias and new-onset diabetes. The authors hypothesize that in open necrosectomy, there is potentially viable pancreatic parenchyma that is unintentionally debrided leading to diabetes as a late complication. This is potentially the reason why other minimally invasive techniques have shown good outcomes. It is important to note, that the PANTER trial is not a direct comparison between open necrosectomy and minimally invasive necrosectomy, rather a comparison between treatment strategies. Perhaps this is a better model moving forward in contrast to doing direct comparisons between open necrosectomies or among the different types of minimally invasive approaches. It is likely that these pancreatic fluid collections are amenable to multiple approaches. Complications of severe acute pancreatitis remain a complex problem to treat. We advocate a multidisciplinary approach with interventional radiologists, gastroenterologists, intensivists, and hepatobiliary surgeons at tertiary care centers. Since comparison data is limited, the minimally invasive approach should be based on location of lesion and individual patient presentation. Challenges moving forward will be learning curves, technology improvements, and product cycles of new techniques. This report provides a brief overview of the evolving minimally invasive approaches as alternatives to open necrosectomy. COMMENTS Background A variety of minimally invasive approaches have been described for treatment of infected pancreatic necrosis and pancreatic abscess. Research frontiers Early, open necrosectomy for infected pancreatitis was the traditional mainstay of surgical therapy for these critically ill patients. Current practice has shifted toward, with intervention ideally delayed for a minimum of two and preferably four weeks from the onset of illness. Moreover, various minimally invasive surgical, endoscopic, and percutaneous drainage/debridement strategies have emerged as alternatives to open operation. Innovations and breakthroughs This systematic review summarized studies published between 1996 and 2010 with greater than 10 patients. The quality of these published reports is limited by their retrospective design, selection bias, and possible publication bias. Applications Treatment algorithms for infected pancreatic necrosis should be multidisciplinary, reflective of local expertise and experience, and tailored to the individual presenting circumstances of each patient. Terminology Pancreatic necrosectomy refers to the debridement and drainage of necrotic pancreatic and peripancreatic tissue. Peer review An exhaustive review of the literature leading to the best summary of minimally invasive surgical approaches to infected pancreatic necrosis. REFERENCES 1 Bradley EL. A clinically based classification system for acute pancreatitis. Summary of the International Symposium on Acute Pancreatitis, Atlanta, Ga, September 11 through 13, Arch Surg 1993; 128: Uhl W, Warshaw A, Imrie C, Bassi C, McKay CJ, Lankisch PG, Carter R, Di Magno E, Banks PA, Whitcomb DC, Dervenis C, Ulrich CD, Satake K, Ghaneh P, Hartwig W, Werner J, McEntee G, Neoptolemos JP, Büchler MW. IAP Guidelines for the Surgical Management of Acute Pancreatitis. Pancreatology 2002; 2: Mier J, León EL, Castillo A, Robledo F, Blanco R. Early versus late necrosectomy in severe necrotizing pancreatitis. Am J Surg 1997; 173: Besselink MG, de Bruijn MT, Rutten JP, Boermeester MA, Hofker HS, Gooszen HG. Surgical intervention in patients with necrotizing pancreatitis. Br J Surg 2006; 93: Babu BI, Sheen AJ, Lee SH, O Shea S, Eddleston JM, Siriwardena AK. Open pancreatic necrosectomy in the multidisciplinary management of postinflammatory necrosis. Ann Surg 2010; 251: Rodriguez JR, Razo AO, Targarona J, Thayer SP, Rattner DW, Warshaw AL, Fernández-del Castillo C. Debridement and closed packing for sterile or infected necrotizing pancreatitis: insights into indications and outcomes in 167 patients. Ann Surg 2008; 247: Besselink MG, Verwer TJ, Schoenmaeckers EJ, Buskens E, Ridwan BU, Visser MR, Nieuwenhuijs VB, Gooszen HG. Timing of surgical intervention in necrotizing pancreatitis. Arch Surg 2007; 142: Freeny PC, Hauptmann E, Althaus SJ, Traverso LW, Sinanan M. Percutaneous CT-guided catheter drainage of infected acute necrotizing pancreatitis: techniques and results. AJR Am J Roentgenol 1998; 170: Echenique AM, Sleeman D, Yrizarry J, Scagnelli T, Guerra JJ, Casillas VJ, Huson H, Russell E. Percutaneous catheterdirected debridement of infected pancreatic necrosis: results in 20 patients. J Vasc Interv Radiol 1998; 9: Gagner M. Laparoscopic Treatment of Acute Necrotizing Pancreatitis. Semin Laparosc Surg 1996; 3: van Santvoort HC, Besselink MG, Bakker OJ, Hofker HS, Boermeester MA, Dejong CH, van Goor H, Schaapherder AF, van Eijck CH, Bollen TL, van Ramshorst B, Nieuwenhuijs VB, Timmer R, Laméris JS, Kruyt PM, Manusama ER, van der Harst E, van der Schelling GP, Karsten T, Hesselink EJ, van Laarhoven CJ, Rosman C, Bosscha K, de Wit RJ, Houdijk AP, van Leeuwen MS, Buskens E, Gooszen HG. A step-up approach or open necrosectomy for necrotizing pancreatitis. N Engl J Med 2010; 362: Rocha FG, Benoit E, Zinner MJ, Whang EE, Banks PA, Ashley SW, Mortele KJ. Impact of radiologic intervention on mortality in necrotizing pancreatitis: the role of organ failure. Arch Surg 2009; 144: Mortelé KJ, Girshman J, Szejnfeld D, Ashley SW, Erturk SM, Banks PA, Silverman SG. CT-guided percutaneous catheter drainage of acute necrotizing pancreatitis: clinical experience and observations in patients with sterile and infected necrosis. AJR Am J Roentgenol 2009; 192: Bruennler T, Langgartner J, Lang S, Wrede CE, Klebl F, Zierhut S, Siebig S, Mandraka F, Rockmann F, Salzberger B, Feuerbach S, Schoelmerich J, Hamer OW. Outcome of patients with acute, necrotizing pancreatitis requiring drainage-does drainage size matter? World J Gastroenterol 2008; 14: Lee JK, Kwak KK, Park JK, Yoon WJ, Lee SH, Ryu JK, Kim YT, Yoon YB. The efficacy of nonsurgical treatment of infected pancreatic necrosis. Pancreas 2007; 34: Baril NB, Ralls PW, Wren SM, Selby RR, Radin R, Parekh D, Jabbour N, Stain SC. Does an infected peripancreatic fluid collection or abscess mandate operation? Ann Surg 2000; 231: Gambiez LP, Denimal FA, Porte HL, Saudemont A, Chambon JP, Quandalle PA. Retroperitoneal approach and endoscopic management of peripancreatic necrosis collections. 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156 Bello B et al. Minimally invasive treatment of pancreatic necrosis 18 Seifert H, Biermer M, Schmitt W, Jürgensen C, Will U, Gerlach R, Kreitmair C, Meining A, Wehrmann T, Rösch T. Transluminal endoscopic necrosectomy after acute pancreatitis: a multicentre study with long-term follow-up (the GEPARD Study). Gut 2009; 58: Coelho D, Ardengh JC, Eulálio JM, Manso JE, Mönkemüller K, Coelho JF. Management of infected and sterile pancreatic necrosis by programmed endoscopic necrosectomy. Dig Dis 2008; 26: Escourrou J, Shehab H, Buscail L, Bournet B, Andrau P, Moreau J, Fourtanier G. Peroral transgastric/transduodenal necrosectomy: success in the treatment of infected pancreatic necrosis. Ann Surg 2008; 248: Lopes CV, Pesenti C, Bories E, Caillol F, Giovannini M. Endoscopic-ultrasound-guided endoscopic transmural drainage of pancreatic pseudocysts and abscesses. Scand J Gastroenterol 2007; 42: Voermans RP, Veldkamp MC, Rauws EA, Bruno MJ, Fockens P. Endoscopic transmural debridement of symptomatic organized pancreatic necrosis (with videos). Gastrointest Endosc 2007; 66: Papachristou GI, Takahashi N, Chahal P, Sarr MG, Baron TH. Peroral endoscopic drainage/debridement of walledoff pancreatic necrosis. Ann Surg 2007; 245: Hookey LC, Debroux S, Delhaye M, Arvanitakis M, Le Moine O, Devière J. Endoscopic drainage of pancreatic-fluid collections in 116 patients: a comparison of etiologies, drainage techniques, and outcomes. Gastrointest Endosc 2006; 63: Charnley RM, Lochan R, Gray H, O Sullivan CB, Scott J, Oppong KE. Endoscopic necrosectomy as primary therapy in the management of infected pancreatic necrosis. Endoscopy 2006; 38: Seewald S, Groth S, Omar S, Imazu H, Seitz U, de Weerth A, Soetikno R, Zhong Y, Sriram PV, Ponnudurai R, Sikka S, Thonke F, Soehendra N. Aggressive endoscopic therapy for pancreatic necrosis and pancreatic abscess: a new safe and effective treatment algorithm (videos). Gastrointest Endosc 2005; 62: Baron TH, Harewood GC, Morgan DE, Yates MR. Outcome differences after endoscopic drainage of pancreatic necrosis, acute pancreatic pseudocysts, and chronic pancreatic pseudocysts. Gastrointest Endosc 2002; 56: Parekh D. Laparoscopic-assisted pancreatic necrosectomy: A new surgical option for treatment of severe necrotizing pancreatitis. Arch Surg 2006; 141: ; discussion Zhu JF, Fan XH, Zhang XH. Laparoscopic treatment of severe acute pancreatitis. Surg Endosc 2001; 15: Raraty MG, Halloran CM, Dodd S, Ghaneh P, Connor S, Evans J, Sutton R, Neoptolemos JP. Minimal access retroperitoneal pancreatic necrosectomy: improvement in morbidity and mortality with a less invasive approach. Ann Surg 2010; 251: Chang YC, Tsai HM, Lin XZ, Chang CH, Chuang JP. No debridement is necessary for symptomatic or infected acute necrotizing pancreatitis: delayed, mini-retroperitoneal drainage for acute necrotizing pancreatitis without debridement and irrigation. Dig Dis Sci 2006; 51: Castellanos G, Piñero A, Serrano A, Llamas C, Fuster M, Fernandez JA, Parrilla P. Translumbar retroperitoneal endoscopy: an alternative in the follow-up and management of drained infected pancreatic necrosis. Arch Surg 2005; 140: Carter CR, McKay CJ, Imrie CW. Percutaneous necrosectomy and sinus tract endoscopy in the management of infected pancreatic necrosis: an initial experience. Ann Surg 2000; 232: Horvath K, Freeny P, Escallon J, Heagerty P, Comstock B, Glickerman DJ, Bulger E, Sinanan M, Langdale L, Kolokythas O, Andrews RT. Safety and efficacy of videoassisted retroperitoneal debridement for infected pancreatic collections: a multicenter, prospective, single-arm phase 2 study. Arch Surg 2010; 145: S- Editor Gou SX L- Editor A E- Editor Xiong L 6835 December 14, 2012 Volume 18 Issue 46

157 Online Submissions: doi: /wjg.v18.i World J Gastroenterol 2012 December 14; 18(46): ISSN (print) ISSN (online) 2012 Baishideng. All rights reserved. BRIEF ARTICLE Dynamic magnetic resonance defecography in 10 asymptomatic volunteers Andreas G Schreyer, Christian Paetzel, Alois Fürst, Lena M Dendl, Elisabeth Hutzel, René Müller-Wille, Philipp Wiggermann, Stephan Schleder, Christian Stroszczynski, Patrick Hoffstetter Andreas G Schreyer, Lena M Dendl, René Müller-Wille, Philipp Wiggermann, Stephan Schleder, Christian Stroszczynski, Patrick Hoffstetter, Department of Radiology, University Medical Center Regensburg, Regensburg, Germany Christian Paetzel, Department of Radiology, Weiden Hospital, Weiden, Germany Alois Fürst, Elisabeth Hutzel, Department of Surgery, St. Josef Hospital, Regensburg, Germany Author contributions: Schreyer AG and Paetzel C contributed equally as first authors; Schreyer AG, Paetzel C, Fürst A and Hutzel E designed the research; Hoffstetter P, Schleder S, Wiggermann P and Hutzel E performed the research; Dendl LM, Müller-Wille R, Stroszczynski C and Schleder S analyzed the data; Schreyer AG, Hoffstetter P and Paetzel C wrote the paper. Correspondence to: Andreas G Schreyer, Professor, MD, MBA, Department of Radiology, University Medical Center Regensburg, Regensburg, Germany. [email protected] Telephone: Fax: Received: December 7, 2011 Revised: March 20, 2012 Accepted: May 13, 2012 Published online: December 14, 2012 Abstract AIM: Evaluation of the wide range of normal findings in asymptomatic women undergoing dynamic magnetic resonance (MR) defecography. METHODS: MR defecography of 10 healthy female volunteers (median age: 31 years) without previous pregnancies or history of surgery were evaluated. The rectum was filled with 180 ml gadolinium ultrasound gel mixture. MR defecography was performed in the supine position. The pelvic floor was visualized with a dynamic T2-weighted sagittal plane where all relevant pelvic floor organs were acquired during defecation. The volunteers were instructed to relax and then to perform straining maneuvers to empty the rectum. The pubococcygeal line (PCGL) was used as the line of reference. The movement of pelvic floor organs was measured as the vertical distance to this reference line. Data were recorded in the resting position as well as during the defecation process with maximal straining. Examinations were performed and evaluated by two experienced abdominal radiologists without knowledge of patient history. RESULTS: Average position of the anorectal junction was located at -5.3 mm at rest and mm during straining. The anorectal angle widened significantly from 93 at rest to 109 during defecation. A rectocele was diagnosed in eight out of 10 volunteers showing an average diameter of 25.9 mm. The bladder base was located at a position of +23 mm at rest and descended to -8.1 mm during defecation in relation to the PCGL. The bladder base moved below the PCGL in six out of 10 volunteers, which was formally defined as a cystocele. The uterocervical junction was located at an average level of mm at rest and at +7.9 mm during straining. The uterocervical junction of three volunteers fell below the PCGL; described formally as uterocervical prolapse. CONCLUSION: Based on the range of standard values in asymptomatic volunteers, MR defecography values for pathological changes have to be re-evaluated Baishideng. All rights reserved. Key words: Magnetic resonance imaging; Defecography; Standard values Peer reviewers: Edward J Ciaccio, PhD, Research Scientist, Department of Medicine, HP 804, Columbia University, 180 Fort Washington Avenue, New York, NY 10032, United States; Nageshwar D Reddy, Professor, Asian Institute of Gastroenterology, , Somajiguda, Hyderabad , India Schreyer AG, Paetzel C, Fürst A, Dendl LM, Hutzel E, Müller- Wille R, Wiggermann P, Schleder S, Stroszczynski C, Hoffstetter 6836 December 14, 2012 Volume 18 Issue 46

158 Schreyer AG et al. MRI defecography in healthy volunteers P. Dynamic magnetic resonance defecography in 10 asymptomatic volunteers. World J Gastroenterol 2012; 18(46): Available from: URL: v18/i46/6836.htm DOI: i INTRODUCTION As a result of the complex anatomy and synergism of pelvic organs and their muscular structures, there is a wide variety of static and functional disorders. Outlet obstruction, anism, dyskinesia of the puborectal muscle, intussusception, prolapse of the anus and rectum, vaginal prolapse, rectocele, cystocele, and enterocele represent common diagnoses in proctology, urology and gynecology. Besides constipation, fecal or urinary incontinence is the most common symptom, with serious problems for the patients that have a major negative impact on quality of life. Women are affected at a significantly higher rate with a ratio of 9:1 [1]. Patients usually attend gynecologists, urologists and proctologists. Therefore, a comprehensive interdisciplinary approach for diagnosis and therapy is the most promising strategy. A systematic medical history as well as a thorough proctological examination (inspection, palpation, rectoscopy, and proctoscopy) along with manometry and endosonography is the basic diagnostic approach for complex pelvic floor disorders. Furthermore, radiological imaging with evacuation proctography can give important additional information [2]. With the development of magnetic resonance imaging (MRI) technology, dynamic MRI of the pelvic floor has become an important alternative for the diagnosis of complex combined pelvic floor disorders. Since its first introduction by Yang et al [3] and Kruyt et al [4] in 1991, MRI has increasingly replaced evacuation proctography, which was first described in 1952 by Walldén [5], for evaluation of outlet obstruction. For the evaluation of extraluminal pelvic disorders such as enterocele or utero-vaginal prolapse, MRI of the pelvic floor is favorable in several aspects compared to clinical examination and conventional evacuation proctography, which does not depict extraluminal structures [3,4,6-10]. Additionally, MRI examination can be repeated because of the total lack of ionizing radiation. This may increase the chances of detecting pathological findings in some patients [9]. The horizontal position of the patient during MR defecography may be a disadvantage, because it could influence the pelvic floor physiology as well as the dynamic defecation process. Therefore, some authors consider that videoproctography is still superior to MRI for assessment of an enterocele or rectocele [11,12]. So far, patient preparation, examination technique, as well as reference lines for the evaluation of MRI are still not standardized and findings differ widely in the current literature. Most of the previously performed studies did not examine the defecation process itself. Normal findings and values were defined in small control groups and therefore were only applicable within the particular study setting. The aim of the present study was to show the wide range of normal findings in asymptomatic female volunteers and therefore the necessity of obtaining common standards not only in terms of patient preparation, but also in the evaluation of numeric values for the definition of pathological and normal dynamic MR examinations of the pelvic floor. MATERIALS AND METHODS Volunteers Ten healthy female volunteers (median age: 31 years, range: years) without previous pregnancy or history of any gynecological, urological or proctological surgery were evaluated by dynamic pelvic MRI. The volunteers had no symptoms of incontinence, constipation or other stool evacuation problems. None of the volunteers had any contraindications for MRI. Informed consent was obtained from all volunteers. We applied a uniform admission questionnaire for standardized documentation of patient history. The study was approved by the local ethics committee. Dynamic MRI of the pelvic floor All healthy volunteers were asked to empty their bladder 3 h before the examination to achieve a medium filling of the urinary bladder during MRI. The rectum was filled with 180 ml ultrasound gel mixture (1% Gd- DTPA-GEL-Mixture). MRI (1.5 Tesla MRI, Magnetom Symphony; Siemens, Erlangen, Germany) was performed in the supine position with hips and knees bent at 45. The pelvic floor was visualized in three planes (transversal, coronal, sagittal, T1 and T2) to find the appropriate sagittal plane in which all relevant pelvic floor organs were acquired during defecation over 55 s at a frequency of one shot per 1.1 s [True FISP (True Fast Imaging with Steady State Precession), TR: 5.8 ms, TE: 2.8 ms). Slice thickness was 7 mm (field of view: 270 mm 270 mm, matrix ). During the examination, healthy volunteers were instructed via headphones. They first were asked to relax and then to perform straining maneuvers to empty the rectum as completely as possible. The sequences were acquired digitally and analyzed cinematographically. MRI evaluation The pubococcygeal line (PCGL) was defined as the line of reference (Figure 1). Movement of pelvic floor organs was measured as the vertical distance with reference to this line. The location of structures above the PCGL were marked with positive values (+), structures located below this line were marked negative (-). Data were recorded in the resting position as well as during the defecation process with maximal straining. All data represents mean values with corresponding SD December 14, 2012 Volume 18 Issue 46

159 Schreyer AG et al. MRI defecography in healthy volunteers Pubococcygeal line Cystocele Rectocele uterovaginal prolapse Figure 1 The pubococcygeal line (black) according to Yang et al [3] from the most inferior part of the pubic symphysis to the last coccygeal joint; furthermore, gray dots show (from left to right) bladder base, uterocervical junction, and anorectal junction. Figure 2 Dynamic magnetic resonance imaging of a 36-year-old healthy female volunteer. At the end of defection, a rectocele, cystocele and uterovaginal prolapse were visualized. The anorectal junction was defined as the intersection point of the central axis of the anal canal and a line along the posterior rectal wall. The anorectal angle was measured between these two lines [3,7,9]. According to the current literature, based on different evaluation techniques, a rectocele is defined as a bulge of the anterior rectal wall of > mm [11,13]. For our data analysis we applied the method described by Delemarre, who defined the distance from the anorectal junction to the tip of the protrusion of the anterior rectal wall as the correct measurement [11]. MRI revealed data about the position of the bladder base, the uterocervical junction and the vagina. Additionally the location of small bowel in relation to the PCGL was assessed. A cystocele and a uterovaginal prolapse were diagnosed if the bladder base or the uterocervical junction fell below the PCGL during defecation [9,10]. Widening of the rectovaginal space or a descent of mesenteric parts, small bowel or sigmoid colon beyond the PCGL during defecation was defined as an enterocele [8,10]. All examinations were performed and evaluated in consensus by two experienced board-certified abdominal radiologists without any knowledge of the volunteers history or age. RESULTS All 10 volunteers were able to hold the rectal enema and perform defecation within the MR scanner according to the examination protocol. The average position of the anorectal junction was located at -5.3 mm (± 9.9 mm) at rest and descended to mm (± 10.3 mm) during maximal straining. The average anorectal angle widened significantly from 93 (± 4.8 ) at rest to (± 14.7 ) at maximal straining during defecation. A rectocele based on the current definition of an anterior bulging of the anterior rectum wall of at least 20 mm was diagnosed in eight of 10 volunteers, showing an average size of 26 mm (± 6 mm). The bladder base was located at an average position Table 1 Results of magnetic resonance defecography of 10 healthy asymptomatic volunteers Anatomic structure Rest Straining Relative movement Annorectal junction (mm) -5.3 (± 9.9) (± 10.3) Anorectal angle ( ) (± 4.8) (± 14. 7) 15.7 Bladder base (mm) 23.0 (± 4.6) -8.1 (± 11.1) Uterovaginal junction (mm) 43.1 (± 7.8) 7.9 (± 16.5) A positive value represents the lowest position of the pelvic structure above the pubococcygeal line, a negative value marks structures located below the pubococcygeal line. The values represent the median measured distance in millimeters; the numbers in brackets represent the range. of +23 mm (± 4.6 mm) at rest and descended to -8.1 mm (± 11.1 mm) during defecation. The bladder base moved below the PCGL in six of 10 healthy volunteers during maximal straining, which is defined as a cystocele (Figure 2). The uterocervical junction or vaginal vault was located at an average level of mm (± 7.8 mm) at rest and at +7.9 mm (± 16.5 mm) during maximal straining. The uterocervical junction of three volunteers descended below the PCGL at maximal straining during defecation, described as uterocervical prolapse according to the common definition in the literature (Figure 2). No enterocele or other additional pathological findings were detected in any volunteers. Table 1 shows an overview of all results and findings of the dynamic MRI examinations. DISCUSSION Dynamic MRI of the pelvic floor, also known as MR defecography, is increasingly used for the diagnosis of complex pelvic floor disorders and replaces conventional videoproctography. Different medical specialties such as radiology, urology, gynecology and surgery are involved in the interpretation and evaluation of findings and therefore need an accepted definition of normal values and clinical practical reference lines for image assessment December 14, 2012 Volume 18 Issue 46

