Acute Exacerbation of Chronic Hepatitis B: Volume 1. Definition, Research Technology, Virology, Genetics and Immunology [1st ed.] 978-94-024-1604-6;978-94-024-1606-0

Table of contents :
Front Matter ....Pages i-xxiii
Introduction to Acute Exacerbation of Chronic Hepatitis B (AECHB) (Qin Ning, Di Wu, Wei Guo, Wei-Na Li, Xiao-Jing Wang, Ke Ma)....Pages 1-47
Research Methods and Techniques for Acute Exacerbation of Chronic Hepatitis B (Zhi Chen, Dong Xi, Tao Chen, Dao-Feng Yang, Yi-Min Mao)....Pages 49-118
Virological Factors Involved in AECHB (Hong Tang, Mei-Fang Han, Ji-Ming Zhang)....Pages 119-157
Host Genetic Characters of Acute Exacerbation of Chronic Hepatitis B (AECHB) (Yu-Ming Wang, Jun-Qi Niu, Guo-Hong Deng, Ying-Ren Zhao)....Pages 159-221
Immunological Features of AECHB (Ping Lei, Guan-Xin Shen, Fu-Sheng Wang, Qin Ning, Hong Ren, Wei-Ming Yan et al.)....Pages 223-314
Other Precipitating Factors for AECHB (Bao-Hong Wang, Jing Guo, Lan-Juan Li, Tao Chen, Chun-Xia Guo, Yong-Wen He)....Pages 315-369
Correction to: Acute Exacerbation of Chronic Hepatitis B (Qin Ning)....Pages C1-C1

Citation preview

Acute Exacerbation of Chronic Hepatitis B Volume 1. Definition, Research Technology, Virology, Genetics and Immunology Qin Ning Editor

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Acute Exacerbation of Chronic Hepatitis B

Qin Ning Editor

Acute Exacerbation of Chronic Hepatitis B Volume 1. Definition, Research Technology, Virology, Genetics and Immunology

Editor Qin Ning Department of Infectious Disease Tongji Hospital Wuhan, China

ISBN 978-94-024-1604-6    ISBN 978-94-024-1606-0 (eBook) https://doi.org/10.1007/978-94-024-1606-0 Library of Congress Control Number: 2019930050 © Springer Nature B.V. and Huazhong University of Science and Technology Press 2019 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature B.V. The registered company address is: Van Godewijckstraat 30, 3311 GX Dordrecht, The Netherlands

My dedications go to the intelligent, committed ones Wei Che and Wen Yu who served in the editorial process. A special thanks to Dr. Dong Xu, who helped to collect manuscripts in a timely fashion from all parts of the world. My dedications to all my teachers and my mentors Dr. Yongsui Dong and Dr. Gary Levy. I would like to dedicate this book to my family for their love, patience, and support. To my parents Dihua and Jinping who have stood by me through thick and thin. To my children Jianing (Jenny) and Fengning (Fred), adorable individuals who know that knowledge is no substitute for wisdom. To my husband Xiaoping for that I know you are always with me near and far, and for your constant support of my professional endeavors. To my sisters Qiao, Yuan, and Huan for your understandings and encouragements. To all my students and my secretary Ms. Jinshang Hu, you are part of my life and family.

Foreword

Acute-on-chronic liver failure (ACLF) secondary to hepatitis B virus infection is now recognized as an important worldwide life-threatening disease with a high mortality. The work described in this book by experts in the field provides important information to the reader on its pathogenesis, clinical manifestations and current and future management strategies. The work provides important new advances in the science of HBV replication and the host response. With major advances in our understanding of the virology and immunology of HBV infection, this book gives reason for cautious optimism that we will soon be able to provide exciting new therapies for this disorder. To date, with the exception of liver replacement therapy (transplantation), there are few therapeutic options for patients who develop ACLF secondary to HBV. However, advances in diagnosis as well as management strategies including introduction of antiviral agents and inhibitors of pro-inflammatory cytokines offer the hope of better short- and long-term outcomes. The advances in the basic science of ACLF and the development of small animal models outlined in this book give hope that new therapeutic approaches will lead to the control or eradication of HBV and amelioration of inflammatory disease lessening the need for liver transplantation. The work described in this book strongly supports that clinical research in ACLF should build on the findings of basic science research and be directed to carefully controlled studies with well-characterized cohorts of patients so that we can evaluate the potential of new therapeutic approaches. The use of exciting new approaches detailed here will not only provide important new therapeutics but also insights into the mechanism of disease. The findings described in this book strongly support that we are approaching an exciting new era for therapy for patients with ACLF. Toronto, ON