160 Schreyer AG et al. MRI defecography in healthy volunteers Back in 1991, Kruyt et al [4] were the first authors using MRI to study functional aspects of the anorectal region. In their study, the anatomy of the anorectum and the anorectal angle was examined in 10 healthy volunteers placed in the prone position within the MRI scanner. The symphysiosacral line reaching from the most superior part of the pubic symphysis to the lower part of the sacrum, was taken as the reference line to evaluate the mobility of the pelvic organs. Dynamics were measured during contraction and relaxation of the pelvic floor and during straining. In this study, the defecation process itself was not visualized because no contrast medium or enema was applied to the rectum. Also in 1991, Yang et al [3] introduced dynamic MRI as a new method for the diagnosis of the descending perineum in women. They compared 26 patients with 16 asymptomatic women in the supine position. Values were documented with various degrees of straining maneuvers. Again, defecation itself was not because the rectum was not filled with contrast medium. The PCGL was taken as the reference line to evaluate the degree of descent of the pelvic structures. Another study comparing clinical examination, videoproctography and dynamic MRI in the diagnosis of anterior rectoceles was published by Delemarre et al [11]. In this study patients were examined in the prone position without any rectal enema, which again made evaluation of the defecation process itself impossible. The pubosacral line reaching from the most inferior part of the pubic symphysis to the lower part of the sacrum was chosen as the reference line for MRI. Measurements were performed at rest and during straining for both imaging techniques. The pubosacral line was also used by Goodrich et al [6] who examined five female patients with descending perineum syndrome pre- and postoperatively, as well as 10 asymptomatic female volunteers undergoing MRI. In contrast to the previous study, patients were placed in the supine position. The authors did not state whether the rectum, vagina or bladder had been contrasted. Healy et al [8,14] have analyzed various aspects of pelvic floor disorders in patients and healthy volunteers comparing videoproctography with dynamic MRI with patients placed in the supine position. The defecation maneuver was not acquired. In contrast to other studies [9,10,12], instead of contrast medium, a tampon was placed in the rectum. Measurements were performed during maximal straining, while the lower PCGL was the reference landmark. Tacke [15] has tested a new method of dynamic MRI with radial real-time imaging and a reduced image area for defecography. Patients in the supine position were asked to void a condom filled with a gadolinium-based contrast gel. The authors discussed this form of rectal filling critically as it may mask intussusception or latent incontinence. Vanbeckevoort et al [12] compared colpocystoproctography (videoproctography with opacification of vagina and bladder) and dynamic MRI in supine position. For MRI, the rectum was filled with 100 ml of ultrasound gel, which was not meant to be voided. Measurements were taken during maximal straining using the PCGL as a reference line. Lienemann et al [10] compared colpocystoproctography and MRI for the diagnosis of enteroceles. In MRI, patients and healthy volunteers were placed in a supine position and the rectum was filled with 200 ml of ultrasound gel, which was to be defecated during imaging. The study does not reveal data on frequency and completeness of the defecation process. Reference line was the PCGL. In a technically equal approach, Lienemann et al [9] examined 44 female patients and five asymptomatic volunteers for descent of the pelvic floor. Patients and volunteers were asked not to void the gel. Additionally, there was no discussion of the findings of the healthy volunteers in this study. Hilfiker et al [16] and Schoenberger et al [17] presented an open system MRI where patients were examined in an upright position analogous to videoproctography. In the study of Schoenberger et al [17], the findings of 15 patients examined with videoproctography and this new form of open configured MRI were compared. Five healthy volunteers were included for the definition of normal values, which are not mentioned in the paper. This overview illustrates the problems arising with the introduction of a new examination technique. So far, dynamic MRI is widely accepted as a promising technique for the diagnosis of the pelvic floor [3,4,6,9,10], particularly for functional aspects of pelvic floor disorders because pelvic organs and muscles can be visualized and evaluated without invasive opacification and without any exposure to ionizing radiation. Still, without standardization of patient preparation, examination technique and evaluation of the data according to standardized reference lines and landmarks, it may not yet replace a well-established technique like videoproctography or colpocystoproctography. We believe that a comparison of both procedures has to take place under standardized conditions. In this context, the documentation of the defecation process is most important for subsequent comparability of both techniques. Vanbeckevoort et al [12] have compared the results of 35 patients examined with colpocystoproctography with and without defecation with the findings of dynamic MRI without defecation. For colpocystoproctography the urinary bladder and the small bowel were filled with a contrast medium. In their analysis, colpocystoproctography including defecation was by far superior to the same technique without defecation. Based on the observation that the pelvic floor reached its maximum downward movement only during defecation, and supported by the fact that patients are placed in a horizontal position in MRI, the authors concluded that colpocystoproctography including defecation may also be superior to MRI without defecation. Our results also revealed that the formation of a rectocele and 6839 December 14, 2012 Volume 18 Issue 46

161 Schreyer AG et al. MRI defecography in healthy volunteers Table 2 Patient preparation, evaluation techniques, normal values and findings as described by other authors Yang et al [3] Kruyt et al [4] Vanbeckevoort et al [12] Lienemann et al [9] Healy et al [14] Paetzel et al [19] Defecation No No No No No Yes Rectal contrast No No 100 ml mg 200 ml mg Plastic tube 180 ml mg Bladder contrast No No No 60 ml sodium chloride No No Vaginal contrast No No No 50 ml ug Plastic tube No Position Horizontal Prone Horizontal Horizontal Horizontal Horizontal Reference line Pubococcygeal Symphysiosacral Pubosacral Pubococcygeal Pubococcygeal Pubococcygeal Anorectal junction -25 mm -30 mm to -40 mm -25 mm No information -20 mm -5.3 mm Bladder base -10 mm No information ± 0 ± 0-10 mm +23 mm Uterocervical junction +10 mm No information No information +0 ± mm Rectocele (evaluation) No information No information Yoshioka Yoshioka Yoshioka Delemarre Rectocele (size) No information no information < 30 mm < 30 mm no information 26 mm Anorectal angle No information < 130 (rest) No information No information No information 93 (rest) a cystocele as well as the maximal descent of the anorectal junction and of the uterocervical junction was only completely visible at the end of the defecation process but not during straining alone. In our study, the rectum was filled with 180 ml ultrasound gel with a gadolinium-based contrast medium. Ikenberry et al [18] have shown that varying viscosity of the contrast medium does not significantly influence the findings in videoproctography. Due to the natural differences in signal intensity of the urinary bladder, vagina, small bowel, and peritoneum, the procedure can be kept on a low level of invasivity. The lower PCGL was used to visualize relative movements of the pelvic organs during defecation. The anorectal junction and anorectal angle were determined by the central axis of the anal canal and a line along the posterior wall of the rectum [8-10,14]. According to Delemarre et al [11], rectoceles were measured from the anorectal junction to the tip of protrusion of the anterior rectum wall. Table 2 gives an overview of different techniques and normal values as described in other studies. The position of the anorectal junction related to the PCGL at rest and during defecation is one parameter in the evaluation of a descent of the perineum. In the present study the anorectal junction moved 30 mm on average below the PCGL during defecation. In a previously published study by our group we evaluated symptomatic patients with pelvic floor disorders. In this study the anorectal junction moved an average 49 mm below the PCGL, while in patients suffering from a rectal incontinence, an average of 51 mm was measured [19]. Besides the descent of the anorectal junction, changes in the anorectal angle are commonly used as an indicator for the functional status of the pelvic floor [20]. A narrowing of the anorectal angle may indicate a disorder of the puborectal muscle [21]. This may lead to constipation with subsequent straining leading to rectal intussusception, rectocele and mucosal prolapse with a solitary ulcer of the rectum [21,22]. If the anorectal angle is already widened at rest, this may be a sign of weakness of the pelvic floor and is commonly observed along with incontinence and rectal prolapse [23-25]. Standard values in the literature vary enormously. For videoproctography, Hardcastle et al [26] described normal values between 60 and 105 at rest whereas the findings of other groups [27-29] relying on control groups with up to 150 volunteers show values between 90 and 104 at rest and 103 to 137 during defecation. Comparing the data with our study we determined an average angle of 93 at rest and during straining. Accordingly, the use of changes in the anorectal angle as a diagnostic parameter is difficult not only because data vary significantly but also because findings of patients and asymptomatic volunteers tend to overlap [4,7,8,15,29]. A patient with an anterior rectocele often presents with symptoms of incomplete defecation and are observed along with a descent of the pelvic floor [13]. However, a rectocele can also frequently be found in asymptomatic patients. Therefore, some authors assume that symptoms depend on the diameter of the rectocele [9,29,30]. Due to different approaches in the attempt to measure the expansion of a rectocele, there are still no well-defined normal values available. Delemarre et al [11] used videoproctography and MRI to examine 38 patients in the prone position without rectal filling and without defecation. He concluded that videoproctography was superior to MRI for diagnosis of rectoceles. He postulated that a symptomatic rectocele > 20 mm needs to be operated. Lienemann et al [9] and Yoshioka et al [13] have defined a rectocele as a protrusion of the anterior rectal wall of > 30 mm. Yoshioka et al [13] have found that, in comparison to clinical examination, MRI was superior to colpocystoproctography in detecting rectoceles. Patients were examined in the supine position with rectal filling during maximal straining but without defecation. In contrast, Healy et al [8] rated videoproctography as superior to MRI. They have defined expansion of > 20 mm as pathological. In addition, they found that a rectocele < 13 mm measured by videoproctography was totally missed by MRI. MRI examination again was performed without defecation. According to our observations the horizontal position does not seem to be a disadvantage because a rectocele was found in eight out of 10 healthy volunteers, with an average size of 26 mm (Table 1). This was in accordance with data from the literature [9,14] where the 6840 December 14, 2012 Volume 18 Issue 46

162 Schreyer AG et al. MRI defecography in healthy volunteers incidence of a rectocele in asymptomatic volunteers was about 80%, although these are described as small rectoceles. Incidence and degree of a cystocele and an uterocervical prolapse is usually connected with the number of vaginal deliveries, preceding hysterectomy, and chronic constipation with increased straining maneuvers [12]. Besides clinical examination, imaging techniques make quantification of findings possible [9]. Yang et al [3], Lienemann et al [9], Vanbeckevoort et al [12] and Healy et al [7,14] have used normal values for the descent of the bladder base and uterocervical junction during straining in relation to the PCGL. These values were raised partly in healthy volunteers and partly determined at random. Due to the natural differences in signal intensity of the urinary bladder and the vagina there was no necessity to apply additional contrast media [16]. In our study, six of 10 asymptomatic healthy female volunteers without a history of previous delivery or surgery showed the finding of a cystocele, and in three of 10 a vaginal prolapse was diagnosed. In the studies of Lienemann et al [31] and Sprenger et al [32], 20 and 39 healthy females were examined with dynamic MRI of the pelvic floor, including defecation. A cystocele or vaginal prolapse was detected in none of them. An enterocele is defined as herniation of the peritoneum into the rectovaginal space [33], which may contain small bowel loops or sigmoid colon. They are often accompanied by severe defecation disorders and a sensation of pressure as well as downward movement of the pelvic floor [34]. The prevalence of an enterocele in women is between 18% and 37% [35,36]. They often occur after hysterectomy [33]. No enterocele was detected in any volunteer. Lienemann et al [10] examined 55 patients and 11 asymptomatic volunteers with colpocystoproctography and dynamic MRI, without application of contrast medium into the peritoneum or small bowel. MRI had a clear advantage because the peritoneum and contents of the enterocele were easily identified. He concluded that MRI may replace colpocystoproctography in the diagnosis of an enterocele. It proved to be useful to acquire the complete defecation process after a rectal enema to detect potential pathological changes. A rectocele can easily be identified by dynamic MRI. The pelvic organs of healthy volunteers show a relatively high mobility, therefore, presently suggested normal values for the position of pelvic organs in relation to the PCGL have to be redefined. It is necessary to evaluate normal values under standardized investigation conditions and with large groups of healthy volunteers. COMMENTS Background Pelvic floor disorders are commonly seen in proctology, urology and gynecology, and the patients are mostly women. Due to the complexity of anatomy and physiology of the pelvic floor we have come across a wide variety of disorders. Research frontiers Imaging plays an important role in the diagnostic workup of complex combined pelvic floor disorders. Functional examinations like videoproctography provide valuable information but it has been increasingly replaced by dynamic magnetic resonance imaging (MRI) that can visualize intra- and extraluminal structures. Further strengths of MRI are excellent soft tissue contrast and the lack of ionizing radiation. Innovations and breakthroughs There have been several studies about the diagnostic value of dynamic MRI of the pelvic floor; most of them with symptomatic patients. This investigation is believed to be the first to document the range of normal findings in MR defecography of asymptomatic female volunteers. Applications The wide range of normal findings in this study emphasizes the necessity of obtaining common standards for the evaluation of MR defecography. Terminology MR defecography is a dynamic pelvic MRI examination. After optional application of a rectal enema, defecation is visualized by a series of fast MRI sequences. Peer review This is a good study but the authors should make some revision. REFERENCES 1 Winkler R. Proktologie- Ein Leitfaden für die Praxis. Stuttgart-New York: Georg Thieme Verlag, Herold A, Müller-Lobeck H, Jost WH, Duschka L, Leder D. [Diagnostic evaluation of the rectum and pelvic floor in chronic constipation]. Zentralbl Chir 1999; 124: Yang A, Mostwin JL, Rosenshein NB, Zerhouni EA. Pelvic floor descent in women: dynamic evaluation with fast MR imaging and cinematic display. Radiology 1991; 179: Kruyt RH, Delemarre JB, Doornbos J, Vogel HJ. Normal anorectum: dynamic MR imaging anatomy. Radiology 1991; 179: Wallden L. Defecation block in cases of deep rectogenital pouch. Acta Chir Scand 1952; 103: Goodrich MA, Webb MJ, King BF, Bampton AE, Campeau NG, Riederer SJ. Magnetic resonance imaging of pelvic floor relaxation: dynamic analysis and evaluation of patients before and after surgical repair. Obstet Gynecol 1993; 82: Healy JC, Halligan S, Reznek RH, Watson S, Bartram CI, Kamm MA, Phillips RK, Armstrong P. Magnetic resonance imaging of the pelvic floor in patients with obstructed defaecation. Br J Surg 1997; 84: Healy JC, Halligan S, Reznek RH, Watson S, Bartram CI, Phillips R, Armstrong P. Dynamic MR imaging compared with evacuation proctography when evaluating anorectal configuration and pelvic floor movement. AJR Am J Roentgenol 1997; 169: Lienemann A, Anthuber C, Baron A, Kohz P, Reiser M. Dynamic MR colpocystorectography assessing pelvic-floor descent. Eur Radiol 1997; 7: Lienemann A, Anthuber C, Baron A, Reiser M. Diagnosing enteroceles using dynamic magnetic resonance imaging. Dis Colon Rectum 2000; 43: ; discussion Delemarre JB, Kruyt RH, Doornbos J, Buyze-Westerweel M, Trimbos JB, Hermans J, Gooszen HG. Anterior rectocele: assessment with radiographic defecography, dynamic magnetic resonance imaging, and physical examination. Dis Colon Rectum 1994; 37: Vanbeckevoort D, Van Hoe L, Oyen R, Ponette E, De Ridder D, Deprest J. Pelvic floor descent in females: comparative study of colpocystodefecography and dynamic fast MR imaging. J Magn Reson Imaging 1999; 9: Yoshioka K, Matsui Y, Yamada O, Sakaguchi M, Takada H, Hioki K, Yamamoto M, Kitada M, Sawaragi I. Physiologic 6841 December 14, 2012 Volume 18 Issue 46

163 Schreyer AG et al. MRI defecography in healthy volunteers and anatomic assessment of patients with rectocele. Dis Colon Rectum 1991; 34: Healy JC, Halligan S, Reznek RH, Watson S, Phillips RK, Armstrong P. Patterns of prolapse in women with symptoms of pelvic floor weakness: assessment with MR imaging. Radiology 1997; 203: Tacke J, Nolte-Ernsting C, Glowinski A, Schäffter T, Adam G, Günther RW. [MR defecography at 1.5 Tesla with radial real-time imaging and reduced image field]. Rofo 1999; 171: Hilfiker PR, Debatin JF, Schwizer W, Schoenenberger AW, Fried M, Marincek B. MR defecography: depiction of anorectal anatomy and pathology. J Comput Assist Tomogr 1998; 22: Schoenenberger AW, Debatin JF, Guldenschuh I, Hany TF, Steiner P, Krestin GP. Dynamic MR defecography with a superconducting, open-configuration MR system. Radiology 1998; 206: Ikenberry S, Lappas JC, Hana MP, Rex DK. Defecography in healthy subjects: comparison of three contrast media. Radiology 1996; 201: Paetzel C, Strotzer M, Fürst A, Rentsch M, Lenhart M, Feuerbach S. [Dynamic MR defecography for diagnosis of combined functional disorders of the pelvic floor in proctology]. Rofo 2001; 173: Yang XM, Partanen K, Farin P, Soimakallio S. Defecography. Acta Radiol 1995; 36: Johansson C, Ihre T, Holmström B, Nordström E, Dolk A, Brodén G. A combined electromyographic and cineradiologic investigation in patients with defecation disorders. Dis Colon Rectum 1990; 33: Goei R, Baeten C, Janevski B, van Engelshoven J. The solitary rectal ulcer syndrome: diagnosis with defecography. AJR Am J Roentgenol 1987; 149: Goei R. Anorectal function in patients with defecation disorders and asymptomatic subjects: evaluation with defecography. Radiology 1990; 174: Hardcastle JD. The descending perineum syndrome. Practitioner 1969; 203: Porter NH. A physiological study of the pelvic floor in rectal prolapse. Ann R Coll Surg Engl 1962; 31: Hardcastle JD, Parks AG. A study of anal incontinence and some principles of surgical treatment. Proc R Soc Med 1970; 63 Suppl: Mahieu P, Pringot J, Bodart P. Defecography: I. Description of a new procedure and results in normal patients. Gastrointest Radiol 1984; 9: Piloni V, Fioravanti P, Spazzafumo L, Rossi B. Measurement of the anorectal angle by defecography for the diagnosis of fecal incontinence. Int J Colorectal Dis 1999; 14: Shorvon PJ, McHugh S, Diamant NE, Somers S, Stevenson GW. Defecography in normal volunteers: results and implications. Gut 1989; 30: Kelvin FM, Maglinte DD, Benson JT. Evacuation proctography (defecography): an aid to the investigation of pelvic floor disorders. Obstet Gynecol 1994; 83: Lienemann A, Sprenger D, Janssen U, Anthuber C, Reiser M. [Functional MRI of the pelvic floor. The methods and reference values]. Radiologe 2000; 40: Sprenger D, Lienemann A, Anthuber C, Reiser M. [Functional MRI of the pelvic floor: its normal anatomy and pathological findings]. Radiologe 2000; 40: Holley RL. Enterocele: a review. Obstet Gynecol Surv 1994; 49: Shull BL. Clinical evaluation of women with pelvic support defects. Clin Obstet Gynecol 1993; 36: Maglinte DD, Kelvin FM, Hale DS, Benson JT. Dynamic cystoproctography: a unifying diagnostic approach to pelvic floor and anorectal dysfunction. AJR Am J Roentgenol 1997; 169: Mellgren A, Johansson C, Dolk A, Anzén B, Bremmer S, Nilsson BY, Holmström B. Enterocele demonstrated by defaecography is associated with other pelvic floor disorders. Int J Colorectal Dis 1994; 9: S- Editor Cheng JX L- Editor Kerr C E- Editor Xiong L 6842 December 14, 2012 Volume 18 Issue 46