Gary Levy

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Preface

It is now recognized that as a consequence of chronic HBV infection, many patients with or without established cirrhosis will develop acute decompensation and multi-­ organ failure, a syndrome known as acute-on-chronic liver failure (ACLF). Once patients develop ACLF, they are at high risk of death. A number of triggers including reactivation of HBV, coinfection of hepatitis A or E virus, onset of bacterial infection, gastrointestinal bleeding and development of renal dysfunction can precipitate the development of ACLF in patients who have been previously stable. ACLF is prevalent in Asia where many patients have incubative chronic hepatitis B virus (HBV) infection. For the past decade, with an increasing understanding of the disease mechanisms and improved general internal medications, the overall mortality has significantly decreased due to HBV infection-related ACLF (HBV-ACLF) in Chinese patients. Here we have assembled a group of hepatologists and scientists from academic hospitals and universities to explore the current understanding of the clinical, genetic, virologic and immunologic factors that contribute to ACLF. In this book of 12 chapters, we have explored the current state of knowledge of HBV infection with a specific focus on the natural history and the clinical course to define important host and viral factors to the development of ACLF, sharing our profound experience and clinical procedures in early diagnosis and treatment of HBV-ACLF patients and its complications. All together about 2649 references have been cited, of which 754 were since 2012. At the beginning of the book, there is a complete table of contents, which together with the general index makes it possible for the reader to find specific topics easily. In each chapter, there is an abstract for the reader to gain a quick information of the chapter. We have also used 55 coloured figures to make the illustrations even more visual. We enlisted the helpful advice of friends, colleagues and senior experts to supplement or confirm our own interpretations. The contacts arising from these discussions have been immensely benignant to me. Here my special thanks to Prof. Gary Levy, Prof. Didier Samuel, Prof. Gyongyi Szabo, Prof. Lanjuan Li, Prof. Zhimeng Lu, Prof. Shiv Kumar Sarin, Prof. Stephen Locarnini, Prof. Xinhua Weng, Prof. Yuquan Wei and Prof. Hui Zhuang.

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Finally I should express my gratitude to the employees at HUST Press and Springer Publishing House (Mr. James Hu) for their professional help in completing this book, especially to Ms. Lian-Di Wang, senior editor, and Mr. Wei Che, projector manager, who gave their kind support at all times. Wuhan, China

Qin Ning

Acknowledgements

We would like to thank the consultants, Editorial Committee and other staff members who have contributed to the compilation of the book “Acute Exacerbation of Chronic Hepatitis B (Chinese Version)”. Consultant (In Alphabetic Order) Yu-Mei Wen Fudan University Shanghai Medical College Ling-Xia Zhang 302 Military Hospital of China Editorial Board Member (In Alphabetic Order) Cheng-Wei Chen 85TH Hospital of People’s Liberation Army Hong-Song Chen Peking University People’s Hospital Xin-Wen Chen Wuhan Institute of Virology, Chinese Academy of Sciences Jun Cheng Beijing Ditan Hospital Capital Medical University Zhong-Ping Duan Beijing Youan Hospital Capital Medical University Xue-Gong Fan Xiangya Hospital Central South University Jin-Lin Hou Nanfang Hospital Ji-Dong Jia Beijing Friendship Hospital Capital Medical University Xiao-Hui Miao Shanghai Changzheng Hospital Ji-Fang Sheng The First Affiliated Hospital Zhejiang University Guang-Feng Shi Huashan Hospital De-Ming Tan XiangYa Hospital Central South University De-Ying Tian Tongji Medical College Huazhong University of Science & Technology Mo-Bin Wan Changhai Hospital Gui-Qiang Wang Peking University First Hospital Lai Wei Peking University People’s Hospital Qing Xie Shanghai Jiao Tong University School of Medicine Sheng-Long Ye Zhongshan Hospital Xin-Xin Zhang Shanghai Jiao Tong University School of Medicine

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Acknowledgements

xii Other Staff Member (In Alphabetic Order) Min Chen Ming-Quan Chen Hong Du Ning-Ling Ge Xiao-Meng Hu Man Xie Ling-Bo Liang Feng Liu Chun-Chen Wu Hang-Di Xu Qiao Yang Yi-Jun Zeng Heng-Hui Zhang Li Zhou Rong-Rong Zhou Peng Zhu

Yu-Ping Ding Xin-Wu Guo Jing-Lan Jin Yu Shi Xiang-Sheng Xu Lin Zhang Guang-De Zhou Yue-Ke Zhu

Jing Dong Xiao-Feng Hang Chen Li Zhan-Hui Wang Xu-Wen Xu Wei Zhang Yan Zhuang

Contents

1 Introduction to Acute Exacerbation of Chronic Hepatitis B (AECHB)����������������������������������������������������������������������������������   1 Qin Ning, Di Wu, Wei Guo, Wei-Na Li, Xiao-Jing Wang, and Ke Ma 2 Research Methods and Techniques for Acute Exacerbation of Chronic Hepatitis B ������������������������������������������������������������������������������������  49 Zhi Chen, Dong Xu, Tao Chen, Dao-Feng Yang, and Yi-Min Mao 3 Virological Factors Involved in AECHB���������������������������������������������������� 119 Hong Tang, Mei-Fang Han, and Yi-Ming Zhang 4 Host Genetic Characters of Acute Exacerbation of Chronic Hepatitis B (AECHB)������������������������������������������������������������������� 159 Yu-Ming Wang, Jun-Qi Niu, Guo-Hong Deng, and Ying-Ren Zhao 5 Immunological Features of AECHB���������������������������������������������������������� 223 Ping Lei, Guan-Xin Shen, Fu-Sheng Wang, Qin Ning, Hong Ren, Wei-Ming Yan, and Di Wu 6 Other Precipitating Factors for AECHB �������������������������������������������������� 315 Bao-Hong Wang, Jing Guo, Lan-Juan Li, Tao Chen, Chun-­Xia Guo, and Yong-Wen He

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Contributors

Chief Editor Qin  Ning  Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China

Associate Editor Zhi Chen  The First Affiliated Hospital, Zhejiang University, Zhejiang, China Yu-Ming Wang  Southwest Hospital, Army Medical University, Chongqing, China Guan-Xin  Shen  Tongji Medical College, Huazhong University of Science and Technology, Hubei, China

Advisory Board (In Alphabetic Order) Didier Samuel  Department of Hepatology and Gastroenterology, Université ParisSud, Villejuif, France Gary  Levy  Multi Organ Transplant Program, Transplant Institute, University of Toronto, Toronto, ON, Canada Gyongyi  Szabo  University of Massachusetts Medical School, Worcester, MA, USA Hui Zhuang  Peking University Health Science Center, Beijing, China Lan-Juan Li  The First Affiliated Hospital, Zhejiang University, Zhejiang, China Shiv  Kumar  Sarin  Department of Hepatology, Institute of Liver and Biliary Sciences (ILBS), New Delhi, India Stephen  Locarnini  Victorian Infectious Diseases Melbourne Health, Melbourne, VIC, Australia

Reference

Laboratory,

Xin-Hua Weng  Huashan Hospital, Fudan University, Shanghai, China xv

xvi

Contributors

Yu-Quan  Wei  National Key Laboratory of Biotherapy, Sichuan University, Sichuan, China Zhi-Meng Lu  Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

Editorial Board Members (In Alphabetic Order) Chuan-Long  Zhu  Jiangsu Province Hospital, The First Affiliated Hospital of Nanjing Medical University, Jiangsu, China Da-Zhi Zhang  The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China Dao-Feng Yang  Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China Dong  Xu  Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China Gary  Levy  Multi Organ Transplant Program, University of Toronto Transplant Institute, Toronto, ON, Canada Guan-Xin  Shen  Tongji Medical College, Huazhong University of Science and Technology, Hubei, China Guo-Hong  Deng  Southwest Hospital, Army Medical University, Chongqing, China Hai-Bing Su  The Fifth Medical Center of PLA General Hospital, Beijing, China Hong Ren  Chongqing Medical University, Sichuan, China Hong Tang  West China Hospital, Sichuan University, Sichuan, China Yi-Ming Zhang  Huashan Hospital, Fudan University, Shanghai, China Jia Shang  Henan Provincial People’s Hospital, Henan, China Jia-Quan Huang  Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China Jian-Xin Song  Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China Jin-Hua Hu  The Fifth Medical Center of PLA General Hospital, Beijing, China Jin-Ming  Zhao  The Fifth Medical Center of PLA General Hospital, Beijing, China Jun-Qi Niu  The First Hospital of Jilin University, Jilin, China

Contributors

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Jun-Ying Qi  Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China Ke Li  The Fifth Medical Center of PLA General Hospital, Beijing, China Lan-Juan Li  The First Affiliated Hospital, Zhejiang University, Zhejiang, China Mei-Fang Han  Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China Qin  Ning  Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China Tao  Chen  Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China Xi-Ping Zhao  Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China Xiao-Guang  Dou  Shengjing Hospital of China Medical University, Liaoning, China Xiao-Jing Wang  Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China Xiong  Ma  Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Xue-Fan Bai  Tangdu Hospital, Air Force Medical University, Shanxi, China Yi-Min Mao  Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Ying-Ren Zhao  The First Affiliated Hospital of Xi’an Jiaotong University, Shanxi, China Yong-Wen He  Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Hubei, China Yu-Ming Wang  Southwest Hospital, Army Medical University, Chongqing, China Yuan-Cheng  Huang  Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China Zhi Chen  The First Affiliated Hospital, Zhejiang University, Zhejiang, China Zhi-Liang Gao  The Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China Zhi-Shui Chen  Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China

Editors

Chief Editor Associate Editors

Qin Ning Zhi Chen

Yu-Ming Wang

Qin Ning

Zhi Chen

Yu-Ming Wang

Guan-Xin Shen

Guan-Xin Shen

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Editors

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Advisory Board (In Alphabetic Order) Didier Samuel Department of Hepatology and Gastroenterology, Université Paris-Sud, Villejuif, France Gary Levy Multi Organ Transplant Program, Transplant Institute, University of Toronto, Toronto, ON, Canada Gyongyi Szabo University of Massachusetts Medical School, Worcester, MA, USA Hui Zhuang Peking University Health Science Center, Beijing, China Lan-Juan Li The First Affiliated Hospital, Zhejiang University, Zhejiang, China Shiv Kumar Department of Hepatology. Institute of Liver and Biliary Sciences (ILBS), Sarin New Delhi, India Stephen Victorian Infectious Diseases Reference Laboratory, Melbourne Health, Locarnini Melbourne, VIC, Australia Xin-Hua Weng Huashan Hospital, Fudan University, Shanghai, China Yu-Quan Wei National Key Laboratory of Biotherapy, Sichuan University, Sichuan, China Zhi-Meng Lu Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