164 Online Submissions: doi: /wjg.v18.i World J Gastroenterol 2012 December 14; 18(46): ISSN (print) ISSN (online) 2012 Baishideng. All rights reserved. BRIEF ARTICLE Improved techniques for double-balloon-enteroscopyassisted endoscopic retrograde cholangiopancreatography Takashi Osoegawa, Yasuaki Motomura, Kazuya Akahoshi, Naomi Higuchi, Yoshimasa Tanaka, Terumasa Hisano, Souichi Itaba, Junya Gibo, Mariko Yamada, Masaru Kubokawa, Yorinobu Sumida, Hirotada Akiho, Eikichi Ihara, Kazuhiko Nakamura Takashi Osoegawa, Yasuaki Motomura, Kazuya Akahoshi, Naomi Higuchi, Yoshimasa Tanaka, Terumasa Hisano, Souichi Itaba, Junya Gibo, Mariko Yamada, Masaru Kubokawa, Department of Gastroenterology, Aso Iizuka Hospital, Iizuka , Japan Yorinobu Sumida, Hirotada Akiho, Eikichi Ihara, Kazuhiko Nakamura, Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka , Japan. Author contributions: Osoegawa T, Motomura Y, and Higuchi N designed the research; Osoegawa T, Motomura Y, Higuchi N, Tanaka Y, Hisano T, Itaba S, Gibo J, Yamada M, Kubokawa M, Sumida Y, and Akiho H analyzed and interpreted the data; Osoegawa T and Motomura Y drafted the article; Akahoshi K, Ihara E, and Nakamura K revised the article for important intellectual content. Correspondence to: Kazuya Akahoshi, MD, PhD, Department of Gastroenterology, Aso Iizuka Hospital, 3-83 Yoshiomachi, Iizuka , Japan. [email protected] Telephone: Fax: Received: May 11, 2012 Revised: September 27, 2012 Accepted: October 16, 2012 Published online: December 14, 2012 Abstract AIM: To investigate the clinical outcome of double balloon enteroscopy (DBE)-assisted endoscopic retrograde cholangiopancreatography (DB-ERCP) in patients with altered gastrointestinal anatomy. METHODS: Between September 2006 and April 2011, 47 procedures of DB-ERCP were performed in 28 patients with a Roux-en-Y total gastrectomy (n = 11), Billroth Ⅱ gastrectomy (n = 15), or Roux-en-Y anastomosis with hepaticojejunostomy (n = 2). DB-ERCP was performed using a short-type DBE combined with several technical innovations such as using an endoscope attachment, marking by submucosal tattooing, selectively applying contrast medium, and CO2 insufflations. RESULTS: The papilla of Vater or hepaticojejunostomy site was reached in its entirety with a 96% success rate (45/47 procedures). There were no significant differences in the success rate of reaching the blind end with a DBE among Roux-en-Y total gastrectomy (96%), Billroth Ⅱ reconstruction (94%), or pancreatoduodenectomy (100%), respectively (P = 0.91). The total successful rate of cannulation and contrast enhancement of the target bile duct in patients whom the blind end was reached with a DBE was 40/45 procedures (89%). Again, there were no significant differences in the success rate of cannulation and contrast enhancement of the target bile duct with a DBE among Roux-en-Y total gastrectomy (88 %), Billroth Ⅱ reconstruction (89%), or pancreatoduodenectomy (100%), respectively (P = 0.67). Treatment was achieved in all 40 procedures (100%) in patients whom the contrast enhancement of the bile duct was successful. Common endoscopic treatments were endoscopic biliary drainage (24 procedures) and extraction of stones (14 procedures). Biliary drainage was done by placement of plastic stents. Stones extraction was done by lithotomy with the mechanical lithotripter followed by extraction with a basket or by the balloon pull-through method. Endoscopic sphincterotomy was performed in 14 procedures with a needle precutting knife using a guidewire. The mean total duration of the procedure was 93.6 ± 6.8 min and the mean time required to reach the papilla was 30.5 ± 3.7 min. The mean time required to reach the papilla tended to be shorter in Billroth Ⅱ reconstruction (20.9 ± 5.8 min) than that in Roux-en-Y total gastrectomy (37.1 ± 4.9 min) but there was no significant difference (P = 0.09). A major complication occurred in one patient (3.5%); perforation of the long limb in a patient with Billroth Ⅱ anastomosis December 14, 2012 Volume 18 Issue 46

165 Osoegawa T et al. Technical tips for DB-ERCP CONCLUSION: Short-type DBE combined with several technical innovations enabled us to perform ERCP in most patients with altered gastrointestinal anatomy Baishideng. All rights reserved. Key words: Double-balloon enteroscopy; Endoscopic retrograde cholangiopancreatography; Pathological anatomy; Pancreatobiliary disease Peer reviewers: William Dickey, Departments of Gastroenterology and Histopathology, Altnagelvin Hospital, Londonderry, Northern Ireland BT47 6SB, United Kingdom; Dr. Masayoshi Ito, Department of Endoscopy, Yotsuya Medical Cube, 7-7 Nibancho, Chiyoda-ku, Tokyo , Japan Osoegawa T, Motomura Y, Akahoshi K, Higuchi N, Tanaka Y, Hisano T, Itaba S, Gibo J, Yamada M, Kubokawa M, Sumida Y, Akiho H, Ihara E, Nakamura K. Improved techniques for doubleballoon-enteroscopy-assisted endoscopic retrograde cholangiopancreatography. World J Gastroenterol 2012; 18(46): Available from: URL: v18/i46/6843.htm DOI: i INTRODUCTION Patients who have undergone bowel reconstruction have been considered unsuitable for endoscopic retrograde cholangiopancreatography (ERCP) using conventional endoscopy [1-6]. This is because the endoscopic approach to the afferent loop, blind end, and the choledochojejunostomy site using a conventional endoscope is difficult because of the distance from the gastrojejunal anastomosis site, the unusual anatomical features of the intestine such as its winding form, and postoperative adhesions. Recently, double balloon enteroscopy (DBE) has been reported to be of significant benefit for the diagnosis and treatment of biliary and pancreatic diseases in such patients [7-13]. DBE enables us to advance much deeper into the small intestine than using a conventional push-enteroscope [14-16]. It is still difficult, however, not only to reach the papilla of Vater or the site of choledochojejunostomy, but also to cannulate selectively into the pancreatic and/or biliary duct in ERCP using DBE (DB-ERCP). Therefore, further contrivances including both instrumental and technical innovations have been required to improve the outcome of DB-ERCP. One of the most significant instrumental innovations is a shorttype DBE, which has been recently developed, and contributes to increased success rates not only for reaching the papilla and blind end but also for ERCP-related interventions because of its short working length and the availability of various accessory devices [17,18]. There has still been a need for developing the technical innovations to obtain more favorable outcome of the DB-ERCP. This study describes several technical innovations and tips in the use of a short-type DBE to improve clinical outcomes of DB-ERCP. MATERIALS AND METHODS Patient population Between September 2006 and April 2011, 48 ERCP procedures were analyzed in 28 patients (10 female, 18 male; age years, mean 74.0 years) with pancreatobiliary diseases who had previously undergone bowel reconstruction. The bowel reconstruction methods included Roux-en-Y total gastrectomy in 11 patients (26 procedures), Billroth Ⅱ gastrectomy in 15 patients (18 procedures), and Roux-en-Y anastomosis with pancreatoduodenectomy in two patients (three procedures). Patients with Roux-en-Y total gastrectomy or Billroth Ⅱ gastrectomy had naïve papilla (n = 26), and those with pancreatoduodenectomy had hepaticojejunostomy (n = 2). Twenty-eight consecutive postsurgical patients were included in this study after written informed consent was obtained. Endoscope and accessories We used short-type DBE, EC-450B15 (Fujifilm, Saitama, Japan). The DBE system is a high-resolution videoendoscope with a flexible overtube. The videoendoscope has a 2.8-mm working channel and a working length of 1520 mm, and a detachable balloon at its tip. It is used with a soft overtube measuring 1050 mm in length with another balloon at the distal end. The endoscope and overtube balloons are made from latex, which is 0.1 mm thick and very soft. The balloons can be inflated or deflated by a specially designed air pump controller with one-touch controls, while monitoring the air pressure. A soft transparent hood (DH-17EN; Fujifilm) was attached to the tip of a scope in all procedures. DB-ERCP All procedures were performed during conscious sedation by giving 35 mg pethidine hydrochloride and mg of flunitrazepam intravenously. Scopolamine butylbromide was only used after reaching the end of the afferent loop. DBE was performed using a standard technique as described by Yamamoto et al [14], Kita et al [15] and May et al [16], and DB-ERCP was carried out as follows. When an endoscopist was not sure whether the endoscope was in the afferent loop, selective contrast enhancement in the intestine was performed with a balloon inflated on the endoscope tip, allowing the endoscopist to confirm the direction of the afferent loop under fluoroscopy [17,18]. Although formerly we used standard air insufflations during the procedure, since April 2009 we have used CO2 instead of standard air insufflations to improve intubation depth during DBE, as well as to reduce postprocedural pain [19]. In the first procedure, the beginning of the Roux limb was marked by submucosal tattooing (Indian ink) in patients with a Roux-en-Y anastomosis (Figure 1). The time taken for this procedure and the whole procedure was recorded. Once the endoscope reached the papilla or ductal anastomosis, appropriate stabilization of the enteroscope with the overtube and/or enteroscope balloon 6844 December 14, 2012 Volume 18 Issue 46

166 Osoegawa T et al. Technical tips for DB-ERCP A B C D Figure 1 In the first procedure, the beginning of the Roux limb was marked by submucosal tattooing in patients with a Roux-en-Y anastomosis. A B Hood Papilla C D Figure 2 A soft transparent hood (DH-17EN; Fujifilm) was attached to the tip of a scope in all procedures. was often required prior to ERCP. Using DBE, the endoscopist could stabilize the endoscope by the deep insertion of an overtube, and immobilize the intestine in a position that allowed the bile duct to run in the 11 o clock direction, as in the conventional ERCP view. A soft transparent hood was attached to the tip of a scope in all procedures. The attachment hood was useful to fix the ampulla (Figure 2). Selective biliary cannulation 6845 December 14, 2012 Volume 18 Issue 46

167 Osoegawa T et al. Technical tips for DB-ERCP Table 1 Success rate of double balloon enteroscopy-assisted endoscopic retrograde cholangiopancreatography n (%) Reaching the papilla of Vater Contrast enhancement of target bile duct Treatment of pancreatic and/or biliary duct Roux-en-Y reconstruction 24 (96) 21 (88) 21 (100) Billroth Ⅱ gastrectomy 18 (95) 16 (89) 16 (100) Pancreatoduodenectomy 3 (100) 3 (100) 3 (100) Total 45 (96) 40 (89) 40 (100) Figure 3 A sphincterotome with rotatable tip (Autotome RX49; Boston Scientific) was also useful for cannulation in some cases, in which biliary cannulation was unsuccessful with a standard cannula. was achieved using a standard cannula or, if necessary, a sphincterotome (Autotome RX49; Boston Scientific, Tokyo, Japan) with a tip rotatable through 360 (Figure 3). Guidewire (Jagwire 0.035; Boston Scientific) was preloaded into the cannula or sphincterotome, and was placed in the bile duct after successful cannulation for the following procedure. Endoscopic sphincterotomy (EST) was performed using a sphincterotome with rotatable tip or a precutting knife (Single Use 3-Lumen Needle knife V KD-V441M; Olympus, Tokyo, Japan). When EST was difficult, endoscopic papillary balloon dilation was performed with an 8- or 10-mm balloon dilation catheter (Hurricane RX 4594; Boston Scientific). When the opening of the choledochojejunostomy was stenotic, balloon dilation using either an 8-10-mm balloon dilation catheter or 12-mm balloon catheter (CRE Wireguided Balloon Dilatation Catheter 5842; Boston Scientific) was performed. Stone extraction was performed with a six-wire basket (MTW) or a mechanical lithotripter (Xemex crusher catheter; Zeon Medical, Tokyo, Japan). After the stones were removed, a retrieval balloon catheter (Extractor ; Boston Scientific) was used with a guidewire to confirm that no small stones remained. In patients with the bile duct winding at sharp angles where the mechanical lithotripter could not be inserted, and in patients with a bulky stone that could not be extracted in one attempt, a 7-Fr plastic stent (Flexima Biliary Stent System 3920; Boston Scientific) was positioned. Statistical analysis All data are expressed as mean ± SE, if applicable. Statistical analyses were performed with JMP statistical software (Version 9.0). Pearson s χ 2 test was performed to compare the success rate of DB-ERCP among Rouxen-Y total gastrectomy, Billroth II reconstruction, and pancreatoduodenectomy. An analysis of variance followed by Tukey s multiple comparison test was carried out to determine statistical significance among these three groups. A value of P < 0.05 was considered statistically significant. RESULTS Reaching the blind end with a DBE Twenty-eight patients had a total of 47 sessions of DBE. Deep insertion of an endoscope to the papilla of Vater was successful in 24 out of 25 procedures (96%) in patients with Roux-en-Y total gastrectomy, in 18 out of 19 procedures (94%) in patients with Billroth Ⅱ reconstruction, and in all three procedures in patients with pancreatoduodenectomy (Table 1). There were no significant differences in the success rate of reaching the blind end with a DBE among Roux-en-Y total gastrectomy, Billroth Ⅱ reconstruction, and pancreatoduodenectomy (P = 0.91). DBE can be a very useful modality for patients with altered gastrointestinal anatomy irrespective of surgical procedures. Cannulation and contrast enhancement of the target bile duct in patients in whom the blind end was reached Overall, the rate of successful cannulation and contrast enhancement of the target bile duct was 40 out of 45 (89%) in patients in whom the blind end was reached; this was made up of 21 out of 24 procedures (88%) in Roux-en-Y total gastrectomy patients, 16 out of 18 procedures (89%) in Billroth Ⅱ patients, and all three procedures in patients with pancreatoduodenectomy. Again, there were no significant differences in the success rate of cannulation and contrast enhancement of the target bile duct in patients whom the blind end was reached with a DBE among Roux-en-Y total gastrectomy, Billroth Ⅱ reconstruction, and pancreatoduodenectomy (P = 0.67). Treatment of the pancreatic and/or biliary duct Treatment was achieved in all 40 procedures (100%) in patients in whom contrast enhancement of the bile duct was successful. The 40 treatment procedures are listed in Table 2, and the details of treatment are given in Table 3. The most common endoscopic treatment was endoscopic biliary drainage (24 procedures), which was done by placement of plastic stents. Extraction of stones (14 procedures) was done by lithotomy with the mechanical lithotripter followed by extraction with a basket or by the balloon pull-through method. EST was performed with a needle precutting knife using a guidewire (14 procedures). The choledochojejunostomy site was dilated by balloon in three procedures December 14, 2012 Volume 18 Issue 46

168 Osoegawa T et al. Technical tips for DB-ERCP Table 2 Treatment procedures for double balloon enteroscopy-assisted endoscopic retrograde cholangiopancreatography Roux-en-Y reconstruction Billroth Ⅱ gastrectomy Pancreatoduodenectomy n EST + drainage 2 1 EST + SE 2 EST + SE + drainage 2 1 BD + SE BD + SE + drainage 1 EST + BD + drainage 1 1 EST + BD + SE 2 EST + BD + SE + drainage 1 1 SE + drainage 1 Drainage 12 5 Other 1 1 Roux-en-Y reconstruction Table 3 Detailed treatment procedures for double balloon enteroscopy-assisted endoscopic retrograde cholangiopancreatography Pancreatoduodenectomy Billroth Ⅱ gastrectomy Total Stone extraction EBD EPD 3 3 Endoscopic sphincterotomy Balloon dilation Cytological diagnosis 1 1 EBD: Endoscopic biliary drainage; EPD: Endoscopic pancreatic duct drainage. BD: Balloon dilation; SE: Stone extraction; EST: Endoscopic sphincterotomy. Time of the procedure The mean total duration of the procedure was 93.6 ± 6.8 min. The mean time required to reach the papilla was 30.5 ± 3.7 min. For a subanalysis, the time required to reach the papilla of Vater was 37.1 ± 4.9 min in Rouxen-Y total gastrectomy, 20.9 ± 5.8 min in Billroth Ⅱ reconstruction, and 33.3 ± 13.4 min in pancreatoduodenectomy. It tended to be shorter in Billroth Ⅱ reconstruction than that in Roux-en-Y total gastrectomy but there was no significant difference (P = 0.09). DB-ERCP seemed to be technically more difficult in patients with Roux-en-Y total gastrectomy than those with Billroth Ⅱ reconstruction. Complications A major complication occurred in one patient (3.5%); perforation of the long limb in a patient with Billroth Ⅱ anastomosis. Perforation occurred during insertion of DBE in this patient s second session. The perforation was successfully resolved surgically. No post-ercp pancreatitis occurred. DISCUSSION The invention of DBE has dramatically changed the endoscopic management of pancreatobiliary diseases in patients who have undergone bowel reconstruction [8-12]. DBE has made it possible to reach the papilla or biliopancreatoenteric anastomosis site even in patients with Roux-en-Y surgical reconstruction. Although patients who have undergone Billroth Ⅱ reconstruction are considered capable of undergoing ERCP with a conventional endoscope, the endoscopic approach to the afferent loop, blind end, and the site of hepaticojejunostomy is difficult in cases where there are severe adhesions or a long afferent loop. In such patients with Billroth Ⅱ reconstruction, DB-ERCP is effective. DB-ERCP has three major steps; the first step is intubation to the papilla or the site of hepaticojejunostomy, the second is cannulation into the bile and/or pancreatic duct, and the third is therapeutic manipulation such as sphincterotomy and/or stone extraction. There have been several reports of DB-ERCP. Although the success rates of reaching the papilla or of cannulation for intact papilla are high (50%-100%, 67%-100%, respectively) [17,18,20-22], they are still lower than for conventional ERCP. Therefore, technical difficulties or instrument limitations are considered likely. Some useful techniques or endoscopic accessories have been reported for conventional endoscopy or DBE, however, there are few reports which describe in detail the technique or the instruments for each step of DB-ERCP. We describe these things in detail, because they may contribute to improving the outcome of DB-ERCP. For the first step, intubation to the papilla or the site of hepaticojejunostomy, short-type DBE may be recommended. Originally, double balloon enteroscopes were 200 cm long, which did not cause a problem with insertion into the papilla, but limited the number of devices for the ERCP procedure [17,18,20,21]. Short-type DBE, which was originally developed for colonic insertion, has resolved this problem [17,18]. Shimatani et al [18] have reported that because of its short working length and the availability of various accessory devices, short-type DBE increases the success rates for reaching the papilla and blind end, and for ERCP-related interventions. Cases in which double balloon enteroscope insertion may be difficult are patients who have jejunojejunostomy such as Roux-en-Y anastomosis or pancreatoduodenectomy. Selective contrast enhancement in the intestine with a balloon inflated on the endoscope tip is useful to confirm the direction of the afferent loop [17,18]. Once the correct way is confirmed, submucosal tattooing at the beginning of the Roux limb may be useful for later sessions (Figure 1) [12,17,21,23,24]. In our series, the blind end was reached in 45 out of 47 procedures (95.7%) with short-type DBE, comprising 18 out of 19 procedures in Billroth Ⅱ patients, all three in pancreatoduodenectomy, and 24 out of 25 procedures in Roux-en-Y patients. One procedure in Billroth Ⅱ patients was aborted because of perforation of the long limb, and one in the Roux-en-Y group failed because of 6847 December 14, 2012 Volume 18 Issue 46

169 Osoegawa T et al. Technical tips for DB-ERCP strong adhesion. We have used CO2 instead of standard air insufflations since April 2009 to improve intubation depth during DBE, as well as to reduce postprocedural pain [19]. The mean duration for reaching the blind end using CO2 insufflation in Roux-en-Y patients tended to be shorter than that using air insufflation, although the difference was not significant (P = 0.18, 40.9 min vs 29.4 min, air vs CO2, respectively). CO2 insufflation could be useful especially in patients with Roux-en-Y total gastrectomy where DB-ERCP seems to be technically difficult compared to Billroth Ⅱ reconstruction. The second step, deep cannulation into the bile and/ or pancreatic duct, is an important precondition for later therapeutic manipulations. In patients with Roux-en-Y reconstruction, the position of the papilla may vary and may be observed by moving the screen in various directions. The manipulation of both the overtube and the endoscope makes it possible to change the position of the papilla in the view field. Double balloon enteroscopes are not provided with an elevator, therefore, cannulation may be difficult, but the attachment hood is useful to fix the ampulla and to align the axes of the cannula and the bile duct, enabling safe endoscopic procedures and selective pancreatic and/or biliary cannulation (Figure 2). We usually use a normal ERCP catheter. If the biliary axis does not fit, we use a sphincterotome with a rotatable tip (Figure 3). Needle-knife precut papillotomy was performed when the conventional biliary cannulation promised to be difficult, as described by Fukatsu et al [25]. In this study, cannulation was successful and contrast enhancement of the target bile duct was achieved in 40 out of 45 (89%) patients in whom the blind end was reached. There were difficult cases in Roux-en-Y patients (21/24, 88%) and in Billroth Ⅱ patients (16/18, 89%). It was easy in all three patients with pancreatoduodenectomy. The third therapeutic step is the endpoint of the DB-ERCP procedure. Deep insertion and placing the guidewire into the biliary and/or pancreatic duct is indispensable for the following procedures. Guidewire stabilizes the papilla and makes it easy to perform sphincterotomy or insert the devices into the duct. Treatment was achieved in all 40 procedures in patients in whom contrast enhancement of the bile duct and guidewire placement were successful. Endoscopic biliary drainage by placement of plastic stents was not difficult. However, the diameter of the stents was limited to < 7 Fr. Sphincterotomy using a sphincterotome is sometimes difficult in DB-ERCP. A rotatable sphincterotome or needle precutting knife may be useful in such cases. If sphincterotomy is impossible or insufficient, balloon dilation should be performed. Extraction of stones was done by lithotomy with the mechanical lithotripter followed by extraction with a basket or by the balloon pull-through method. A few types of mechanical lithotripters and baskets that can go through the DBE working channel are commercially available at the present time. However, most of them do not have the wire-guided mechanism, which means it is sometimes difficult to insert into the duct. DB-ERCP complications in patients who have undergone bowel reconstruction have featured in a limited number of reports. Perforations or emphysema at the time of insertion of an endoscope or endoscopic sphincterotomy have been reported in 2.3%-11.1% of cases. Raithel et al [21] have reported post-ercp pancreatitis in 2.3% and post-interventional bleeding in 1.1% of patients. In our study, complications occurred in only one patient (3.5%); perforation of the long limb in a patient with Billroth Ⅱ anastomosis [17,18,21]. With regard to the insertion of an endoscope and shortening of the intestinal tract, attention should be paid to avoid forced insertion or shortening, which may cause perforation. In conclusion, short-type DBE combined with several technical innovations enabled us to perform ERCP and ERCP-related interventions in a number of patients for whom it was previously impossible. Endoscopic tattooing at the beginning of the limb, using an attachment hood, and CO2 insufflation might facilitate intubation of the papilla and reduce the duration of the procedure. Cannulation into the bile or pancreatic duct is the most difficult part of DB-ERCP. Using an attachment hood and/or a rotatable sphincterotome may facilitate the procedure. Further instrumental improvements such as the widening of the DBE working channel or development of the accessories for use in balloon enteroscope systems are necessary to improve the outcome of DB- ERCP. ACKNOWLEDGMENTS The author thanks Mr. Steve Cotton for English editing. COMMENTS Background Patients who have undergone bowel reconstruction have been considered to be difficult for endoscopic retrograde cholangiopancreatography (ERCP) using conventional endoscopy. Double balloon enteroscopy (DBE) has brought a significant benefit for performing ERCP in patients with such an altered gastrointestinal anatomy. However, there are still some technical difficulties to be resolved. Research frontiers Recently developed short DBE contributes to increased success rates for reaching the papilla and for ERCP-related interventions in DBE-assisted ERCP (DB-ERCP). In the area of DB-ERCP, the research hotspot is how to improve its results for completing therapeutic interventions such as stone extraction and stent insertion of the pancreatobiliary system. Innovations and breakthroughs Although a short DBE is more useful than a conventional long DBE, clinical outcomes of DB-ERCP are still unsatisfactory compared to conventional ERCP. DB-ERCP has three major steps: intubation to the papilla or the site of hepaticojejunostomy, cannulation into the bile and/or pancreatic duct, and therapeutic manipulation. To improve intubation, the authors used CO2 instead of standard air insufflations, selective contrast enhancement in the intestine with a balloon inflated on the endoscope tip to confirm the right direction, and submucosal tattooing in patients with Roux-en-Y anastomosis for the next session. To improve cannulation, we attached a soft transparent hood to the tip of a scope, and used a sphincterotome with a rotatable tip. To improve therapeutic manipulation, we performed guidewire placement into the biliary/pancreatic duct, and needle December 14, 2012 Volume 18 Issue 46