Didier Samuel

Gary Levy

Gyongyi Szabo

Hui Zhuang

Editors

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Lan-Juan Li

Shiv Kumar Sarin

Stephen Locarnini

Xin-Hua Weng

Yu-Quan Wei

Introduction

This book assembles recent achievements in both basic research and clinical management in the field of hepatology, virology, and immunology. It provides up-to-­ date information for clinicians who can apply the relevant knowledge to their daily clinical practice and for researchers who are interested in clinically orientated studies. The updated and detailed technology and state-of-the-art treatment strategies provided in this book serve as references for clinicians and resident physicians in the daily management of ACLF. The rationality and strategies for basic research as well as patient management in this book can also be a valuable reference for other fatal and end-stage liver diseases than HBV-induced ACLF. This Volume 1 has six chapters and focuses on the definition, research technology, virology, genetics, and immunology.

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Introduction to Acute Exacerbation of Chronic Hepatitis B (AECHB) Qin Ning, Di Wu, Wei Guo, Wei-Na Li, Xiao-Jing Wang, and Ke Ma

Abstract

This chapter describes definition, natural history and recent achievement debrief: 1. Although the definition and classification of liver failure have differed, a consensus has been reached regarding the definition, classification and clinical diagnosis of liver failure. 2. Acute exacerbation of chronic hepatitis B (with the most severe form, HBV-­ ACLF) refers to submassive to massive necrosis in the livers of HBV-infected patients with mild or moderate inflammation, taking place over a short period of time and leading to progressive damage of liver function, metabolic disorders, and secondary multiple organ failure without an appropriate management. Clinical manifestations include progressive disturbances in blood coagulation, jaundice, hepatic encephalopathy, and ascites. 3. The natural history of severe hepatitis B is mainly influenced by host factors, including gender, age, precipitating factors, and underlying diseases, and by virological factors, including virus genotype, viral mutations, and viral replication. Severe hepatitis B can be divided into early, middle and late stages according to major clinical indicators, e.g. prothrombin activity. Antiviral treatment and artificial liver support is beneficial to clinical outcomes and prognosis. 4. Recent research on the pathologic mechanism of severe hepatitis B has focused primarily on virology, host immunology, and genetics. No sensitive, reliable early warning parameters have been found to predict the development of severe hepatitis B.  Early antiviral treatment has become an important means to prevent severe hepatitis B. Immune regulation and repair of liver cell damage are expected to become effective intervention measures. Q. Ning (*) · D. Wu · W. Guo · W.-N. Li · X.-J. Wang · K. Ma Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China © Springer Nature B.V. and Huazhong University of Science and Technology Press 2019 Q. Ning (ed.), Acute Exacerbation of Chronic Hepatitis B, https://doi.org/10.1007/978-94-024-1606-0_1

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 efinition and Nomenclature of Acute Exacerbation D of Chronic Hepatitis B

Di Wu, Wei Guo and Qin Ning

1.1.1 Concepts of Acute Exacerbation of Chronic Hepatitis B Chronic hepatitis B virus (HBV) infection and its sequelae continue to be a major global health problem. World Health Organization estimates that approximately 2 billion people worldwide have been infected with HBV, representing nearly a third of the entire world population. Up to240 million are chronically infected, among which nearly 650,000 patients die of liver failure, cirrhosis and hepatocellular carcinoma (HCC) every year [1, 2]. The interaction between HBV replication and the host immune response plays an important role in determining the outcome of HBV infection. After exposure to HBV, initiation of a broad and vigorous immune response to HBV is responsible for an acute self-limited infection, leading to acute hepatitis, while an aberrant immune response may result in fulminant hepatitis [3]. Patients who fail to amount efficient immune responses against HBV develop chronic hepatitis B [4, 5]. HBV persists in the nucleus of infected hepatocytes as a stable non-integrated covalently closed circular DNA (cccDNA) even after patients’ serological recovery from acute hepatitis B [6]. Breakdown of immune balance with too vigorous immune pressure can induce reactivation or acute exacerbation of chronic hepatitis B (AECHB) in patients who have been infected with HBV [7]. The incidence of AECHB is found to be directly proportional to the prevalence of chronic HBV infection in a region, making this phenomenon common in countries or areas with high or intermediate endemicity. AECHB (with the most severe form, HBV-related acute-on-chronic liver failure (ACLF))is a unique presentation with a rapid deterioration of liver function in patients with HBV-related chronic liver disease, characterized by high ALT levels, jaundice, coagulopathy, hepatic encephalopathy (HE) and ascites, which may lead to hepatic decompensation and subsequent hepatic and/or or extrahepatic organ failure. Submassive and massive necrosis of hepatocytes is a typical presentation of AECHB.  According to animal studies and clinical observations, an upsurge of serum HBV DNA always precedes or coincides with the abrupt elevation of alanine aminotransferase (ALT) levels and occurrence of AECHB [8–10]. Although, there is a lack of consensus definition of AECHB, and different parameters and cut-off values have been used in different studies, this clinical entity is characterized by sudden elevation of ALT level and the abrupt increase or re-emergence of serum HBV DNA, caused by HBV flare or reactivation in a patient with chronic inactive HBV and resolved HBV infection, always due to an imbalance between virus replication and host immune responses. According to the recent Chinese guideline of prevention and treatment for chronic hepatitis acute exacerbation or flare of hepatitis B refers to elevation of serum ALT level to more than 10-times the upper limit of normal (ULN) after excluding other factors resulting in liver injury [11]. The

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updated guidelines released by the Asian Pacific Association for the Study of the Liver (APASL) defines acute exacerbation or flare of hepatitis in chronic HBV-­ infected patient as intermittent elevations of serum aminotransferase level to more than five times the ULN and more than twice the baseline value [12]. A proportion of patients with chronic hepatitis B presents very high ALT level accompanied by jaundice and hepatic decompensation, namely severe AECHB, which may progress to HBV-ACLF [8]. HBV-ACLF, also terms as severe hepatitis B in China, is a severe clinical entity with high short-term mortality in chronic hepatitis B patient, presenting with a rapid deterioration of liver function and evolving multi-organ failure, with the highest incidences being in the Asia-Pacific and African regions [13, 14]. Both host and virus factors contribute to the mechanisms underlying the pathogenesis of severe AECHB, including excessive immune response, HBV genotype, etc. Reactivation of hepatitis B in HBV Genotype C patients may have a higher risk of progression to liver cirrhosis [15]. HBV DNA kinetics after initiating therapy can predict the severity of AECHB [16]. During AECHB, the cut-­ off value of 1.55  ×  109 copies/mL for serum HBV DNA may identify HBeAg-­ positive chronic hepatitis B patients eligible for immediate antiviral therapy [17]. For patients with severe AECHB, prothrombin activities and serum bilirubin are important predictors of clinical outcome. Once hepatic encephalopathy (HE) develops in patients with AECHB, the mortality is very high, however, unlike patients with decompensated liver cirrhosis, some patients with severe AECHB can resume almost normal liver function. AECHB should be differentiated from acute hepatitis caused by HBV and other etiologies.

1.1.2 AECHB Causing Liver Function Derangements AECHB causing Liver function derangements may be found not only in hepatitis B flare during immune clearance phase, but also as a HBV reactivation in patients with HBsAg carriers with normal ALT levels or even in patients with previously resolved HBV infection who have lost serum HBsAg, but are positive for antibody to the hepatitis B core antigen (anti-HBc), particularly when they are receiving immunosuppressive therapy, chemotherapy or organ transplantation. Several viral factors (such as HBV genotype and drug resistant mutants) and host factors (including serological and immunological status of patients, the use of immunosuppressive therapy, the existence of underlying diseases) may be associated with the occurrence of AECHB.  For instance, HBeAg positive chronic hepatitis B patients are at higher risks of AECHB than those who are anti-HBe positive. Evidence showed that more than 90% of AECHB in HBeAg positive patients resulted from spontaneous viral activation during immune clearance phase, whereas only half of HBeAg negative patients developed AECHB due to spontaneous HBV reactivation, the remaining cases resulting from super infection by other hepatitis viruses [18]. The immunological mechanisms responsible for AECHB have not been completely elucidated. Disruption of host immune surveillance plays a more significant

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role in breakdown of immune tolerance than HBV genomic variations [7]. During the immune tolerant phase, impaired cytotoxic T lymphocyte (CTL) function and IFN-γ production are inadequate to eradicate HBV infection, but continually induce cytolysis of hepatocytes. In the immune reactive phase, spontaneous HBeAg seroconversion usually accompany with mild transient liver function disturbances [19, 20]. However, in a proportion of patients, the immune system activation may lead to severe hepatic dysfunction and sometimes liver failure. Hepatitis B flare is related to enhanced reactivity of HBV-specific CD4+ T cells. Spontaneous reactivation of HBV is associated with elevated numbers of HBV-specific CD8+ T cells, which can also cause immune-mediated liver injury. By contrast, HBV reactivation in patients undergoing chemotherapy or immunosuppressive therapy is due to markedly impaired immune response. Reduction of immunosuppression in these patients lead to the renewed HBV replication and increased HBV-specific T cells, which may result in AECHB [21]. Except liver function derangements resulting from HBV clearance and reactivation, AECHB can occur during or after antiviral treatment, which can be caused by development of nucleoside analogs (NAs)-resistant mutants, withdrawal of NAs, IFN-induced immune stimulation. Besides, liver function derangements due to other possible causes, including the emergence of HBV genotypic variations, such as core promoter mutant and HBV DNA polymerase mutant, may also cause AECHB [22, 23].