170 Osoegawa T et al. Technical tips for DB-ERCP knife precutting and/or balloon dilation to make a large enough opening of the orifice. Applications In this study, the authors showed the possibility that short-type DBE combined with several technical innovations enabled us to perform ERCP and ERCPrelated interventions more efficiently in a number of patients with altered gastrointestinal anatomy for whom it was previously impossible. Terminology DBE has been developed for visualization of and intervention in the entire small intestine. Currently, two types of double-balloon endoscope are available including a conventional type (200 cm working length) and a short type (152 cm). Short-type DBE has great practical use because of its short working length and the availability of various accessory devices. Roux-en-Y reconstruction of the small bowel is a general surgical technique in gastrointestinal oncology surgery, hepatobiliary and pancreatic surgery, and bariatric surgery. For the endoscopist, accessing the ampulla is technically difficult in a patient needing Roux-en-Y reconstruction, because of the long length of bowel that the endoscope must pass through, and the acute angle of the afferent limb and Roux limb anastomosis. Peer review This is an important paper describing difficult ERCP cases after gastric surgery. This is a useful report of DB-ERCP techniques. The authors modifications of the technique are particularly helpful. REFERENCES 1 Elton E, Hanson BL, Qaseem T, Howell DA. Diagnostic and therapeutic ERCP using an enteroscope and a pediatric colonoscope in long-limb surgical bypass patients. Gastrointest Endosc 1998; 47: Gostout CJ, Bender CE. Cholangiopancreatography, sphincterotomy, and common duct stone removal via Roux-en-Y limb enteroscopy. Gastroenterology 1988; 95: Alberti-Flor JJ, Hernandez ME, Ferrer JP. 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ERCP plus papillotomy by use of double-balloon enteroscopy after Billroth II gastrectomy. Gastrointest Endosc 2007; 66: Haruta H, Yamamoto H, Mizuta K, Kita Y, Uno T, Egami S, Hishikawa S, Sugano K, Kawarasaki H. A case of successful enteroscopic balloon dilation for late anastomotic stricture of choledochojejunostomy after living donor liver transplantation. Liver Transpl 2005; 11: Chahal P, Baron TH, Topazian MD, Petersen BT, Levy MJ, Gostout CJ. Endoscopic retrograde cholangiopancreatography in post-whipple patients. Endoscopy 2006; 38: Mönkemüller K, Fry LC, Bellutti M, Neumann H, Malfertheiner P. ERCP with the double balloon enteroscope in patients with Roux-en-Y anastomosis. Surg Endosc 2009; 23: Pohl J, May A, Aschmoneit I, Ell C. Double-balloon endoscopy for retrograde cholangiography in patients with choledochojejunostomy and Roux-en-Y reconstruction. Z Gastroenterol 2009; 47: Aabakken L, Bretthauer M, Line PD. Double-balloon enteroscopy for endoscopic retrograde cholangiography in patients with a Roux-en-Y anastomosis. Endoscopy 2007; 39: Yamamoto H, Sekine Y, Sato Y, Higashizawa T, Miyata T, Iino S, Ido K, Sugano K. Total enteroscopy with a nonsurgical steerable double-balloon method. Gastrointest Endosc 2001; 53: Kita H, Yamamoto H. New indications of double balloon endoscopy. Gastrointest Endosc 2007; 66: S57-S59 16 May A, Nachbar L, Wardak A, Yamamoto H, Ell C. Doubleballoon enteroscopy: preliminary experience in patients with obscure gastrointestinal bleeding or chronic abdominal pain. Endoscopy 2003; 35: Shimatani M, Matsushita M, Takaoka M, Koyabu M, Ikeura T, Kato K, Fukui T, Uchida K, Okazaki K. Effective short double-balloon enteroscope for diagnostic and therapeutic ERCP in patients with altered gastrointestinal anatomy: a large case series. Endoscopy 2009; 41: Itoi T, Ishii K, Sofuni A, Itokawa F, Tsuchiya T, Kurihara T, Tsuji S, Ikeuchi N, Fukuzawa K, Moriyasu F, Tsuchida A. Long- and short-type double-balloon enteroscopy-assisted therapeutic ERCP for intact papilla in patients with a Rouxen-Y anastomosis. Surg Endosc 2011; 25: Domagk D, Bretthauer M, Lenz P, Aabakken L, Ullerich H, Maaser C, Domschke W, Kucharzik T. Carbon dioxide insufflation improves intubation depth in double-balloon enteroscopy: a randomized, controlled, double-blind trial. Endoscopy 2007; 39: Ryozawa S, Iwamoto S, Iwano H, Ishigaki N, Taba K, Sakaida I. ERCP using double-balloon endoscopes in patients with Roux-en-Y anastomosis. J Hepatobiliary Pancreat Surg 2009; 16: Raithel M, Dormann H, Naegel A, Boxberger F, Hahn EG, Neurath MF, Maiss J. Double-balloon-enteroscopy-based endoscopic retrograde cholangiopancreatography in postsurgical patients. World J Gastroenterol 2011; 17: Cho S, Kamalaporn P, Kandel G, Kortan P, Marcon N, May G. Short double-balloon enteroscope endoscopic retrograde cholangiopancreatography in patients with a surgically altered upper gastrointestinal tract. Can J Gastroenterol 2011; 25: 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: Kuga R, Furuya CK, Hondo FY, Ide E, Ishioka S, Sakai P. ERCP using double-balloon enteroscopy in patients with Roux-en-Y anatomy. Dig Dis 2008; 26: Fukatsu H, Kawamoto H, Kato H, Hirao K, Kurihara N, Nakanishi T, Mizuno O, Okamoto Y, Ogawa T, Ishida E, Okada H, Sakaguchi K. Evaluation of needle-knife precut papillotomy after unsuccessful biliary cannulation, especially with regard to postoperative anatomic factors. Surg Endosc 2008; 22: S- Editor Gou SX L- Editor Kerr C E- Editor Xiong L 6849 December 14, 2012 Volume 18 Issue 46

171 Online Submissions: doi: /wjg.v18.i World J Gastroenterol 2012 December 14; 18(46): ISSN (print) ISSN (online) 2012 Baishideng. All rights reserved. Is laparoscopy equal to laparotomy in detecting and treating small bowel injuries in a porcine model? BRIEF ARTICLE Cheng-Xiang Shan, Chong Ni, Ming Qiu, Dao-Zhen Jiang Cheng-Xiang Shan, Chong Ni, Ming Qiu, Dao-Zhen Jiang, Department of General Surgery, Chang Zheng Hospital Affiliated to the Second Military Medical University, Shanghai , China Author contributions: Shan CX and Ni C contributed equally to this work; Shan CX and Qiu M designed the research; Shan CX, Ni C and Jiang DZ performed the experiments; Shan CX and Qiu M wrote the paper. Correspondence to: Dao-Zhen Jiang, MD, Department of General Surgery, Chang Zheng Hospital Affiliated to the Second Military Medical University, No. 415 Fengyang Road, Shanghai , China. [email protected] Telephone: Fax: Received: May 23, 2012 Revised: September 23, 2012 Accepted: October 16, 2012 Published online: December 14, 2012 Abstract AIM: To evaluate the safety and effectiveness of laparoscopy compared with laparotomy for diagnosing and treating small bowel injuries (SBIs) in a porcine model. METHODS: Twenty-eight female pigs were anesthetized and laid in the left recumbent position. The SBI model was established by shooting at the right lower quadrant of the abdomen. The pigs were then randomized into either the laparotomy group or the laparoscopy group. All pigs underwent routine exploratory laparotomy or laparoscopy to evaluate the abdominal injuries, particularly the types, sites, and numbers of SBIs. Traditional open surgery or therapeutic laparoscopy was then performed. All pigs were kept alive within the observational period (postoperative 72 h). The postoperative recovery of each pig was carefully observed. RESULTS: The vital signs of all pigs were stable within 1-2 h after shooting and none of the pigs died from gunshot wounds or SBIs immediately. The SBI model was successfully established in all pigs and definitively diagnosed with single or multiple SBIs either by exploratory laparotomy or laparoscopy. Compared with exploratory laparotomy, laparoscopy took a significantly longer time for diagnosis (41.27 ± min vs ± min, P = 0.02), but the time for therapeutic laparoscopy was similar to that of open surgery. The length of incision was significantly reduced in the laparoscopy group compared with the laparotomy group (5.27 ± 1.86 cm vs ± 1.06 cm, P < 0.01). In the final post-mortem examination 72 h after surgery, both laparotomy and laparoscopy offered a definitive diagnosis with no missed injuries. Postoperative complications occurred in four cases (three following laparotomy and one following laparoscopy, P = 0.326). The average recovery period for bowel function, vital appearance, and food re-intake after laparoscopy was ± 4.72 h, ± 3.14 h, and ± 7.11 h, respectively. All of these were significantly shorter than after laparotomy (21.27 ± h, P = 0.004; ± 9.61 h, P < 0.001; and ± 9.72 h, respectively, P = 0.016). CONCLUSION: Compared with laparotomy, laparoscopy offers equivalent efficacy for diagnosing and treating SBIs, and reduces postoperative complications as well as recovery period Baishideng. All rights reserved. Key words: Laparoscopy; Laparotomy; Small bowel injury; Porcine model; Diagnosis; Treatment; Penetrating injury; Firearm injury Peer reviewers: Lygia Stewart, MD, Professor of Clinical Surgery, University of California San Francisco, 4150 Clement Street, San Francisco, CA 94121, United States; Keiji Hirata, MD, Surgery 1, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu , Japan; Dr. Olivier Detry, Department of Abdominal Surgery and Transplantation, University of Liège, CHU Sart Tilman B35, B-4000 Liège, Belgium Shan CX, Ni C, Qiu M, Jiang DZ. Is laparoscopy equal to 6850 December 14, 2012 Volume 18 Issue 46

172 Shan CX et al. Laparoscopy for small bowel injuries laparotomy in detecting and treating small bowel injuries in a porcine model? World J Gastroenterol 2012; 18(46): Available from: URL: v18/i46/6850.htm DOI: i INTRODUCTION The vast anatomic space occupied by the gastrointestinal tract prediposes it to penetrating injuries. In penetrating trauma, the small bowel is most frequently injured, followed by the large intestine and stomach [1]. Small bowel injury (SBI) is seldom diagnosed preoperatively especially when there are no frank signs of hemoperitoneum or peritonitis [2,3]. Laparotomy is considered the gold standard for evaluation of intra-abdominal injuries sustained from trauma [4]. However, complications following negative or nontherapeutic laparotomy can be as high as 20%-40% [5-7]. Therefore, it is advantageous to avoid a negative laparotomy while providing a reliable and accurate alternative diagnostic procedure [8]. While laparoscopy has become a standard component of diagnosis and therapy for many conditions in general surgery, its role in trauma remains controversial. Many concerns about the safety, sensitivity, and specificity of laparoscopy have limited its application in abdominal trauma [9-12], particularly in detecting SBIs [13-15]. It was reported that laparoscopy in trauma initially resulted in a high rate of missed injuries (41%-77%) and considerable criticism of laparoscopy as a diagnostic tool [9,10]. Studies in 1993 and 2006 showed little statistical change in its reliability; only 20% of SBIs were correctly identified by laparoscopy, and sensitivity was 25% for diagnosis of hollow viscus and retroperitoneal injuries [16,17]. The high proportion of missed occult SBIs with laparoscopy in trauma (LIT) is a major reason why some surgeons still preclude LIT use today [18]. These considerations conflict with the considerable advances that have been made in LIT. In this present study we mimicked injuries to the small intestines by firing bullets into the abdomen of anesthetized pigs. We tried to provide a reproducible and hemodynamically stable porcine model with multiple SBIs. We also aimed to evaluate the safety and effectiveness of diagnostic and therapeutic laparoscopy compared with the laparotomy for SBI in the porcine model. MATERIALS AND METHODS Experimental design The study protocol was approved by the Institutional Review Board of the Research Institute of Surgery (RIS), which was affiliated to the Third Military Medical University in Chong Qing (People s Republic of China). Animal welfare and experimental procedures were carried out strictly in accordance with the guide for the Animal Care and Use Committee of RIS. A prospective, randomized, comparative study was conducted between February and July Enrolled in this research were 28 consecutive healthy pigs native to Chong Qing (all females). The pigs were provided and fed by staff from the Medical Animal Research Center of Da Ping Hospital (Affiliated to the Third Military Medical University). After mastering the skilled techniques gained from previous experience in modeling and treating SBIs, we carried out this study to ascertain if laparoscopy alone could replace laparotomy in diagnosing and treating SBIs in this porcine model. Preoperative preparation All 28 pigs had free access to food and tap water without oral intake of antibiotics. After premedication with intramuscular administration of azaperone 4 mg/kg, ketamine 10 mg/kg, and atropine 0.02 mg/kg, general anesthesia was induced by 3% pentobarbital (1 ml/kg) via the left ear vein; an additional 3-5 ml of 3% pentobarbital was given if the pigs became restless. A 6.5-F endotracheal tube was applied, and pigs allowed to breathe spontaneously. They were ventilated with room air using a standard ventilator if necessary. Most of the animals were in the anesthetic plane between medium and deep anesthesia [muscles relaxed; most reflexes (palpebral, corneal) absent; pupillary light reflex slow or absent]. SBI modeling procedures: After anesthesia, the pigs were transferred to the shooting cabin to establish the model. Pigs were laid in the left lateral recumbent position; the right forelimb and hindlimb were abducted and suspended on the shooting shelf whereas the left forelimb and hindlimb were fixed horizontally. The entry point was 2 cm medial and 3 cm cephalad from the point of the right hip (this position was determined according to our previously experience). Once the entry point was defined, the location of the predicted exit point was obtained by ensuring that both points were in the right quadrant of the abdomen and the line between the two points was horizontal. The predicted exit point was equivalent to the entry point moving medially, and was 4 cm lateral to the abdominal midline (Figure 1). The injury was inflicted by a 56-type military firearm. It fired 7.62-mm steel-core bullets weighing 7.9 g at a shooting distance of 5 meters. The military firearm was used after fine adjustment of the ballistic trajectory according to the position of animal. All devices and the professional marksman were provided by the RIS. Surgical procedures: After shooting at the abdomen, pigs were rapidly transferred to the operating room. Immediately before surgery, all pigs were randomized to one of the two surgical approaches (laparotomy or laparoscopy). The interval between shooting and surgery was similar in each pig: approximately min. Laparotomy group: Pigs were placed in the supine 6851 December 14, 2012 Volume 18 Issue 46

173 Shan CX et al. Laparoscopy for small bowel injuries A B Figure 1 Animal model. The experimenters fixed the position of the anesthetized pig. The black arrows point at the entry and exit wounds of the abdomen. position. Access to the abdominal cavity was gained through a midline incision ranging from approximately 7 cm above the umbilicus to approximately 9 cm below the umbilicus. The abdominal wall was drawn back with a metal retractor. Viscera (particularly the intestinal tract) were exposed manually. The sequence of exploration was from the major vessels, liver, spleen, the gyri centripetales and gyri centrifugales of the colon to the left kidney and left segment of the pancreas; this was continued from the sigmoid colon, rectum, bladder, and cecum to the ileum and jejunum; then continued from the stomach, duodenum, and the pancreatic head to the right segment of the pancreas and right kidney. After exploration, surgical treatment was performed for intraabdominal injuries. Intestinal resection and anastomoses were undertaken if the diameter of the intestinal rupture was longer than 50% of the intestine circumference, otherwise simple repair with edge trimming was applied; end-to-end single-layer anastomoses were conducted with running number 0 silk sutures; Laparoscopy group: A pneumoperitoneum of 13 mmhg with CO2 insufflation was established after insertion of a Veress needle. A 10-mm camera access port was introduced 1 cm above the umbilicus. A 30 laparoscope connected to a camera allowed endoscopic visualization. Two additional 5-mm or 10-mm ports were inserted in the left lower quadrant of the abdomen in right-angled triangular fashion under laparoscopic guidance. Pigs were positioned in a Trendelenburg angle or a reverse angle to facilitate free access to the intestinal tract and other viscera. The sequence of exploration and some of the specific rules of treatment during surgery were the same as those mentioned in the laparotomy. The small bowel was examined for traumatic injuries by two 5-mm atraumatic bowel graspers from the ileocecal valve to the ligament of Treitz (running the bowel). Once exploration was accomplished, effective therapeutic laparoscopic procedures were undertaken to treat the injuries. Larger bleedings were controlled through application of a laparoscopic clip or with bipolar forceps. If the intestinal injuries were not severe (injury scale lower than Grade Ⅱ [19] ) with the exception of ruptures of the colon or rectum, single repair with single-layer interrupted sutures was undertaken entirely under the laparoscopic view (total laparoscopy). Otherwise, intestinal resection and anastomoses were performed through an extended 5-cm incision of the exit wound (video-assisted laparoscopy). After anastomoses and debridement, the abdominal cavity was irrigated with approximately 2000 ml normal (0.9%) saline, and a drainage tube placed in the pelvic cavity. The exit wound was managed with sharp debridement and two layers of interrupted sutures for the deep fascia and skin and the surgical incision. Administration of anesthetics was stopped. Both laparoscopy and laparotomy were performed by the same surgeons in our study, all of whom trained at RIS and used similar techniques. Postoperative management Postoperatively, a clean dry dressing was sutured with the abdominal skin to prevent contamination of the incision. Each pig was transferred to the feeding room, kept in a single cage, and injected with 25 mg pethidine 3 h after surgery. All pigs received 500 ml 5% glucose solution containing 1 g cefradine and 0.5 g metronidazole per day through a peripheral vein. The eating habits and physical activities of the pigs were closely monitored three times daily to detect signs of peritonitis and generalized sepsis. The recovery of bowel function was recorded as the time of return of bowel sounds. At 72 h after surgery, all animals were sacrificed for a thorough exploration of the abdomen to check for missed injuries. Statistical analysis Statistical analysis were carried out with SPSS software (SPSS/PC ; SPSS, Chicago, IL, United States). Values are presented as mean ± standard deviation; P < 0.05 was considered significant. Statistical comparisons of data were carried out by the Student t-test, the Chisquare test or repeated measures as appropriate. In addition, Fisher s exact test was applied if the sample was < 5. RESULTS SBI model The point of the entry wound was cm medial (mean, 2.60 ± 0.19 cm) and cm cephalad (mean, 6852 December 14, 2012 Volume 18 Issue 46