1.1.3 Differentiating AECHB from Acute Viral Hepatitis B Because some patients with chronic HBV infection are asymptomatic or have mild nonspecific symptoms, AECHB may often be present as the first clinical manifestation of HBV infection, thus, this condition may be mistaken as acute hepatitis B (AHB) [24, 25]. AECHB is difficult to differentiate from AHB without accurate history of chronic HBV infection or recent infection with HBV. It is estimated that more than half of patients presenting AECHB may be misdiagnosed as AHB in endemic areas. Misdiagnosis is essentially due to the facts that clinical, biochemical and serological characteristics of AECHB closely resemble those of AHB, including the abrupt onset of severe liver injury, development of advanced grades of HE, high ALT levels and elevated international normalized ratio (INR), and HBV DNA, HBsAg and HBcAb IgM seropositivity. A combination of high HBV DNA levels, low HBcAb IgM titers, evidence of preexisting HBV-related chronic liver disease could be helpful in differentiating severe AECHB and AHB [9, 10]. Differentiation between AECHB and AHB is very important and necessary because these two distinct clinical entities require different therapeutic strategies and have differential prognosis. Most patients with AHB may spontaneously resolve and only few patients who develop fulminant hepatitis B will need therapy. On the contrary, patients with AECHB often require treatment since the liver dysfunction may result in severe acute exacerbation and subsequent hepatic decompensation and organ failure.

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1.1.4 The Concept and Classification of Liver Failure Liver failure (LF) is considered a life-threatening condition with significant morbidity and mortality induced by various causes and is defined as severe hepatic dysfunction of synthesis, metabolism and detoxification, characterized by coagulopathy, jaundice, HE and ascites. LF can be classified into acute liver failure (ALF) [26], chronic liver failure (CLF) and acute-on-chronic liver failure (ACLF) [27]. ALF is a critical condition with rapid deterioration in liver function characterized by abrupt onset of jaundice, coagulation disturbance, and HE, in the absence of preexisting liver disease. The natural course of ALF proceeds with rapid hepatic dysfunction, resulting in multiple organ failure and eventually death. The overall mortality rate remains as high as 80%. Based on the time interval from onset of first hepatic symptoms (e.g. jaundice) to onset of HE, different subdivisions of ALF exist. One classification of ALF defines hyperacute as within 7 days, acute as 8–28 days, and subacute as 4–24 weeks [28].CLF usually occurs in the context of cirrhosis characterized by progressive, irreversible deterioration in liver function. Compensated cirrhosis is not usually symptomatic and clinically detectable. As patients develop more advanced liver fibrosis, pressure in the portal vein increases, potentially leading to the development of cirrhosis-related complications, including those associated with hepatic insufficiency (e.g. jaundice, hypoalbuminemia), and those associated with portal hypertension (e.g. peripheral edema, ascites, variceal bleeding or HE), which is called decompensated cirrhosis. Generally, survival in patients with decompensated cirrhosis is poor, only treatment being timely liver transplantation. Recently, increasing attention has been given to a third form of liver failure, known as ACLF.  The essential characteristics of ACLF include preexisting liver disease, precipitating factors, severe but possibly reversible liver dysfunction (different from CLF), multiple organ failure, and high short-term mortality. ACLF is a severe condition where an acute insult superimposed on an underlying chronic compensated (known or unknown) liver disease due to the precipitating events, manifested as jaundice, coagulopathy and HE, with development of subsequent extra-hepatic organ failure involving dysfunction of brain, kidney, respiratory, circulatory, coagulation, and usually accompanied by sepsis, resulting in high mortality. ACLF is particularly frequent in alcoholic liver disease and hepatitis B-related cirrhosis. The prognosis of ACLF is determined not by the severity of preexisting liver disease, but by the severity of end-stage organ failure. ACLF should be distinguished from both ALF and CLF, because ACLF of preexisting chronic liver disease leads to significantly higher short-term mortality than decompensated cirrhosis, ACLF is often due to precipitating events, Besides, in ACLF, the acute deterioration of hepatic function may be reversible [29, 30].

1.1.5 The Definition of ACLF While ALF and CLF are clearly understood and well defined, there are no universal definition and widely accepted diagnostic criteria for ACLF.  ACLF is a distinct