174 Shan CX et al. Laparoscopy for small bowel injuries A B Figure 2 Porcine model with gastrointestinal injuries. A: Multiple ruptures of the small intestine; B: Injuries to the sigmoid colon. 1.5 ± 0.12 cm) from the point of the right hip. The exit wounds were located in the right lower quadrant of the abdomen, and cm (mean, 4.53 ± 0.51 cm) lateral to the midline. The vital signs of all pigs were stable within 1-2 h after shooting. None of the pigs died from gunshot wounds (GSW) to the abdomen. All 28 pigs underwent routine exploration of the abdominal cavity. All had single or multiple injuries to the small bowel, one (3.57%) with sigmoid ruptures (Figure 2), and one (3.57%) with cecum ruptures. Injuries to the liver, spleen, the gyri centripetales and gyri centrifugales of the colon, crucial vessels and retroperitoneal organs were not observed. The details of the SBIs are listed in Table 1; the organ injury scale was based on that of Moore et al [19]. In addition, the severity of SBIs in the two groups was also compared. We considered injury numbers of Grade Ⅱ SBIs < 3 as minor injuries, and injury numbers of Grade Ⅲ or more SBIs 1 as severe injuries. In the laparoscopy group, 5 cases were found with minor injuries and 9 cases with severe injuries, while in the laparotomy group, 4 cases were diagnosed with minor injuries and 10 cases with severe injuries. There was no significant difference between the groups concerning the severity of SBIs. Perioperative outcomes All 28 pigs were randomized into either the laparotomy group or the laparoscopy group (14 in each group). According to the operative findings, both laparotomy and laparoscopy offered a definitive diagnosis with no missed injuries (all confirmed by the final post-mortem examination). Compared with laparotomy, laparoscopy took a significantly longer time for diagnosis (41.27 ± min vs ± min, P = 0.02), but the time of therapeutic laparoscopy was similar to that of open surgery (83.27 ± min vs ± min, P > 0.05). Furthermore, according to our analysis, the overall operative (diagnostic plus therapeutic) time was slightly longer in the laparoscopy group, but the difference did not reach statistical significance (P = 0.12). In addition, the length of the incision was significantly reduced in the laparoscopy group compared with the laparotomy group (5.27 ± 1.86 cm vs ± 1.06 cm, P < 0.01). Although all 28 pigs survived during the 72 h of follow-up, the recovery was not uneventful. Three cases (3/14, 21.5%) in the laparotomy group developed postoperative complications (one small bowel volvulus, one gastric retention, and one abdominal cavity infection and abscess) while only one case (1/14, 7.14%) had a complication of incisional infection in the laparoscopy group. No complications related to the technique (e.g., leakage, obstruction due to tight anastomosis, bleeding) were found. Although the incidence of complications was slightly higher following laparotomy, the difference between groups was not significant (P = 0.326). The average recovery period for bowel function, vital appearance, and food re-intake after laparoscopy was ± 4.72 h, ± 3.14 h, and ± 7.11 h, respectively, following laparoscopy. All of these were significantly shorter than after laparotomy (21.27 ± h, P = 0.004; ± 9.61 h, P < 0.001; and ± 9.72 h, respectively, P = 0.016). DISCUSSION Few studies have been carried out focusing on the methodology of SBI in pigs arising from GSW [20,21]. According to our experience, the entry and exit points were extremely critical in establishing a purely SBI model. Through our work on intra-abdominal anatomy, we found that the distribution pattern was considerably regular in pigs. The small bowel had a centralized distribution in the right abdomen, and the gyri centripetales and gyri centrifugales of the colon were located in the left upper quadrant in a circular fashion. The cecum was approximately 20 cm in length with no obvious mesentery; it was mainly located in the left lower quadrant with its end pointing towards the pelvic cavity. Therefore, we assumed that if both the entry and exit wounds were located on the right lower quadrant of the abdomen, avoiding bony structures such as right ribs, right hip and pubis, there would be injuries only to the small intestine. As predicted, in the present study, all 28 pigs were diagnosed with SBIs. One of the greatest concerns about LIT has been its unreliability in detecting SBIs [13,14], a major reason why some surgeons still preclude LIT today. However, according to the results of the final post-mortem ab December 14, 2012 Volume 18 Issue 46

175 Shan CX et al. Laparoscopy for small bowel injuries Table 1 Summary of the details of small bowel injuries in all pigs after gunshots Case Injury Scale 1 of SB Treatment IH IL Ⅱ Ⅲ Ⅳ Ⅴ Sum Laparotomy Laparoscopy (total) Laparoscopy (total) Laparoscopy (video-assisted) Laparotomy Laparoscopy (video-assisted) Laparotomy Laparotomy Laparoscopy (video-assisted) Laparoscopy (video-assisted) Laparotomy Laparotomy Laparotomy Laparoscopy (total) Laparoscopy (video-assisted) Laparotomy Laparotomy Laparotomy Laparoscopy (total) Laparotomy Laparoscopy (video-assisted) Laparotomy Laparoscopy (video-assisted) Laparotomy Laparoscopy (video-assisted) Laparotomy Laparoscopy (total) Laparoscopy (video-assisted) 1 Based on the reference of Moore et al [19]. IH: Hematoma, contusion or hematoma without devascularization; IL: Laceration, partial thickness, no perforation; Ⅱ: Laceration, laceration < 50% of circumference; Ⅲ: Laceration, laceration > 50% of circumference without transaction; Ⅳ: Laceration, transection of small bowel; Ⅴ: Laceration, transection of small bowel with segmental tissue loss vascular, devascularized segment; SB: Small bowel. dominal examination, the present study demonstrated that laparoscopy could also offer equivalent efficacy for diagnosing SBIs compared with laparotomy, with no missed injuries. According to our experience, the systematic approach for diagnostic laparoscopy exploration of the gastrointestinal tract was very important for avoiding missed SBIs [15], and mainly consisted of all the principles of open exploratory laparotomy for trauma and the technique of running the small bowel. During the exploration, we initially lifted an 8-10-cm segment of the small bowel with two 5-mm atraumatic bowel graspers at the ileocecal valve, and one side of the bowel and mesentery was observed. The graspers were then turned 180 degrees, and the other side of the bowel was visualized. This sequence was repeated until the ligament of Treitz was reached. Therefore, after using this systematic approach, we could correctly identify SBIs, minimizing the potential of a missed injury. Although diagnostic laparoscopy was time-consuming, and took approximately 45 min to diagnose intestinal injuries in each case, which was significantly longer than the laparotomy exploration, the safety and sensitivity of diagnostic laparoscopy for SBIs was good according to our research. While surgeons disputed the disadvantages of laparoscopy for SBIs, the benefits of laparoscopy were evident [22]. First, by directly visualizing the abdominal cavity, laparoscopy allowed the surgeon to exclude the presence of other associated intra-abdominal injuries, and was also able to eliminate the blind zone of visual fields when using a 30 laparoscope. Secondly, following the laparoscopic guidance, the SBIs could be treated directly using a laparoscopic technique, or using a notably shorter incision, which was near the injuries. In the current study, five pigs in the laparosopy group were diagnosed with simple SBIs, and received total laparoscopy for treatment. In addition, video-assisted therapeutic laparoscopy, which utilized a 6-7 cm incision, was successfully carried out in nine pigs with relatively severe SBIs. Therefore, laparoscopy would also be a reliable and accurate alternative therapeutic procedure for severe SBIs [23]. Thirdly, compared with the laparotomy group, the length of incision was significantly shortened in the laparoscopy group. The length of incision is an important factor affecting surgical stress [24]. Thus, given the minimally invasive nature of laparoscopy, there is good reason to assume that laparoscopy is advantageous over conventional laparotomy in reducing surgical trauma. Another benefit of laparoscopy was the significantly reduced recovery period after surgery. The almost 50% reduction in recovery periods of bowel function, vital appearance, and food re-intake in the present research was not surprising considering the less invasive nature of the procedure. Therefore, laparoscopy could be a promising minimally invasive approach for treating SBIs [25,26]. Limitation One overwhelming limitation of this study was that a major proportion of patients with GSW to the abdomen would have significant injuries involving not only the small intestines, but also many other kinds of abdominal organs, which would make the conditions of the patients very complicated. Thus, therapeutic laparoscopy might not be the first choice for patients with GSW. However, we believe that, in the hemodynamically stable patient with abdominal penetrating injury and without extensive intra-abdominal adhesions, a thorough laparoscopic examination is possible and feasible. The results of diagnostic laparoscopy would be a useful guidance, and directly influence the modality of further treatment. Moreover, we aimed to evaluate the feasibility and validity of diagnostic and therapeutic laparoscopy for SBI, which was the chief objective of our study, and we did obtain convincing evidence supporting the use of laparoscopy for SBIs. In conclusion, our research indicates that laparoscopy could be a minimally invasive approach for diagnosing and treating SBIs. Compared with laparotomy, laparoscopy could offer equivalent efficacy for SBIs, and reduces postoperative complications as well as recovery period December 14, 2012 Volume 18 Issue 46

176 Shan CX et al. Laparoscopy for small bowel injuries ACKNOWLEDGMENTS Jian-Min Wang, Xiao-Xia Li, Liang-Chao Zhang, and Jian-Yi Kang are thanked for the skilled technical assistance when establishing the animal model (RIS, Third Military Medical University, Chongqing, China). COMMENTS Background While laparoscopy has become a standard component of diagnosis and therapy for many conditions in general surgery, its role in abdominal trauma remains controversial. Many concerns about the safety, sensitivity, and specificity of the laparoscopy have limited its application in abdominal trauma, particularly in detecting small bowel injuries (SBIs). Research frontiers One of the greatest concerns about laparoscopy in trauma (LIT) has been its unreliability in detecting SBIs, a major reason why some surgeons still preclude LIT today. This study aimed to evaluate the safety and effectiveness of diagnostic and therapeutic laparoscopy compared with laparotomy for SBIs in a porcine model. Innovations and breakthroughs Few studies have been carried out focusing on the value of laparoscopy for SBIs due to gunshot wounds in pigs. To the best of the authors knowledge, the present study was the most detailed and systematic study establishing a purely SBI porcine model. Furthermore, the study also provided a comprehensive picture of the safety and effectiveness of laparoscopy and laparotomy in trauma. Applications In the hemodynamically stable patient with abdominal penetrating injury, a thorough diagnostic and therapeutic laparoscopy was possible and feasible. Compared with laparotomy, laparoscopy offers equivalent efficacy for diagnosing and treating SBIs, and reduces postoperative complications as well as recovery period. Peer review The author compared the safety and efficacy of laparoscopy for diagnosing and treating small bowel injuries in a trauma model using pigs. The study is well designed, and the article is also important and interesting. REFERENCES 1 Nicholas JM, Rix EP, Easley KA, Feliciano DV, Cava RA, Ingram WL, Parry NG, Rozycki GS, Salomone JP, Tremblay LN. Changing patterns in the management of penetrating abdominal trauma: the more things change, the more they stay the same. J Trauma 2003; 55: ; discussion Watts DD, Fakhry SM. Incidence of hollow viscus injury in blunt trauma: an analysis from 275,557 trauma admissions from the East multi-institutional trial. J Trauma 2003; 54: Busić Z, Lovrić Z, Amić E, Busić D, Lovrić L. Small bowel injuries in penetrating abdominal trauma during war: tenyear follow-up findings. Mil Med 2004; 169: Lee WC, Uddo JF, Nance FC. Surgical judgment in the management of abdominal stab wounds. Utilizing clinical criteria from a 10-year experience. Ann Surg 1984; 199: Ertekin C, Yanar H, Taviloglu K, Güloglu R, Alimoglu O. Unnecessary laparotomy by using physical examination and different diagnostic modalities for penetrating abdominal stab wounds. Emerg Med J 2005; 22: Renz BM, Feliciano DV. Unnecessary laparotomies for trauma: a prospective study of morbidity. J Trauma 1995; 38: Henderson VJ, Organ CH, Smith RS. Negative trauma celiotomy. Am Surg 1993; 59: Lin HF, Wu JM, Tu CC, Chen HA, Shih HC. Value of diagnostic and therapeutic laparoscopy for abdominal stab wounds. World J Surg 2010; 34: Leppäniemi A, Haapiainen R. Diagnostic laparoscopy in abdominal stab wounds: a prospective, randomized study. J Trauma 2003; 55: Elliott DC, Rodriguez A, Moncure M, Myers RA, Shillinglaw W, Davis F, Goldberg A, Mitchell K, McRitchie D. The accuracy of diagnostic laparoscopy in trauma patients: a prospective, controlled study. Int Surg 1998; 83: Simon RJ, Rabin J, Kuhls D. Impact of increased use of laparoscopy on negative laparotomy rates after penetrating trauma. J Trauma 2002; 53: ; discussion Villavicencio RT, Aucar JA. Analysis of laparoscopy in trauma. J Am Coll Surg 1999; 189: Streck CJ, Lobe TE, Pietsch JB, Lovvorn HN. Laparoscopic repair of traumatic bowel injury in children. J Pediatr Surg 2006; 41: Mathonnet M, Peyrou P, Gainant A, Bouvier S, Cubertafond P. Role of laparoscopy in blunt perforations of the small bowel. Surg Endosc 2003; 17: Kawahara NT, Alster C, Fujimura I, Poggetti RS, Birolini D. Standard examination system for laparoscopy in penetrating abdominal trauma. J Trauma 2009; 67: Ivatury RR, Simon RJ, Stahl WM. A critical evaluation of laparoscopy in penetrating abdominal trauma. J Trauma 1993; 34: ; discussion Becker HP, Willms A, Schwab R. [Laparoscopy for abdominal trauma]. Chirurg 2006; 77: Como JJ, Bokhari F, Chiu WC, Duane TM, Holevar MR, Tandoh MA, Ivatury RR, Scalea TM. Practice management guidelines for selective nonoperative management of penetrating abdominal trauma. J Trauma 2010; 68: Moore EE, Cogbill TH, Malangoni MA, Jurkovich GJ, Champion HR, Gennarelli TA, McAninch JW, Pachter HL, Shackford SR, Trafton PG. Organ injury scaling, II: Pancreas, duodenum, small bowel, colon, and rectum. J Trauma 1990; 30: Olofsson P, Vikström T, Nagelkerke N, Wang J, Abu-Zidan FM. Multiple small bowel ligation compared to conventional primary repair after abdominal gunshot wound with haemorrhagic shock. Scand J Surg 2009; 98: Wang P, Wang J, Zhang W, Li Y, Li J. Effect of the combination of fibrin glue and growth hormone on intestinal anastomoses in a pig model of traumatic shock associated with peritonitis. World J Surg 2009; 33: Barzana DC, Kotwall CA, Clancy TV, Hope WW. Use of laparoscopy in trauma at a level II trauma center. JSLS 2011; 15: Kaban GK, Novitsky YW, Perugini RA, Haveran L, Czerniach D, Kelly JJ, Litwin DE. Use of laparoscopy in evaluation and treatment of penetrating and blunt abdominal injuries. Surg Innov 2008; 15: Ishibashi S, Takeuchi H, Fujii K, Shiraishi N, Adachi Y, Kitano S. Length of laparotomy incision and surgical stress assessed by serum IL-6 level. 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177 Online Submissions: doi: /wjg.v18.i World J Gastroenterol 2012 December 14; 18(46): ISSN (print) ISSN (online) 2012 Baishideng. All rights reserved. BRIEF ARTICLE Analysis of TLR4 and TLR2 polymorphisms in inflammatory bowel disease in a Guangxi Zhuang population Lan Chen, Mei-Jiao Lin, Ling-Ling Zhan, Xiao-Ping Lv Lan Chen, Mei-Jiao Lin, Xiao-Ping Lv, Department of Gastroenterology, the First Affiliated Hospital of Guangxi Medical University (West), Nanning , Guangxi Zhuang Autonomous Region, China Ling-Ling Zhan, Department of Clinical Experimental Medicine, the First Affiliated Hospital of Guangxi Medical University, Nanning , Guangxi Zhuang Autonomous Region, China Author contributions: Chen L performed the experiment and wrote the manuscript; Lin MJ collected the samples; Zhan LL analyzed the data and was also involved in editing the manuscript; Lv XP designed the research and revised the paper. Supported by The Natural Science Foundation of Guangxi Zhuang Autonomous Region, China, No ; the Chinese Traditional Medicine Science Foundation of Guangxi Zhuang Autonomous Region, China, No. GZKZ Correspondence to: Xiao-Ping Lv, Professor, Department of Gastroenterology, the First Affiliated Hospital of Guangxi Medical University (West), Nanning , Guangxi Zhuang Autonomous Region, China. [email protected] Telephone: Fax: Received: September 18, 2012 Revised: October 18, 2012 Accepted: November 6, 2012 Published online: December 14, 2012 Abstract AIM: To study the polymorphisms of toll-like receptor 4 (TLR4 ) gene Asp299Gly, Thr399Ile and TLR2 gene Arg753Gln, Arg677Trp and susceptibility to inflammatory bowel disease (IBD) in the Zhuang population from Guangxi, China. METHODS: A case-control study was performed from February 2007 to October 2011 which included 146 Zhuang patients with IBD in the experimental group and 164 healthy Zhuang subjects who acted as the control group. All patients and healthy subjects were from the Guangxi Zhuang Autonomous Region of China. Genomic DNA was extracted from intestinal tissue by the phenol chloroform method. TLR4 gene Asp299Gly, Thr399Ile and TLR2 gene Arg753Gln, Arg677Trp were amplified by polymerase chain reaction (PCR), and then detected by PCR-restriction fragment length polymorphism (RFLP). RESULTS: The TLR4 gene Asp299Gly was digested using Nco Ⅰ restriction enzyme, and a single band of 249 bp was observed which showed that it was a wild type (AA). The TLR4 gene Thr399Ile was digested using Hinf Ⅰrestriction enzyme and only the wild type (CC) was detected. In addition, the TLR2 gene Arg- 677Trp was digested using Aci Ⅰ restriction enzyme and only the wild type (CC) was detected. The TLR2 gene Arg753Gln was digested using Pst Ⅰ restriction enzyme. Only the wild type (GG) as a single band of 254 bp was observed during RFLP. Overall, no heterozygous or homozygous single nucleotide polymorphism mutations were found in patients with Crohn s disease and ulcerative colitis both in the TLR4 gene Asp299Gly, Thr399Ile and the TLR2 gene Arg677Trp, Arg753Gln in the Zhuang population from the Guangxi Zhuang Autonomous Region of China. CONCLUSION: The TLR4 gene Asp299Gly, Thr399Ile and TLR2 gene Arg753Gln, Arg677Trp polymorphisms may not be associated with IBD in the Zhuang population from the Guangxi Zhuang Autonomous Region of China Baishideng. All rights reserved. Key words: Toll-like receptor 2; Toll-like receptor 4; Inflammatory bowel disease; Gene polymorphism Peer reviewer: Dr. Dieter Kabelitz, Professor, Department of Immunology, Kiel University (CAU). Michaelisstraße 5, Kiel, Germany Chen L, Lin MJ, Zhan LL, Lv XP. Analysis of TLR4 and TLR2 polymorphisms in inflammatory bowel disease in a Guangxi Zhuang population. World J Gastroenterol 2012; 18(46): Available from: URL: com/ /full/v18/i46/6856.htm DOI: org/ /wjg.v18.i December 14, 2012 Volume 18 Issue 46

178 Chen L et al. TLR4 and TLR2 in Guangxi Zhuang population INTRODUCTION Inflammatory bowel disease (IBD) is a chronic disorder caused by multiple factors in genetically susceptible hosts, and includes ulcerative colitis (UC) and Crohn s disease (CD). The molecular basis of the pathogenesis is not completely clear, but involves a complex interaction of factors such as genetics, immunology, environment and infection. The incidence of IBD in Western populations has increased with an estimated incidence of 0.10%-1.00% for UC and 0.35%-1.00% for CD during the past few decades [1]. NOD2/CARD15 was the first verified predisposing gene for CD, where three NOD2/ CARD15 polymorphisms, Arg702Trp, Gly908Arg and Leu1007fsinsC, were found to be significantly associated with CD in Caucasian populations [2,3]. Nevertheless, these single nucleotide polymorphisms (SNPs) were not reported to be associated with CD in Japanese, and Hubei, Zhejiang, or Hong Kong populations in China [4-7], thus, their exact role in CD is controversial. Recently, genome wide association studies, provided evidence for several determinants including Toll-like receptor 2 (TLR2) and TLR4 [8]. TLR4 is upregulated in intestinal epithelial cells, macrophages, and dendritic cells in patients with UC and CD. In contrast, the expression of TLR2 is unchanged. The Asp299Gly and Thr399Ile polymorphisms of the TLR4 gene were shown to be associated with lipopolysaccharide hyporesponsiveness and with both CD and UC in some studies [9,10]. The TLR2 gene Arg753Gln polymorphism frequency is approximately 1%-4% in the Caucasian population, significantly higher than that in Indian patients with IBD [11]. In view of the discrepant data regarding the association between key regulatory genes and IBD susceptibility, the purpose of our study was to investigate whether the known gene polymorphisms in TLR2 and TLR4 determine susceptibility to IBD in the Guangxi Zhuang population from the Guangxi Zhuang Autonomous Region. Guangxi has a large Zhuang population, where genetic diseases and gene polymorphisms are unique. Therefore, research on the relation between TLR2 and TLR4 polymorphisms and IBD in Chinese Zhuang patients from the Guangxi Zhuang Autonomous Region is needed. MATERIALS AND METHODS Patients The study group consisted of 146 IBD Zhuang patients without genetic kinship enrolled in the Gastroenterology Department, First Affiliated Hospital of Guangxi Medical University, from February 2007 to October The control group included 164 healthy Zhuang subjects without genetic kinship who were healthy individuals or patients with functional dyspepsia, and did not have liver or gastrointestinal diseases. All patients had a well-established diagnosis of UC or CD, according to standard clinical criteria based on endoscopic and histopathological examinations. There were no significant differences in age and sex between the study group and the control group. All patients and healthy controls gave informed consent and the study was approved by the ethical committee of the institute. DNA extraction and genotyping of the TLR4 and TLR2 polymorphisms Genomic DNA was extracted according to the modified protocol of Taggart. The primers used to amplify the TLR4 gene (Asp299Gly, Thr399 Ile) were designed according to the National Center for Biotechnology Information gene database, NM_138554, and the TLR2 gene (Arg677Trp, Arg753Gln) with NM _ shown in Table 1 (primers were synthesized in SHENGGONG Biological Technology Co., Ltd., Shanghai, China). Amplification was performed using H2O µl, 2 Taq polymerase chain reaction (PCR) Master-mix 11.5 µl (TIANGEN), DNA 1 µl, and 1 µl in addition to 10 µmol/l of each primer in a total volume of 25 µl. For Asp299Gly and Arg753Gln, cycle conditions were an initial denaturation for 5 min at 95, followed by 28 cycles of denaturing at 95 for 40 s, annealing at 58 for 30 s, primer extension at 72 for 50 s, followed by a final extension at 72 for 10 min. For Thr399Ile and Arg677Trp, cycle conditions were an initial denaturation for 5 min at 95, followed by 28 cycles of denaturing at 95 for 40 s, annealing at 62 for 40 s, primer extension at 72 for 50 s, followed by a final extension at 72 for 10 min (Thermo Electron Corporation, Waltham, MA, United States). All the PCR products were electrophoresed on a 1.5% agarose gel, with 1 tris-borate-edta buffer, V = 100V for 30 min, and visualized under ultraviolet illumination (Bio-Rad Gel Doc-2000, United States). Five µl PCR products of TLR4 gene (Asp299Gly, Thr399Ile) and TLR2 gene (Arg677Trp,Arg753Gln) were digested at 37 overnight with 0.5 µl (10 U) Nco Ⅰ, Hinf Ⅰ, Aci Ⅰ, and Pst Ⅰ restriction enzymes (Fermentas), respectively. After enzymatic digestion, the fragments were separated and visualized by gel electrophoresis (2% Yito Bio-Instrument Company Ltd., Shanghai, China). PCR products of each SNP were purified with a PCR purification kit (QIAGEN, Hilden, Germany) and were sequenced using an ABI Prism 377 DNA sequencer (LIU HE HUA DA GENE Technology Co, Ltd., Beijing, China). Statistical analysis The genetic equilibrium was tested using Hardy-Weinberg. Allele and genotype frequencies in patients and in controls were compared using the χ 2 test with SPSS 13.0, and P values were considered significant at a level of < RESULTS RFLP analysis was performed to assess the status of SNPs in our samples after digestion with restriction enzymes (Figure 1) December 14, 2012 Volume 18 Issue 46