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disease entity involving two injuries, with one being preexisting injury caused by underlying chronic liver disease, and one being superimposed acute injury induced by an acute hepatic insult, subsequently resulting in rapid deterioration of liver function and hepatic failure with or without extra-hepatic organ failure. The term of ACLF was first introduced in 1995 to describe a condition in which ongoing and chronic liver insult and acute hepatic insult were operating simultaneously [31]. Several societies of hepatology have conducted extensive studies in order to standardize the definition and diagnostic criteria for ACLF. To date, more than a dozen definitions of ACLF have been developed, however, these definitions differ from each other, causing a great deal of confusion. Although the general aspects of this clinical entity have been vaguely defined, the lack of a precise definition limits research regarding ACLF and its clinical application. The reason for the absence of universally accepted and employed definition and diagnostic criterion for ACLF is that underlying liver disease, precipitating events, and clinical manifestations are quite diverse. A universal consensus of ACLF can not only help us understand mechanisms of pathogenesis as well as natural history, but more importantly, may allow earlier identification of patients at increased risk of deterioration and short-term mortality. Recently two representative consensus definitions have been commonly accepted and widely used. One was proposed by the Asia-Pacific Association for the Study of the Liver (APASL) in 2009, the other one was developed by the European Association for the Study of the Liver (EASL) -Chronic Liver Failure (EASL-CLIF) Consortium in 2013 [32, 33].

1.1.6 APASL Definition of ACLF In 2009, APASL first established a consensus diagnostic criterion for ACLF through analyzing data from 200 patients, describing ACLF as “acute hepatic insult manifesting as jaundice and coagulopathy, complicated within 4 weeks by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease”. Then APASL ACLF Research Consortium (AARC) was formed in 2012, which collated and analyzed data from a large cohort of patients in the Asia-Pacific region, subsequently released an updated and revised consensus guideline in 2014,which defines ACLF as “an acute hepatic insult manifesting as jaundice (serum bilirubin ≥5 mg/dl (85micromol/l) and coagulopathy (INR ≥1.5 or prothrombin activity1.50); (5) ascites; (6) with or without HE. According to this criterion, patients with ACLF were subdivided into three stages, namely, early-stage, intermediate-stage and late-­stage ACLF [13]. This diagnostic criterion has been widely used in China. The definitions of ACLF and HBV-ACLF are listed in Table 1.1. AECHB is a unique presentation with a rapid deterioration of liver function in patients with HBV-related chronic liver disease, characterized by high ALT levels, jaundice, coagulopathy, hepatic encephalopathy and ascites, which may lead to hepatic decompensation and subsequent hepatic and/or or extra hepatic organ failure. The most severe form of AECHB, HBV-ACLF is a life-threatening clinical condition leading to a high short-term mortality. Early and long-term NA treatment and artificial liver support may help improve the prognosis. Liver transplantation should be considered for patients who develop ACLF secondary to severe AECHB although availability of donor livers constitutes a major limitation. Therefore, there is an urgent need to develop more effective treatments and explore new therapeutic targets for HBV-ACLF. Recently, granulocyte-colony stimulating factor (G-CSF) has shown promising results as treatment for ACLF which warrants further validation. Because of the diversity of HBV-related chronic liver diseases and precipitating factors, there is no universal definition and diagnostic criteria for AECHB and HBV-­ACLF.  Current research focus is gradually shifting towards AECHB and HBV-­ACLF in the Asia-Pacific regions, aiming at pathogenesis, new and effective treatment options as well as improving the outcome. In order to establish a consensus on the definition of AECHB, develop more effective treatments, and establish a prognostic scoring system, large prospective controlled studies with comprehensive evaluation of the risk factors including virus mutations, host

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Table 1.1  Definitions and diagnostic criteria for ACLF and HBV-ACLF Working party or Consortium APASL ACLF (2009, 2014) EASL-­ AASLD ACLF (2011) EASL-­ CLIF ACLF (2013)

CMA ACLF (2006)

Definition and diagnostic criteria An acute hepatic insult manifesting as jaundice (serum bilirubin ≥5 mg/dl (85 micromol/l) and coagulopathy (INR ≥1.5 or prothrombin activity  2.5 and/or a platelet count of 20x109/L; circulatory failure was defined by the use of dopamine, dobutamine, or terlipressin; respiratory failure was defined by a PaO2/FiO2 ≤ 200 or an SpO2/FiO2 ≤ 200. (1) acute deterioration of preexisting chronic liver disease; (2) extreme fatigue with severe digestive symptoms, such as obvious anorexia, abdominal distension, nausea and vomiting; (3) progressively worsening jaundice within a short period (serum total bilirubin level ≥ 10 mg/dL or a daily elevation ≥1 mg/ dL); (4) an obvious hemorrhagic tendency with PTA ≤40% (or international normalized ratio > 1.50); (5) ascites; (6) with or without hepatic encephalopathy.

ACLF acute-on-chronic liver failure, APASL Asia–Pacific Association for the Study of the Liver, AASLD-EASL American Association for the Study of Liver Disease-European Association for the Study of the Liver, EASL-CLIF European Association for the Study of the Liver -Chronic Liver Failure, Chronic Liver Failure-Sequential Organ Failure Assessment, CMA Chinese Medical Association

immune response, susceptibility genes on the progression of AECHB, are urgently required.