179 Chen L et al. TLR4 and TLR2 in Guangxi Zhuang population Table 1 Specific polymerase chain reaction primers and restriction enzymes for each single nucleotide polymorphism Gene Amino acid substitution Sequence of primers( 5' 3') Fragments (bp) Enzymes TLR4 Asp299Gly F-GATTAGCATACTTAGACTACTACCTCCATG 249 NcoⅠ R-GATCAACTTCTGAAAAAGCATTCCCAC Thr399Ile F-GGTTGCTGTTCTCAAAGTGATTTTGGGAGAA 406 HinfⅠ R-ACCTGAAGACTGGAGAGTGAGTTAAATGCT TLR2 Arg677Trp F-GCCTACTGGGTGGAGAACCTT R-CCAGTTCATACTTGCACCACTC 199 AciⅠ Arg753Gln F-CCTGGCAAGTGGATCATTGAC R-GGCCACTCCAGGTAGGTCTT 254 PstⅠ TLR: Toll-like receptor. A 600 bp B 600 bp 406 bp 249 bp bp 100 bp M M C 600 bp D 600 bp 124 bp bp bp M 75 bp 100 bp M Figure 1 Polymerase chain reaction-restriction fragment length polymorphism analysis. A: Electrophoresis of Asp299Gly restriction fragment length polymorphism (AA type); B: Electrophoresis of Thr399Ile restriction fragment length polymorphism (CC type); C: Electrophoresis of Arg677Trp restriction fragment length polymorphism (CC type); D: Electrophoresis of Arg753Gln restriction fragment length polymorphism (GG type). M: Marker; 1: Ulcerative colitis; 2: Crohn s disease; 3: Control. TLR4 gene Asp299Gly PCR-RFLP Nco Ⅰ restriction enzyme identified the sequence of CCATGG. For the heterozygous type (AG), this could be generated when digested with Asp299Gly and three bands of 249 bp, 223 bp and 26 bp, where one chain was cut, and the others were not. For the homozygous type (GG), a transite to G, Nco Ⅰ identified the mutated site, and the mutated DNA was visible as a double band of 223 bp and 26 bp, whereas a single band of 249 bp was observed in the wild type (AA), as the site was not cut. In this study, only the wild type (AA) was detected, and no other types (Figure 1A). TLR4 gene Thr399Ile PCR-RFLP Hinf Ⅰ restriction enzyme identified the sequence of GANTC. For the heterozygous type (CT), this could be generated when digested with Thr399Ile and three bands of 406 bp, 377 bp and 29 bp, where only one chain was cut, and the others were not. For the homozygous type (TT), C transite to T, Hinf Ⅰ identified the mutated site, and the mutated DNA was visible as a double band of 377 bp and 29 bp, whereas a single band of 406 bp was observed in the wild type (CC), as the site was not cut. In this study, only the wild type (CC) was detected, and no other types (Figure 1B). TLR2 gene Arg677Trp PCR-RFLP Aci Ⅰ restriction enzyme identified the sequence of CCGC. For the heterozygous type (CT), this could be generated when digested with Arg677Trp and three bands of 199 bp, 124 bp and 75 bp, where one chain was cut, and the others were not. For the homozy December 14, 2012 Volume 18 Issue 46

180 Chen L et al. TLR4 and TLR2 in Guangxi Zhuang population gous type (TT), C transite to T, the site was not cut by Aci Ⅰ restriction enzyme, and a single band of 199 bp was observed, for the wild type (CC), it identified the site, and the DNA was visible as a double band of 124 bp and 75 bp. In this study, only the wild type (CC) was detected, and no other types (Figure 1C). TLR2 gene Arg753Gln PCR-RFLP Pst Ⅰ restriction enzyme identified the sequence of CTGCAG. For the heterozygous type (GA), this could be generated when digested with Arg753Gln and three bands of 254 bp, 214 bp and 40 bp, where one chain was cut, and the others were not. For the homozygous type (AA), G transite to A, Pst Ⅰ identified the mutated site, and the mutated DNA was visible as a double band of 214 bp and 40 bp, whereas a single band of 254 bp was observed in the wild type (GG), as the site was not cut. In this study, only the wild type (GG) was detected, and no other types (Figure 1D). DISCUSSION UC and CD are multifactorial diseases of unknown etiology. Despite being disorders of the gastrointestinal tract, an abnormal immune response directed against the gut microflora has been postulated as a possible explanation in genetically susceptible hosts [12]. Numerous studies have been performed on the frequency of NOD2/CARD15 mutations in IBD populations in Western Europe and Northern America. In CD patients from Northern America or Western Europe, allele frequencies ranged from 9.1%-12.9%, 6.6%-16.0% and 3.3%-6.0% for Arg702Trp, 1007finsC, and Gly908Arg, respectively [13]. Interestingly, a recent study reported lower allele frequencies in Finnish IBD patients for all three NOD2/CARD15 variants in CD patients compared with the above-mentioned studies and similar frequencies in patients with UC [14]. Additionally, in Asian IBD populations, NOD2/CARD15 variants were not detected at all [4,5]. Thus, there is a need for more studies in different populations with IBD. Human TLRs participate in the innate immune response and signal the activation of adaptive immunity. Therefore, these TLRs may be important in autoimmune diseases such as IBD, rheumatoid arthritis and systemic lupus erythematosus [15]. This is the first study to report the TLR4 (Asp299Gly, Thr399Ile) and TLR2 (Arg677Trp, Arg753Gln) gene mutations in patients with IBD from the Guangxi Zhuang population of China, where the ethnic background is heterogeneous, and includes Han, Zhuang and Rao. In this study, the mutation genotypes of the TLR4 gene Asp299Gly, Thr399Ile and TLR2 gene Arg677Trp, Arg- 753Gln were not found in the Zhuang population with IBD. Our results are in agreement with those from studies on Asian patients from Hong Kong, Hubei, Zhejiang, Shanghai and Japan [4-7]. In recent years, several studies have reported divergent results [16]. The allele frequency of the Asp299Gly variant ranges from 0%-10% in UC, from 8%-13% in CD and from 3%-15% in healthy controls [17]. Franchimont et al [10] reported that the TLR4 SNP was associated with UC and CD in a Belgian study. This association was in accordance with Dutch, Greek, Australian and German populations with CD, and an association with colonic disease has been described [18-21]. In one German cohort, an association between the TLR4 Thr399Ile SNP and UC was demonstrated [22]. However, because of substantial heterogeneity between populations, no association was noted in Scottish CD patients [23]. The TLR2 gene Arg753Gln variant frequency was 9.4% in Germany, and 1% in Spain [24], the TLR2 gene Arg677Trp was 30.3% in Tunisia [25], where the difference was significant. In addition, Pierik et al [26] reported that an association was found between non-synonymous variants and extensive colonic disease with UC and CD in the TLR2 genes. In our study, we did not find mutations in TLR4 gene Asp299Gly, Thr399Ile and TLR2 gene Arg677Trp, Arg753Gln in the Zhuang population with IBD. These findings were different from those in Tunisia, Germany and Spain. Our results showed that the TLR4 gene Asp- 299Gly, Thr399 Ile and TLR2 gene may not be associated with IBD patients from the Guangxi Zhuang Autonomous Region of China. This is possibly due to the existence of racial and geographic differences. The present study supports the notion that genetics, immunology, environment and infection may be vital in the pathogenesis of IBD. However, the heterogeneity in the small number of available studies limited the ability to draw conclusions. Further studies using a larger cohort of patients with IBD are warranted to identity the risk factors and genetic susceptibility to IBD. COMMENTS Background The pathogenesis of inflammatory bowel disease (IBD) is not completely clear, however, contributing factors may include immunology, genetics, environment and infection. It has been reported that the NOD2/CARD15 polymorphisms (single nucleotide polymorphisms, SNPs) were found to be associated with Crohn s disease (CD) in Caucasian populations. However, the SNPs were not found to be associated with CD in Japan and China. In addition, the TLR2 and TLR4 were reported to provide evidence for several determinants. The study assessed whether the known gene polymorphisms in the toll-like receptor 2 (TLR2) and TLR4 genes determined susceptibility for IBD in the Guangxi Zhuang population. Since Guangxi has a large Zhuang population, genetic diseases and gene polymorphisms are unique. Research frontiers TLR4 (Asp299Gly, Thr399Ile) and TLR2 (Arg677Trp, Arg753Gln) were found to be associated with IBD in Tunisia, Germany and Spain, but not in Hong Kong, Hubei, Zhengjiang, Shanghai and Japan. This is possibly due to the existence of racial and geographic differences. This study determined whether the TLR4 (Asp299Gly, Thr399Ile) and TLR2 (Arg677Trp, Arg753Gln) were associated with IBD in the Guangxi Zhuang population from the Guangxi Zhuang Autonomous Region of China. Innovations and breakthroughs Only a few studies have investigated the TLR2 and TLR4 gene polymorphisms in China. Moreover, all the subjects in these studies were from the Han population. This is the first study to report that TLR4 gene Asp299Gly, Thr399Ile and TLR2 gene Arg753Gln, Arg753Gln polymorphisms may not be associated with IBD in the Zhuang population from the Guangxi Zhuang Autonomous Region of China December 14, 2012 Volume 18 Issue 46

181 Chen L et al. TLR4 and TLR2 in Guangxi Zhuang population Applications TLR2 and TLR4 variants may be rare or non-existent in the Zhuang population from the Guangxi Zhuang Autonomous Region of China. Terminology TLRs play a key role in microbial recognition in innate immunity and control the adaptive immune responses. Among the TLRs, TLR2 recognizes bacterial components such as lipoprotein, lipoteichoic acids, peptido-glycan and zymozan. TLR4 requires CD14 to form the lipopolysaccharide (LPS) receptor. LPS, found in the outer membrane of Gram-negative bacteria, is opsonized by LPS-binding protein, and recognized by CD14. The LPS-LPS binding protein-cd14 complex activates TLR4, which results in the activation of NF-κB. TLR2 and TLR4 gene mutations or deletions can induce abnormal immune responses. Peer review This study demonstrates no association of TLR2 and/or TLR4 polymorphism with IBD in a patient cohort within a restricted chinese population. Although this is a negative result, it adds to the impact of genetic and/or ethnical predisposition to IBD. REFERENCES 1 Branch CMAD. Guidelines for diagnosis and management of inflammatory bowel disease. Zhonghua Xiaohua Zazhi 2001; 21: Hugot JP, Chamaillard M, Zouali H, Lesage S, Cézard JP, Belaiche J, Almer S, Tysk C, O Morain CA, Gassull M, Binder V, Finkel Y, Cortot A, Modigliani R, Laurent-Puig P, Gower-Rousseau C, Macry J, Colombel JF, Sahbatou M, Thomas G. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn s disease. Nature 2001; 411: Hampe J, Cuthbert A, Croucher PJ, Mirza MM, Mascheretti S, Fisher S, Frenzel H, King K, Hasselmeyer A, MacPherson AJ, Bridger S, van Deventer S, Forbes A, Nikolaus S, Lennard-Jones JE, Foelsch UR, Krawczak M, Lewis C, Schreiber S, Mathew CG. Association between insertion mutation in NOD2 gene and Crohn s disease in German and British populations. Lancet 2001; 357: Inoue N, Tamura K, Kinouchi Y, Fukuda Y, Takahashi S, Ogura Y, Inohara N, Núñez G, Kishi Y, Koike Y, Shimosegawa T, Shimoyama T, Hibi T. Lack of common NOD2 variants in Japanese patients with Crohn s disease. Gastroenterology 2002; 123: Leong RW, Armuzzi A, Ahmad T, Wong ML, Tse P, Jewell DP, Sung JJ. NOD2/CARD15 gene polymorphisms and Crohn s disease in the Chinese population. Aliment Pharmacol Ther 2003; 17: Wang ZW, Ji F, Teng WJ, Yuan XG, Ye XM. Risk factors and gene polymorphisms of inflammatory bowel disease in population of Zhejiang, China. World J Gastroenterol 2011; 17: Xie ZQ, Lv B, Li-Jun Zou, Xue-Lan Shang,Yi-Kai Zhou. Genotyping of Toll-like Receptor 4 in Hubei Population of Chinese. J Huazhong Univ Sci Technol 2004; 33: Mathew CG. New links to the pathogenesis of Crohn disease provided by genome-wide association scans. Nat Rev Genet 2008; 9: Okayama N, Fujimura K, Suehiro Y, Hamanaka Y, Fujiwara M, Matsubara T, Maekawa T, Hazama S, Oka M, Nohara H, Kayano K, Okita K, Hinoda Y. Simple genotype analysis of the Asp299Gly polymorphism of the Toll-like receptor-4 gene that is associated with lipopolysaccharide hyporesponsiveness. J Clin Lab Anal 2002; 16: Franchimont D, Vermeire S, El Housni H, Pierik M, Van Steen K, Gustot T, Quertinmont E, Abramowicz M, Van Gossum A, Devière J, Rutgeerts P. Deficient host-bacteria interactions in inflammatory bowel disease? The toll-like receptor (TLR)-4 Asp299gly polymorphism is associated with Crohn s disease and ulcerative colitis. Gut 2004; 53: Biswas D, Gupta SK, Sindhwani G, Patras A. TLR2 polymorphisms, Arg753Gln and Arg677Trp, are not associated with increased burden of tuberculosis in Indian patients. BMC Res Notes 2009; 2: Fiocchi C. Inflammatory bowel disease: etiology and pathogenesis. Gastroenterology 1998; 115: Bonen DK, Cho JH. The genetics of inflammatory bowel disease. Gastroenterology 2003; 124: Heliö T, Halme L, Lappalainen M, Fodstad H, Paavola- Sakki P, Turunen U, Färkkilä M, Krusius T, Kontula K. CARD15/NOD2 gene variants are associated with familially occurring and complicated forms of Crohn s disease. Gut 2003; 52: Huang Q, Pope RM. Toll-like receptor signaling: a potential link among rheumatoid arthritis, systemic lupus, and atherosclerosis. J Leukoc Biol 2010; 88: Riis L, Vind I, Vermeire S, Wolters F, Katsanos K, Politi P, Freitas J, Mouzas IA, O Morain C, Ruiz-Ochoa V, Odes S, Binder V, Munkholm P, Moum B, Stockbrügger R, Langholz E. The prevalence of genetic and serologic markers in an unselected European population-based cohort of IBD patients. Inflamm Bowel Dis 2007; 13: Russell RK, Nimmo ER, Satsangi J. Molecular genetics of Crohn s disease. Curr Opin Genet Dev 2004; 14: Brand S, Staudinger T, Schnitzler F, Pfennig S, Hofbauer K, Dambacher J, Seiderer J, Tillack C, Konrad A, Crispin A, Göke B, Lohse P, Ochsenkühn T. The role of Toll-like receptor 4 Asp299Gly and Thr399Ile polymorphisms and CARD15/NOD2 mutations in the susceptibility and phenotype of Crohn s disease. Inflamm Bowel Dis 2005; 11: Gazouli M, Mantzaris G, Kotsinas A, Zacharatos P, Papalambros E, Archimandritis A, Ikonomopoulos J, Gorgoulis VG. Association between polymorphisms in the Toll-like receptor 4, CD14, and CARD15/NOD2 and inflammatory bowel disease in the Greek population. World J Gastroenterol 2005; 11: Hume GE, Fowler EV, Doecke J, Simms LA, Huang N, Palmieri O, Griffiths LR, Florin TH, Annese V, Radford- Smith GL. Novel NOD2 haplotype strengthens the association between TLR4 Asp299gly and Crohn s disease in an Australian population. Inflamm Bowel Dis 2008; 14: Oostenbrug LE, Drenth JP, de Jong DJ, Nolte IM, Oosterom E, van Dullemen HM, van der Linde K, te Meerman GJ, van der Steege G, Kleibeuker JH, Jansen PL. Association between Toll-like receptor 4 and inflammatory bowel disease. Inflamm Bowel Dis 2005; 11: Török HP, Glas J, Tonenchi L, Mussack T, Folwaczny C. Polymorphisms of the lipopolysaccharide-signaling complex in inflammatory bowel disease: association of a mutation in the Toll-like receptor 4 gene with ulcerative colitis. Clin Immunol 2004; 112: Cuthbert AP, Fisher SA, Mirza MM, King K, Hampe J, Croucher PJ, Mascheretti S, Sanderson J, Forbes A, Mansfield J, Schreiber S, Lewis CM, Mathew CG. The contribution of NOD2 gene mutations to the risk and site of disease in inflammatory bowel disease. Gastroenterology 2002; 122: Sánchez E, Orozco G, López-Nevot MA, Jiménez-Alonso J, Martín J. Polymorphisms of toll-like receptor 2 and 4 genes in rheumatoid arthritis and systemic lupus erythematosus. Tissue Antigens 2004; 63: Ben-Ali M, Barbouche MR, Bousnina S, Chabbou A, Dellagi K. Toll-like receptor 2 Arg677Trp polymorphism is associated with susceptibility to tuberculosis in Tunisian patients. Clin Diagn Lab Immunol 2004; 11: Pierik M, Joossens S, Van Steen K, Van Schuerbeek N, Vlietinck R, Rutgeerts P, Vermeire S. Toll-like receptor-1, -2, and -6 polymorphisms influence disease extension in inflammatory bowel diseases. Inflamm Bowel Dis 2006; 12: 1-8 S- Editor Song XX L- Editor A E- Editor Xiong L 6860 December 14, 2012 Volume 18 Issue 46

182 Online Submissions: doi: /wjg.v18.i World J Gastroenterol 2012 December 14; 18(46): ISSN (print) ISSN (online) 2012 Baishideng. All rights reserved. CASE REPORT Puncture injection of para-toluenesulfonamide combined with chemoembolization for advanced hepatocellular carcinoma Qing He, An-Ren Kuang, Yong-Song Guan, Yue-Qing Liu Qing He, Yong-Song Guan, Yue-Qing Liu, Department of Oncology, West China Hospital of Sichuan University, Chengdu , Sichuan Province, China An-Ren Kuang, Department of Nuclear Medicine, West China Hospital of Sichuan University, Chengdu , Sichuan Province, China Author contributions: He Q and Kuang AR conceived and designed the study and prepared the manuscript; Guan YS and Liu YQ acquired and analyzed the data. Correspondence to: Dr. An-Ren Kuang, Department of Nuclear Medicine, West China Hospital of Sichuan University, Chengdu , Sichuan Province, China. [email protected] Telephone: Fax: Received: July 22, 2012 Revised: September 11, 2012 Accepted: September 19, 2012 Published online: December 14, 2012 Abstract Hepatocellular carcinoma (HCC) is difficult to eradicate due to its resilient nature. Portal vein is often involved in tumors of large size, which exclude the patient from surgical resection and local ablative therapy, such as percutaneous ethanol injection (PEI) and radiofrequency ablation (RFA) because they were considered neither effective nor safe. Currently, there is almost no effective treatment for HCC of such condition. As a unique antitumor agent in form of lipophilic fluid for local injection, para-toluenesulfonamide (PTS) produces mild side effects while necrotizing the tumor tissues quickly and efficiently. Being largely different from both PEI and RFA therapies, PTS can disseminate itself in tumors more easily than other caustic agents, such as alcohol. So PTS may offer additional benefit to HCCs with vascular involvement. We herein describe a 70-year-old HCC patient who was treated with the combination of PTS injection and transcatheter arterial chemoembolization, resulting in a significantly improved clinical prognosis Baishideng. All rights reserved. Key words: Hepatocellular carcinoma; Para-toluenesulfonamide; Antitumor agent; Transcatheter arterial chemoembolization; Therapy Peer reviewers: Alessandro Cucchetti, MD, Liver and Multiorgan Transplant Unit, Policlinico S.Orsola-Malpighi, University of Bologna, PAD 25., Via Massarenti 9, Bologna, Italy; Zenichi Morise, MD, PhD, Professor, Chairman, Department of Surgery Banbuntane Houtokukai Hospital, Fujita Health University School of Medicine, Otobashi Nakagawa-ku, Nagoya, Aichi , Japan He Q, Kuang AR, Guan YS, Liu YQ. Puncture injection of para-toluenesulfonamide combined with chemoembolization for advanced hepatocellular carcinoma. World J Gastroenterol 2012; 18(46): Available from: URL: DOI: org/ /wjg.v18.i INTRODUCTION Transcatheter arterial chemoembolization (TACE) has become the standard treatment for unresectable hepatocellular carcinoma (HCC). Nonetheless, clinical outcomes are often unsatisfactory, especially for recurrent cases. As a novel anticancer agent, para-toluenesulfonamide (PTS) is completely different from genetic, classical chemical and molecular targeted drugs, and has shown amazing antitumor effect in animal HCC experiment [1,2]. Primary pharmacological studies suggest that PTS inhibits tumor growth by acting as a tumor necrotizing agent [1,2]. PTS may strengthen the effect of TACE in advanced HCC. We herein report a patient with refractory HCC who was treated with PTS injection after TACE, which resulted in a very good clinical prognosis. CASE REPORT The patient was a 70-year-old man. A mass was found in his right lobe of the liver in May He refused 6861 December 14, 2012 Volume 18 Issue 46

183 He Q et al. PTS for advanced HCC to receive further diagnosis and treatment until January 2006 when he began to suffer from anorexia and dull abdominal pain. Computer tomography (CT) detected a large mass (about 9.5 cm 8.5 cm 8.5 cm) in the right lobe of the liver with significant enhancement (Figure 1A). Alpha-fetoprotein (AFP) level was > 1210 ng/ml. He was diagnosed with a histologically proven HCC. Due to the large tumor size and invasion to the portal vein, the patient underwent two rounds of TACE with an interval of one month. The drugs used were 5fluoro2deoxyuridine (1.0 g), epirubicin (40 mg) and lipiodol (10 ml). In May 2006, three months after the second TACE, a routine postoperative CT scan found a minimal decrease in tumor size with necrosis < 25% (Figure 1B), while the AFP level was still > 1210 ng/ml. The second angiography revealed hepatic artery-portal vein fistula and hepatic arteriovenous fistula. Although additional gelfoam pieces were used and the fistula disappeared after embolization, a poor prognosis was still predicted. Consequently, PTS (Beijing Vision Drugs Development Ltd., Beijing, China) was used to enhance the effect of TACE after informed consent was obtained from the patient. The mass was punctured percutaneously with a fine needle under CT guidance. After the tip of the needle was manipulated into the mass, about 10 ml of PTS was injected intratumorally in a multi-point fashion. This injection was repeated four times. The interval between each procedure was between three and seven days according to the patient s clinical condition. When all five injections were completed, the AFP level dropped to 1106 ng/ml. Two months later, a routine follow-up CT showed moderate improvement, no blood being supplied to the tumor (Figure 2A), and the AFP level dropped further down to 185 ng/ml. In an attempt to investigate the increased efficacy, PTS injections were repeated three more times. Following a routine check two months later, a CT examination of the abdomen demonstrated complete deposit of oil and no signs of recurrence or tumor embolism (Figure 2B). Furthermore, the AFP was within the normal reference range (4.44 ng/ml). The only side effect of this therapy was abdominal pain, which occurred after the first two procedures, but subsided shortly thereafter in approximately 10 min. No other discomfort was noted post-procedurally. Four years following the last PTS treatment, the patient exhibited no evidence of recurrence and no other abnormal liver function serum values, and he had a normal serum AFP level. The AFP level changes in the course of the treatment are summarized in Table 1. DISCUSSION Despite the fact that TACE has become the standard treatment for unresectable HCC, it is frequently unsuccessful. Rate of local recurrence following tumor resection is also unacceptably high [3-6]. In addition, TACE alone fails to control the tumor completely, often leading Table 1 Changes of alpha-fetoprotein in the course of therapy Time (yr-mo) APF (ng/ml) TACE PTS injections > 1210 Yes > 1210 Yes > 1210 No 5 times No No 3 times No No No No A sharp decrease in alpha-fetoprotein (AFP) levels occurred right after para-toluenesulfonamide (PTS) injections. The initial series of transcatheter arterial chemoembolization (TACE) failed to control the tumor (although the TACE s effect on fistula embolization was irreplaceable). PTS treatment appeared to be effective and well-tolerated. A 4-year follow-up showed no sign of reoccurrence. to a poor prognosis. In this report, the patient had received two sessions of TACE and embolized the fistula successfully before admission. Although little effect was got on HCC, the TACE did restrict the tumor s growth and metastasis that made it possible for PTS to eradicate the tumor later. Due to the large tumor size and involvement of the portal vein, this lesion was inoperable, and other local ablative therapies, such as percutaneous ethanol injection, microwave coagulation therapy and radiofrequency ablation (RFA) were considered neither effective nor safe. Complications caused by these modalities may result in a high mortality rate. Therefore, we attempted to treat the patient with the combined administration of PTS and TACE. Consequently, the patient s condition was significantly improved with satisfactory tumor control and without severe complications. Para-toluenesulfonamide (P-TSA) is the active ingredient in PTS. The P-TSA is a white, odorless, crystalline substance that has a very low solubility in water. The molecular formula is C7H9NO2S. The injection solution is a clear, colorless, oil liquid with a characteristic odor and contains 330 mg/ml P-TSA. PTS has been approved for clinical trial injection in both 3 ml ampoule and 5 ml ampoule. Recommended storage temperature is 25 (77 F), although a range of (50-90 F) is acceptable. Long-term exposure to light should be avoided. PTS produces mild side effects while necrotizing the tumor tissues effectively and thoroughly. However, PTS is still in the phase of clinical trial, and the mechanism of the antitumor activity of PTS is still unclear. Primary pharmacological studies suggest that PTS inhibits tumor growth by acting as a tumor necrotizing agent [1,2]. It has been shown that PTS does not cause serious side effects that have been observed frequently in conventional chemotherapy and locoregional therapy (e.g., RFA, alcohol injection, etc.), such as fever, bone marrow suppression, stomach discomfort, hemorrhage, needletrack seeding, lesion abscess, liver failure, biloma, biliary 6862 December 14, 2012 Volume 18 Issue 46

184 He Q et al. PTS for advanced HCC A A B B Figure 1 Transcatheter arterial chemoembolization had little effect on the hepatocellular carcinoma, but restrict the size of tumor mass. A: Contrast computer tomography (CT) before the procedure showed a huge hypodense mass with remarkable enhancement and portal vein embolus; B: Two months after the initial 2 series of transcatheter arterial chemoembolization, contrast CT still found a hypodense nodule with partial contrast enhancement and portal vein invasion. Figure 2 The following para-toluenesulfonamide injections resulted in a significantly improved clinical prognosis. A: Contrast computer tomography (CT) after the first 5 para-toluenesulfonamide (PTS) injections demonstrated a decreased nodule with partial retention of lipiodol. Remarkable necrosis and no blood supply were found in the rest part of the tumor; B: Two months after the last 3 PTS injections, contrast CT revealed homogeneous dense retention of lipiodol within the entire tumoral mass and no recurrent lesion was identified. stricture, portal vein thrombosis, and hemothorax [7-9]. Because most anticancer drugs are corrosive and extremely toxic, they will destroy both cancer cells and normal cells when given locally at high concentrations. PTS is a local therapeutic drug that is injected directly into the tumors and has been shown to cause selectively necrosis in a variety of cancers with minimal damage to normal tissues [2,10]. Local ablative therapies share similar difficulties with surgical resection. The size, site and number of tumors, vascular and extrahepatic involvement as well as liver function of the patient pose a relatively minor effect on the usage of PTS [8,9]. PTS is a more readily available alternative to the local ablative therapies. PTS, in form of lipophilic fluid, kills tumor cells by a rodent mode. Local and intratumoral injection is the optimal route of PTS delivery. Being largely different from both alcohol and RFA therapies, PTS can disseminate itself in tumors more easily than other caustic agents, such as alcohol. Therefore, a successful PTS administration is to approach to the anatomically dangerous or hard-toreach areas and diffuse to the target area and induce injury to the tumor tissues. This might be the mechanism as to why PTS combined with TACE could effectively treat the HCC with vascular invasion. As a locoregional antitumor agent, PTS is safe [1,2,10]. But up to date, PTS is still only a locally-used antitumor agent. It is intended mainly for the treatment of a limited number of detectable tumors. PTS is not suitable to be used alone for the treatment of multifocal HCCs. This case report demonstrated that PTS is effective in treating liver cancers by intratumoral injection, which was hypothesized to enhance the effect of TACE. This combined therapy may prove to be useful in the treatment of patients with refractory and recurrent HCC. Therefore, subsequent large, multi-center, randomized controlled studies are needed to facilitate the introduction of PTS as a novel modality for the treatment of cancers. REFERENCES 1 Li MY, Meng H, Zhu WL, Zhou SZ, Li HY, Zhang JR. [Doseeffect relationship of para-toluenesulfonamide for treatment of hepatocellular carcinoma in rats]. Nanfang Yike Daxue Xuebao 2008; 28: Meng H, Li MY, Zhu WL, Zhang JR. [Therapeutic effect of para-toluenesulfonamide on transplanted hepatocarcinoma in nude mice]. Nanfang Yike Daxue Xuebao 2009; 29: Qu XD, Chen CS, Wang JH, Yan ZP, Chen JM, Gong GQ, Liu QX, Luo JJ, Liu LX, Liu R, Qian S. The efficacy of TACE combined sorafenib in advanced stages hepatocellullar carcinoma. BMC Cancer 2012; 12: December 14, 2012 Volume 18 Issue 46

185 He Q et al. PTS for advanced HCC 4 Llovet JM, Real MI, Montaña X, Planas R, Coll S, Aponte J, Ayuso C, Sala M, Muchart J, Solà R, Rodés J, Bruix J. Arterial embolisation or chemoembolisation versus symptomatic treatment in patients with unresectable hepatocellular carcinoma: a randomised controlled trial. Lancet 2002; 359: Llovet JM, Bruix J. Systematic review of randomized trials for unresectable hepatocellular carcinoma: Chemoembolization improves survival. Hepatology 2003; 37: He Q, Liu Y, Zou Q, Guan YS. Transarterial injection of H101 in combination with chemoembolization overcomes recurrent hepatocellular carcinoma. World J Gastroenterol 2011; 17: Minami Y, Kudo M. Radiofrequency ablation of hepatocellular carcinoma: a literature review. Int J Hepatol 2011; 2011: Ansari D, Andersson R. Radiofrequency ablation or percutaneous ethanol injection for the treatment of liver tumors. World J Gastroenterol 2012; 18: Signoriello S, Annunziata A, Lama N, Signoriello G, Chiodini P, De Sio I, Daniele B, Di Costanzo GG, Calise F, Olivieri G, Castaldo V, Lanzetta R, Piai G, Marone G, Visconti M, Fusco M, Di Maio M, Perrone F, Gallo C, Gaeta GB. Survival after Locoregional Treatments for Hepatocellular Carcinoma: A Cohort Study in Real-World Patients. Scientific World Journal 2012; 2012: Zhou JQ, Tang ZQ, Zhang JN, Tang JC. Metabolism and effect of para-toluene-sulfonamide on rat liver microsomal cytochrome P450 from in vivo and in vitro studies. Acta Pharmacol Sin 2006; 27: S- Editor Gou SX L- Editor A E- Editor Xiong L 6864 December 14, 2012 Volume 18 Issue 46

186 Online Submissions: World J Gastroenterol 2012 December 14; 18(46): I ISSN (print) ISSN (online) 2012 Baishideng. All rights reserved. ACKNOWLEDGMENTS Acknowledgments to reviewers of World Journal of Gastroenterology We acknowledge our sincere thanks to our reviewers. Many reviewers have contributed their expertise and time to the peer review, a critical process to ensure the quality of our World Series Journals. Both the editors of the journals and authors of the manuscripts submitted to the journals are grateful to the following reviewers for reviewing the articles (either published or rejected) over the past period of time. Paolo Aurello, MD, PhD, Department of Surgery, University of Rome La Sapienza, Faculty of Medicine 2, Via di Grottarossa 1035, Rome, Italy Dr. BS Anand, Professor, Digestive Diseases Section (111D), VA Medical Center, 2002 Holcombe Blvd., Houston, TX 77030, United States Josep M Bordas, MD, Department of Gastroenterology IMD, Hospital Clinic, Llusanes at, Barcelona, Spain Giovanni D De Palma, Professor, Department of Surgery and Advanced Technologies, University of Naples Federico II, School of Medicine, Naples, Italy Yasuhiro Fujino, MD, PhD, Director, Department of Surgery, Hyogo Cancer Center, Kitaoji-cho, Akashi , Japan Po-Shiuan Hsieh, MD, PhD, Head of Department of Physiology and Biophysics, National Defense Medical Center, Taipei 114, Taiwan, China Dr. Abdel-Majid Khatib, PhD, INSERM, UMRS 940, Equipe Avenir, Cibles Thérapeutiques, IGM 27 rue Juliette Dodu, Paris, France Jan Kulig, Professor, MD, Head, 1st Department of General and GI Surgery, Jagiellonian University Medical College, 40 Kopernika St., Kraków, Poland Jae J Kim, MD, PhD, Associate Professor, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50, Irwon-dong, Gangnam-gu, Seoul , South Korea Ian C Lawrance, MB, BS (Hons), PhD, FRACP, Professor, Director, Centre for Inflammatory Bowel Disease, School of Medicine and Pharmacology, University of Western Australia, Centre for Inflammatory Bowel Disease, Fremantle Hospital, T Block, Alma Street, Fremantle, WA 6160, Australia Michael Linnebacher, PhD, Molekulare Onkologie and Immuntherapie, Allgemeine Chirurgie, Universitätsklinikum Rostock, Schillingallee 35, Rostock, Germany Nobuyuki Matsuhashi, MD, Department of Gastroenterology, Kanto Medical Center, NTT East, Higashi-gotanda, Shinagawa-ku, Tokyo , Japan Nageshwar D Reddy, Professor, Asian Institute of Gastroenterology, , Somajiguda, Hyderabad , India Dr. Rupjyoti Talukdar, MD, Department of Gastroenterology and Hepatology, Mayo Clinic, 200 1st Street SW, Rochester, MN 55905, United States Tomohiko Shimatani, Assistant Professor, Department of General Medicine, Hiroshima University Hospital, Kasumi, Minamiku, Hiroshima , Japan Cristiano Simone, PhD, Laboratory of Signal-dependent Transcription, Department of Translational Pharmacology, Consorzio Mario Negri Sud, Via Nazionale 8/A, Santa Maria Imbaro, Italy Santhi Swaroop Vege, MDFACG, FACP, Professor of Medicine, Miles and Shirley Fiterman Center for Digestive Diseases, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, United States Satoshi Yamagiwa, MD, PhD, Division of Gastroenterology and Hepatology, Niigata University Graduate School of Medical and Dental Sciences, 757 Asahimachi-dori, Chuo-ku, Niigata , Japan I December 14, 2012 Volume 18 Issue 46

187 Online Submissions: World J Gastroenterol 2012 December 14; 18(46): I ISSN (print) ISSN (online) 2012 Baishideng. All rights reserved. MEETINGS Events Calendar 2012 January 13-15, 2012 Asian Pacific Helicobacter pylori Meeting 2012 Kuala Lumpur, Malaysia January 19-21, 2012 American Society of Clinical Oncology 2012 Gastrointestinal Cancers Symposium San Francisco, CA 3000, United States January 19-21, Gastrointestinal Cancers Symposium San Francisco, CA 94103, United States January 20-21, 2012 American Gastroenterological Association Clinical Congress of Gastroenterology and Hepatology Miami Beach, FL 33141, United States February 3, 2012 The Future of Obesity Treatment London, United Kingdom February 16-17, th United Kingdom Swallowing Research Group Conference London, United Kingdom February 23, 2012 Management of Barretts Oesophagus: Everything you need to know Cambridge, United Kingdom February 24-27, 2012 Canadian Digestive Diseases Week 2012 Montreal, Canada March 1-3, 2012 International Conference on Nutrition and Growth 2012 Paris, France March 7-10, 2012 Society of American Gastrointestinal and Endoscopic Surgeons Annual Meeting San Diego, CA 92121, United States March 12-14, 2012 World Congress on Gastroenterology and Urology Omaha, NE 68197, United States March 17-20, 2012 Mayo Clinic Gastroenterology and Hepatology Orlando, FL 32808, United States March 26-27, th Annual New Treatments in Chronic Liver Disease San Diego, CA 92121, United States March 30-April 2, 2012 Mayo Clinic Gastroenterology and Hepatology San Antonio, TX 78249, United States March 31-April 1, th Annual New Treatments in Chronic Liver Disease San Diego, CA 92121, United States April 8-10, th International Symposium on Functional GI Disorders Milwaukee, WI 53202, United States April 13-15, 2012 Asian Oncology Summit 2012 Singapore, Singapore April 15-17, 2012 European Multidisciplinary Colorectal Cancer Congress 2012 Prague, Czech April 18-20, 2012 The International Liver Congress 2012 Barcelona, Spain April 19-21, 2012 Internal Medicine 2012 New Orleans, LA 70166, United States April 20-22, 2012 Diffuse Small Bowel and Liver Diseases Melbourne, Australia April 22-24, 2012 EUROSON 2012 EFSUMB Annual Meeting Madrid, Spain April 28, 2012 Issues in Pediatric Oncology Kiev, Ukraine May 3-5, th Congress of The Jordanian Society of Gastroenterology Amman, Jordan May 7-10, 2012 Digestive Diseases Week Chicago, IL 60601, United States May 17-21, ASCRS Annual Meeting- American Society of Colon and Rectal Surgeons Hollywood, FL 1300, United States May 18-19, 2012 Pancreas Club Meeting San Diego, CA 92101, United States May 18-23, 2012 SGNA: Society of Gastroenterology Nurses and Associates Annual Course Phoenix, AZ 85001, United States May 19-22, Digestive Disease Week San Diego, CA 92121, United States June 2-6, 2012 American Society of Colon and Rectal Surgeons Annual Meeting San Antonio, TX 78249, United States June 18-21, 2012 Pancreatic Cancer: Progress and Challenges Lake Tahoe, NV 89101, United States July 25-26, 2012 PancreasFest 2012 Pittsburgh, PA 15260, United States September 1-4, 2012 OESO 11th World Conference Como, Italy September 6-8, Joint International Neurogastroenterology and Motility Meeting Bologna, Italy September 7-9, 2012 The Viral Hepatitis Congress Frankfurt, Germany September 8-9, 2012 New Advances in Inflammatory Bowel Disease La Jolla, CA 92093, United States September 8-9, 2012 Florida Gastroenterologic Society 2012 Annual Meeting Boca Raton, FL 33498, United States September 15-16, 2012 Current Problems of Gastroenterology and Abdominal Surgery Kiev, Ukraine September 20-22, st World Congress on Controversies in the Management of Viral Hepatitis Prague, Czech October 19-24, 2012 American College of Gastroenterology 77th Annual Scientific Meeting and Postgraduate Course Las Vegas, NV 89085, United States November 3-4, 2012 Modern Technologies in Diagnosis and Treatment of Gastroenterological Patients Dnepropetrovsk, Ukraine November 4-8, 2012 The Liver Meeting San Francisco, CA 94101, United States November 9-13, 2012 American Association for the Study of Liver Diseases Boston, MA 02298, United States December 1-4, 2012 Advances in Inflammatory Bowel Diseases Hollywood, FL 33028, United States I December 14, 2012 Volume 18 Issue 46

188 Online Submissions: World J Gastroenterol 2012 December 14; 18(46): I-VI ISSN (print) ISSN (online) 2012 Baishideng. All rights reserved. INSTRUCTIONS TO AUTHORS GENERAL INFORMATION World Journal of Gastroenterology (World J Gastroenterol, WJG, print ISSN , online ISSN , DOI: ) is a weekly, open-access (OA), peer-reviewed journal supported by an editorial board of 1352 experts in gastroenterology and hepatology from 64 countries. The biggest advantage of the OA model is that it provides free, full-text articles in PDF and other formats for experts and the public without registration, which eliminates the obstacle that traditional journals possess and usually delays the speed of the propagation and communication of scientific research results. The open access model has been proven to be a true approach that may achieve the ultimate goal of the journals, i.e. the maximization of the value to the readers, authors and society. Maximization of personal benefits The role of academic journals is to exhibit the scientific levels of a country, a university, a center, a department, and even a scientist, and build an important bridge for communication between scientists and the public. As we all know, the significance of the publication of scientific articles lies not only in disseminating and communicating innovative scientific achievements and academic views, as well as promoting the application of scientific achievements, but also in formally recognizing the priority and copyright of innovative achievements published, as well as evaluating research performance and academic levels. So, to realize these desired attributes of WJG and create a well-recognized journal, the following four types of personal benefits should be maximized. The maximization of personal benefits refers to the pursuit of the maximum personal benefits in a well-considered optimal manner without violation of the laws, ethical rules and the benefits of others. (1) Maximization of the benefits of editorial board members: The primary task of editorial board members is to give a peer review of an unpublished scientific article via online office system to evaluate its innovativeness, scientific and practical values and determine whether it should be published or not. During peer review, editorial board members can also obtain cutting-edge information in that field at first hand. As leaders in their field, they have priority to be invited to write articles and publish commentary articles. We will put peer reviewers names and affiliations along with the article they reviewed in the journal to acknowledge their contribution; (2) Maximization of the benefits of authors: Since WJG is an open-access journal, readers around the world can immediately download and read, free of charge, high-quality, peer-reviewed articles from WJG official website, thereby realizing the goals and significance of the communication between authors and peers as well as public reading; (3) Maximization of the benefits of readers: Readers can read or use, free of charge, high-quality peer-reviewed articles without any limits, and cite the arguments, viewpoints, concepts, theories, methods, results, conclusion or facts and data of pertinent literature so as to validate the innovativeness, scientific and practical values of their own research achievements, thus ensuring that their articles have novel arguments or viewpoints, solid evidence and correct conclusion; and (4) Maximization of the benefits of employees: It is an iron law that a first-class journal is unable to exist without first-class editors, and only first-class editors can create a first-class academic journal. We insist on strengthening our team cultivation and construction so that every employee, in an open, fair and transparent environment, could contribute their wisdom to edit and publish high-quality articles, thereby realizing the maximization of the personal benefits of editorial board members, authors and readers, and yielding the greatest social and economic benefits. Aims and scope The major task of WJG is to report rapidly the most recent results in basic and clinical research on esophageal, gastrointestinal, liver, pancreas and biliary tract diseases, Helicobacter pylori, endoscopy and gastrointestinal surgery, including: gastroesophageal reflux disease, gastrointestinal bleeding, infection and tumors; gastric and duodenal disorders; intestinal inflammation, microflora and immunity; celiac disease, dyspepsia and nutrition; viral hepatitis, portal hypertension, liver fibrosis, liver cirrhosis, liver transplantation, and metabolic liver disease; molecular and cell biology; geriatric and pediatric gastroenterology; diagnosis and screening, imaging and advanced technology. Columns The columns in the issues of WJG will include: (1) Editorial: To introduce and comment on major advances and developments in the field; (2) Frontier: To review representative achievements, comment on the state of current research, and propose directions for future research; (3) Topic Highlight: This column consists of three formats, including (A) 10 invited review articles on a hot topic, (B) a commentary on common issues of this hot topic, and (C) a commentary on the 10 individual articles; (4) Observation: To update the development of old and new questions, highlight unsolved problems, and provide strategies on how to solve the questions; (5) Guidelines for Basic Research: To provide guidelines for basic research; (6) Guidelines for Clinical Practice: To provide guidelines for clinical diagnosis and treatment; (7) Review: To review systemically progress and unresolved problems in the field, comment on the state of current research, and make suggestions for future work; (8) Original Article: To report innovative and original findings in gastroenterology; (9) Brief Article: To briefly report the novel and innovative findings in gastroenterology and hepatology; (10) Case Report: To report a rare or typical case; (11) Letters to the Editor: To discuss and make reply to the contributions published in WJG, or to introduce and comment on a controversial issue of general interest; (12) Book Reviews: To introduce and comment on quality monographs of gastroenterology and hepatology; and (13) Guidelines: To introduce consensuses and guidelines reached by international and national academic authorities worldwide on basic research and clinical practice gastroenterology and hepatology. Name of journal World Journal of Gastroenterology I December 14, 2012 Volume 18 Issue 46

189 Instructions to authors ISSN and EISSN ISSN (print) ISSN (online) Editor-in-Chief Ferruccio Bonino, MD, PhD, Professor of Gastroenterology, Director of Liver and Digestive Disease Division, Department of Internal Medicine, University of Pisa, Director of General Medicine 2 Unit University Hospital of Pisa, Via Roma 67, Pisa, Italy Myung-Hwan Kim, MD, PhD, Professor, Head, Department of Gastroenterology, Director, Center for Biliary Diseases, University of Ulsan College of Medicine, Asan Medical Center, Pungnap- 2dong, Songpa-gu, Seoul , South Korea Kjell Öberg, MD, PhD, Professor, Department of Endocrine Oncology, Uppsala University Hospital, SE Uppsala, Sweden Matt D Rutter, MBBS, MD, FRCP, Consultant Gastroenterologist, Senior Lecturer, Director, Tees Bowel Cancer Screening Centre, University Hospital of North Tees, Durham University, Stocktonon-Tees, Cleveland TS19 8PE, United Kingdom Andrzej S Tarnawski, MD, PhD, DSc (Med), Professor of Medicine, Chief Gastroenterology, VA Long Beach Health Care System, University of California, Irvine, CA, 5901 E. Seventh Str., Long Beach, CA 90822, United States Editorial office World Journal of Gastroenterology Editorial Department: Room 903, Building D, Ocean International Center, No. 62 Dongsihuan Zhonglu, Chaoyang District, Beijing , China [email protected] Telephone: Fax: Indexed and abstracted in Current Contents /Clinical Medicine, Science Citation Index Expanded (also known as SciSearch ), Journal Citation Reports, Index Medicus, MEDLINE, PubMed, PubMed Central, Digital Object Identifier, and Directory of Open Access Journals. ISI, Thomson Reuters, 2011 Impact Factor: (32/74 Gastroenterology and Hepatology). Published by Baishideng Publishing Group Co., Limited SPECIAL STATEMENT All articles published in this journal represent the viewpoints of the authors except where indicated otherwise. Biostatistical editing Statistical review is performed after peer review. We invite an expert in Biomedical Statistics to evaluate the statistical method used in the paper, including t-test (group or paired comparisons), chisquared test, Ridit, probit, logit, regression (linear, curvilinear, or stepwise), correlation, analysis of variance, analysis of covariance, etc. The reviewing points include: (1) Statistical methods should be described when they are used to verify the results; (2) Whether the statistical techniques are suitable or correct; (3) Only homogeneous data can be averaged. Standard deviations are preferred to standard errors. Give the number of observations and subjects (n). Losses in observations, such as drop-outs from the study should be reported; (4) Values such as ED50, LD50, IC50 should have their 95% confidence limits calculated and compared by weighted probit analysis (Bliss and Finney); and (5) The word significantly should be replaced by its synonyms (if it indicates extent) or the P value (if it indicates statistical significance). 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If human participants were involved, manuscripts must be accompanied by a statement that the experiments were undertaken with the understanding and appropriate informed consent of each. Any personal item or information will not be published without explicit consents from the involved patients. If experimental animals were used, the materials and methods (experimental procedures) section must clearly indicate that appropriate measures were taken to minimize pain or discomfort, and details of animal care should be provided. II December 14, 2012 Volume 18 Issue 46

190 Instructions to authors SUBMISSION OF MANUSCRIPTS Manuscripts should be typed in 1.5 line spacing and 12 pt. Book Antiqua with ample margins. 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 are responsible for the opinions expressed by contributors. Manuscripts formally accepted for publication become the permanent property of Baishideng Publishing Group Co., Limited, 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 copy-edit and put onto our website accepted manuscripts. Authors should follow the relevant guidelines for the care and use of laboratory animals of their institution or national animal welfare committee. For the sake of transparency in regard to the performance and reporting of clinical trials, we endorse the policy of the ICMJE to refuse to publish papers on clinical trial results if the trial was not recorded in a publiclyaccessible registry at its outset. The only register now available, to our knowledge, is sponsored by the United States National Library of Medicine and we encourage all potential contributors to register with it. However, in the case that other registers become available you will be duly notified. A letter of recommendation from each author s organization should be provided with the contributed article to ensure the privacy and secrecy of research is protected. Authors should retain one copy of the text, tables, photographs and illustrations because rejected manuscripts will not be returned to the author(s) and the editors will not be responsible for loss or damage to photographs and illustrations sustained during mailing. Online submissions Manuscripts should be submitted through the Online Submission System at: Authors are highly recommended to consult the ONLINE INSTRUC- TIONS TO AUTHORS ( g_info_ htm) before attempting to submit online. For assistance, authors encountering problems with the Online Submission System may send an describing the problem to [email protected], or by telephone: If you submit your manuscript online, do not make a postal contribution. Repeated online submission for the same manuscript is strictly prohibited. MANUSCRIPT PREPARATION All contributions should be written in English. All articles must be submitted using word-processing software. All submissions must be typed in 1.5 line spacing and 12 pt. Book Antiqua with ample margins. Style should conform to our house format. Required information for each of the manuscript sections is as follows: Title page Title: Title should be less than 12 words. Running title: A short running title of less than 6 words should be provided. Authorship: Authorship credit should be in accordance with the standard proposed by ICMJE, based on (1) substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; (2) drafting the article or revising it critically for important intellectual content; and (3) final approval of the version to be published. Authors should meet conditions 1, 2, and 3. Institution: Author names should be given first, then the complete name of institution, city, province and postcode. For example, Xu-Chen Zhang, Li-Xin Mei, Department of Pathology, Chengde Medical College, Chengde , Hebei Province, China. One author may be represented from two institutions, for example, George Sgourakis, Department of General, Visceral, and Transplantation Surgery, Essen 45122, Germany; George Sgourakis, 2nd Surgical Department, Korgialenio-Benakio Red Cross Hospital, Athens 15451, Greece. Author contributions: The format of this section should be: Author contributions: Wang CL and Liang L contributed equally to this work; Wang CL, Liang L, Fu JF, Zou CC, Hong F and Wu XM designed the research; Wang CL, Zou CC, Hong F and Wu XM performed the research; Xue JZ and Lu JR contributed new reagents/analytic tools; Wang CL, Liang L and Fu JF analyzed the data; and Wang CL, Liang L and Fu JF wrote the paper. Supportive foundations: The complete name and number of supportive foundations should be provided, e.g. Supported by National Natural Science Foundation of China, No Correspondence to: Only one corresponding address should be provided. Author names should be given first, then author title, affiliation, the complete name of institution, city, postcode, province, country, and . All the letters in the should be in lower case. A space interval should be inserted between country name and address. For example, Montgomery Bissell, MD, Professor of Medicine, Chief, Liver Center, Gastroenterology Division, University of California, Box 0538, San Francisco, CA 94143, United States. [email protected] Telephone and fax: Telephone and fax should consist of +, country number, district number and telephone or fax number, e.g. Telephone: Fax: Peer reviewers: All articles received are subject to peer review. Normally, three experts are invited for each article. Decision on acceptance is made only when at least two experts recommend publication of an article. All peer-reviewers are acknowledged on Express Submission and Peer-review System website. Abstract There are unstructured abstracts (no less than 200 words) and structured abstracts. The specific requirements for structured abstracts are as follows: An informative, structured abstract should accompany each manuscript. Abstracts of original contributions should be structured into the following sections: AIM (no more than 20 words; Only the purpose of the study should be included. Please write the Aim in the form of To investigate/study/ ), METH- ODS (no less than 140 words for Original Articles; and no less than 80 words for Brief Articles), RESULTS (no less than 150 words for Original Articles and no less than 120 words for Brief Articles; You should present P values where appropriate and must provide relevant data to illustrate how they were obtained, e.g ± 3.86 vs 3.61 ± 1.67, P < 0.001), and CONCLUSION (no more than 26 words). Key words Please list 5-10 key words, selected mainly from Index Medicus, which reflect the content of the study. III December 14, 2012 Volume 18 Issue 46

191 Instructions to authors Text For articles of these sections, original articles and brief articles, the main text should be structured into the following sections: INTRODUCTION, MATERIALS AND METHODS, RESULTS and DISCUSSION, and should include appropriate Figures and Tables. Data should be presented in the main text or in Figures and Tables, but not in both. The main text format of these sections, editorial, topic highlight, case report, letters to the editors, can be found at: com/ /g_info_ htm. Illustrations Figures should be numbered as 1, 2, 3, etc., and mentioned clearly in the main text. Provide a brief title for each figure on a separate page. Detailed legends should not be provided under the figures. This part should be added into the text where the figures are applicable. Figures should be either Photoshop or Illustrator files (in tiff, eps, jpeg formats) at high-resolution. Examples can be found at: pdf; wjgnet.com/ /13/4498.pdf. 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Use one horizontal line under the title, a second under column heads, and a third below the Table, above any footnotes. Vertical and italic lines should be omitted. Notes in tables and illustrations Data that are not statistically significant should not be noted. a P < 0.05, b P < 0.01 should be noted (P > 0.05 should not be noted). If there are other series of P values, c P < 0.05 and d P < 0.01 are used. A third series of P values can be expressed as e P < 0.05 and f P < Other notes in tables or under illustrations should be expressed as 1 F, 2 F, 3 F; or sometimes as other symbols with a superscript (Arabic numerals) in the upper left corner. In a multi-curve illustration, each curve should be labeled with,,,,,, etc., in a certain sequence. Acknowledgments Brief acknowledgments of persons who have made genuine contributions to the manuscript and who endorse the data and conclusions should be included. Authors are responsible for obtaining written permission to use any copyrighted text and/or illustrations. REFERENCES Coding system The author should number the references in Arabic numerals according to the citation order in the text. Put reference numbers in square brackets in superscript at the end of citation content or after the cited author s name. For citation content which is part of the narration, the coding number and square brackets should be typeset normally. For example, Crohn s disease (CD) is associated with increased intestinal permeability [1,2]. If references are cited directly in the text, they should be put together within the text, for example, From references [19,22-24], we know that.... When the authors write the references, please ensure that the order in text is the same as in the references section, and also ensure the spelling accuracy of the first author s name. Do not list the same citation twice. 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The surname of all authors should be typed with the initial letter capitalized, followed by their abbreviated middle and first initials. (For example, Lian-Sheng Ma is abbreviated as Ma LS, Bo-Rong Pan as Pan BR) Book title. Publication number. Publication place: Publication press, Year: start page and end page. Format Journals English journal article (list all authors and include the PMID where applicable) 1 Jung EM, Clevert DA, Schreyer AG, Schmitt S, Rennert J, Kubale R, Feuerbach S, Jung F. Evaluation of quantitative contrast harmonic imaging to assess malignancy of liver tumors: A prospective controlled two-center study. World J Gastroenterol 2007; 13: [PMID: DOI: /wjg ] Chinese journal article (list all authors and include the PMID where applicable) 2 Lin GZ, Wang XZ, Wang P, Lin J, Yang FD. Immunologic effect of Jianpi Yishen decoction in treatment of Pixudiarrhoea. Shijie Huaren Xiaohua Zazhi 1999; 7: In press 3 Tian D, Araki H, Stahl E, Bergelson J, Kreitman M. Signature of balancing selection in Arabidopsis. Proc Natl Acad Sci USA 2006; In press Organization as author 4 Diabetes Prevention Program Research Group. Hypertension, insulin, and proinsulin in participants with impaired glucose tolerance. Hypertension 2002; 40: [PMID: PMCID: DOI: /01.HYP ] IV December 14, 2012 Volume 18 Issue 46

192 Instructions to authors Both personal authors and an organization as author 5 Vallancien G, Emberton M, Harving N, van Moorselaar RJ; Alf-One Study Group. Sexual dysfunction in 1, 274 European men suffering from lower urinary tract symptoms. J Urol 2003; 169: [PMID: DOI: /01.ju ] No author given 6 21st century heart solution may have a sting in the tail. BMJ 2002; 325: 184 [PMID: DOI: /bmj ] Volume with supplement 7 Geraud G, Spierings EL, Keywood C. Tolerability and safety of frovatriptan with short- and long-term use for treatment of migraine and in comparison with sumatriptan. Headache 2002; 42 Suppl 2: S93-99 [PMID: DOI: /j s2.7.x] Issue with no volume 8 Banit DM, Kaufer H, Hartford JM. Intraoperative frozen section analysis in revision total joint arthroplasty. Clin Orthop Relat Res 2002; (401): [PMID: DOI: / ] No volume or issue 9 Outreach: Bringing HIV-positive individuals into care. HRSA Careaction 2002; 1-6 [PMID: ] Books Personal author(s) 10 Sherlock S, Dooley J. Diseases of the liver and billiary system. 9th ed. Oxford: Blackwell Sci Pub, 1993: Chapter in a book (list all authors) 11 Lam SK. Academic investigator s perspectives of medical treatment for peptic ulcer. In: Swabb EA, Azabo S. Ulcer disease: investigation and basis for therapy. New York: Marcel Dekker, 1991: Author(s) and editor(s) 12 Breedlove GK, Schorfheide AM. Adolescent pregnancy. 2nd ed. Wieczorek RR, editor. White Plains (NY): March of Dimes Education Services, 2001: Conference proceedings 13 Harnden P, Joffe JK, Jones WG, editors. Germ cell tumours V. Proceedings of the 5th Germ cell tumours Conference; 2001 Sep 13-15; Leeds, UK. New York: Springer, 2002: Conference paper 14 Christensen S, Oppacher F. An analysis of Koza s computational effort statistic for genetic programming. In: Foster JA, Lutton E, Miller J, Ryan C, Tettamanzi AG, editors. Genetic programming. EuroGP 2002: Proceedings of the 5th European Conference on Genetic Programming; 2002 Apr 3-5; Kinsdale, Ireland. Berlin: Springer, 2002: Electronic journal (list all authors) 15 Morse SS. Factors in the emergence of infectious diseases. Emerg Infect Dis serial online, , cited ; 1(1): 24 screens. Available from: URL: Patent (list all authors) 16 Pagedas AC, inventor; Ancel Surgical R&D Inc., assignee. Flexible endoscopic grasping and cutting device and positioning tool assembly. United States patent US Aug 1 Statistical data Write as mean ± SD or mean ± SE. Statistical expression Express t test as t (in italics), F test as F (in italics), chi square test as χ 2 (in Greek), related coefficient as r (in italics), degree of freedom as υ (in Greek), sample number as n (in italics), and probability as P (in italics). Units Use SI units. For example: body mass, m (B) = 78 kg; blood pressure, p (B) = 16.2/12.3 kpa; incubation time, t (incubation) = 96 h, blood glucose concentration, c (glucose) 6.4 ± 2.1 mmol/l; blood CEA mass concentration, p (CEA) = mg/l; CO 2 volume fraction, 50 ml/l CO 2, not 5% CO 2 ; likewise for 40 g/l formaldehyde, not 10% formalin; and mass fraction, 8 ng/g, etc. Arabic numerals such as 23, 243, 641 should be read The format for how to accurately write common units and quantums can be found at: g_info_ htm. Abbreviations Standard abbreviations should be defined in the abstract and on first mention in the text. In general, terms should not be abbreviated unless they are used repeatedly and the abbreviation is helpful to the reader. Permissible abbreviations are listed in Units, Symbols and Abbreviations: A Guide for Biological and Medical Editors and Authors (Ed. Baron DN, 1988) published by The Royal Society of Medicine, London. Certain commonly used abbreviations, such as DNA, RNA, HIV, LD50, PCR, HBV, ECG, WBC, RBC, CT, ESR, CSF, IgG, ELISA, PBS, ATP, EDTA, mab, can be used directly without further explanation. Italics Quantities: t time or temperature, c concentration, A area, l length, m mass, V volume. Genotypes: gyra, arg 1, c myc, c fos, etc. Restriction enzymes: EcoRI, HindI, BamHI, Kbo I, Kpn I, etc. Biology: H. pylori, E coli, etc. Examples for paper writing Editorial: htm Frontier: htm Topic highlight: htm Observation: htm Guidelines for basic research: 27/g_info_ htm Guidelines for clinical practice: Review: htm Original articles: htm Brief articles: htm V December 14, 2012 Volume 18 Issue 46

193 Instructions to authors Case report: htm Letters to the editor: htm Book reviews: htm Guidelines: htm RESUBMISSION OF THE REVISED MANUSCRIPTS Authors must revise their manuscript carefully according to the revision policies of Baishideng Publishing Group Co., Limited. The revised version, along with the signed copyright transfer agreement, responses to the reviewers, and English language Grade B certificate (for non-native speakers of English), should be submitted to the online system via the link contained in the sent by the editor. If you have any questions about the revision, please send to [email protected]. Language evaluation The language of a manuscript will be graded before it is sent for revision. (1) Grade A: priority publishing; (2) Grade B: minor language polishing; (3) Grade C: a great deal of language polishing needed; and (4) Grade D: rejected. Revised articles should reach Grade A or B. Copyright assignment form Please download a Copyright assignment form from Responses to reviewers Please revise your article according to the comments/suggestions provided by the reviewers. The format for responses to the reviewers comments can be found at: com/ /g_info_ htm. Proof of financial support For papers supported by a foundation, authors should provide a copy of the approval document and serial number of the foundation. Links to documents related to the manuscript WJG will be initiating a platform to promote dynamic interactions between the editors, peer reviewers, readers and authors. After a manuscript is published online, links to the PDF version of the submitted manuscript, the peer-reviewers report and the revised manuscript will be put on-line. Readers can make comments on the peer reviewer s report, authors responses to peer reviewers, and the revised manuscript. We hope that authors will benefit from this feedback and be able to revise the manuscript accordingly in a timely manner. Science news releases Authors of accepted original articles are suggested to write science news to promote their articles. The news will be released rapidly at EurekAlert/AAAS ( The title for science news should be less than 90 characters; the summary should be less than 75 words; and main body less than 500 words. Science news should be lawful, ethical, and strictly based on your original content with an attractive title and interesting pictures. Publication fee WJG is an international, peer-reviewed, Open-Access, online journal. Articles published by this journal are distributed under the terms of the Creative Commons Attribution Non-commercial License, which permits use, distribution, and reproduction in any medium, provided the original work is properly cited, the use is non commercial and is otherwise in compliance with the license. Authors of accepted articles must pay a publication fee. The related standards are as follows. Publication fee: 1365 USD per article. Editorial, topic highlights, book reviews and letters to the editor are published free of charge. VI December 14, 2012 Volume 18 Issue 46

194 World Journal of Gastroenterology Volume 18 Number 46 December 14, 2012 Published by Baishideng Publishing Group Co., Limited Room 1701, 17/F, Henan Building, No. 90 Jaffe Road, Wanchai, Hong Kong, China Fax: Telephone: I S S N ISSN CN /R Local Post Offices Code No