1.2

 atural History of Severe Acute Exacerbation of Chronic N Hepatitis B

Wei-Na Li, Xiao-Jing Wang, Ke Ma, and Qin Ning It has been estimated that over 350 million people worldwide are chronically infected with hepatitis B virus (HBV), with over one million deaths per year. Severe hepatitis B or HBV-related liver failure is mainly reported in Asia and accounts for

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more than 80% of acute on chronic liver failure(ACLF)in China [40–42]. HBVACLF is characterized by the acute deterioration of liver function due to the effects of a precipitating event include intra-hepatic or extra-hepatic on the basis of chronic hepatitis B (CHB) [43]. This condition has various clinical manifestations, the most notable being renal dysfunction, hepatic encephalopathy and coagulopathy. Severe acute exacerbation of CHB is characterized by very high alanine aminotransferase levels, accompanied by jaundice and hepatic decompensation [44]. Exacerbation of chronic HBV infection occurs in ∼40–50% of hepatitis B e antigen (HBeAg)positive patients and in 15–30% of patients positive for antibodies against HBeAg (anti-HBe) [45]. One of the first prospective studies investigating the natural history of acute exacerbation of CHB found that 15–47% of patients with CHB developed exacerbations with 4 years [46]. Extensive understanding of the natural history of CHB exacerbation can contribute to optimized management of patients infected with HBV.

1.2.1 Primary Causes 1.2.1.1 Host Factors To date, conflicting results on the relationship between gender and severe acute exacerbation of CHB have been reported. Among all HBV-infected patients, however, the rates of liver cirrhosis (LC), hepatocellular carcinoma (HCC),cirrhosis, and death have been consistently higher in males than females, indicating that male sex was significantly associated with increased risks of HBV related end-stage liver diseases, severe acute exacerbation of CHB, and death [47]. Most studies have reported that the rate of severe acute exacerbation of CHB is two to three-fold higher in males than in females. Rates of occurrence of liver failure, cirrhosis and HCC have been found to be 1.5–7.6-fold higher in men than in women, even after adjusting for important potential confounders such as age, severity of liver disease, and other health-related factors [47, 48]. Age is regarded as significantly related to the prognosis of hepatitis B-related disease. Studies from China and other countries have shown that the likelihood of events such as HCC, cirrhosis, liver failure, and death increased with age [47]. However, the extent to which these associations were due to age at initial infection, duration of infection, and comorbidities more frequent in senior individuals remains unclear. Changes in human microecology have been reported to play significant roles in the occurrence and development of liver diseases [49]. Intestinal microecology is considered related to both liver anatomy and liver function. Studies in animal models demonstrated that enterobacteria and endotoxins increase the numbers of Kupffer cells and improve liver function [50, 51]. Normally, the liver can eliminate various toxins from intestinal tract, including endotoxins, ammonia, indole, phenols, short chain fatty acids, and false neurotransmitters, as well as bacteria and fungi originating in the gut [53]. Severe liver injury results in marked changes in intestinal microecology, secondarily damaging gut barrier function, and causing metabolites and

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gut-origin bacteria to migrate into other organs. This, in turn, may result in over activation of the immune system, resulting in abnormal immune reactions, and leading to hepatocyte apoptosis and necrosis [52]. These linked reactions significantly accelerate the process of CHB exacerbation, as well as inducing complications of cirrhosis such as upper gastrointestinal hemorrhage, hepatorenal syndrome and infection. Moreover, liver diseases may be exacerbated by endotoxemia and dysfunction of the gastrointestinal tract.

1.2.1.2 Virus Factors Besides host factors, virus factors also play pivotal roles in the mechanism of severe exacerbation of CHB. HBV replication, the primary cause of severe exacerbation of hepatitis B, has been observed during early stages of severe hepatitis [53–55]. The coinfection with other virus and any changes in the replication of HBV caused by changes in host transcriptional factors and HBV mutations may exacerbate CHB [56–58]. Many precipitating factors are involved in the severe acute exacerbation of CHB [59]. Coinfection with other viruses, including hepatitis A virus (HAV),hepatitis E virus (HEV),hepatitis C virus (HCV),hepatitis delta virus (HDV), human immunodeficiency virus (HIV), and cytomegalovirus (CMV),has been associated with poorer clinical outcomes [40, 41, 60–62]. The superinfection of HEV was prominent and steady in superinfection rates during the past decade, which was regarded as one of the important precipitating factors of AECHB [41]. Mechanisms responsible for AECHB include cellular immunodeficiency, interference with the process of replication, involvement of cytokines, and HBV mutations. HBV/HCV co-­ infection may precipitate AECHB through an overlapping effect, since both viruses can induce immune reactions. Exacerbation caused by HBV/HDV co-infection is rare. HBV/CMV coinfection has been shown to suppress host immune reactions, resulting in greater HBV replication and affecting the regulatory function of cytokine networks. This makes elimination of HBV more difficult, resulting in the severe acute exacerbation of CHB.  HIV coinfection of patients with HBV can result in the transformation of acute to chronic HBV infection. Conversely, HBV could also affect the elimination of HIV, accelerating deterioration to AIDS. Remarkably, most HBV/HIV coinfected patients do not die of liver failure but of AIDS, especially of opportunistic infections [61]. For CHB patients, an immunocompromised state makes it more difficult to eliminate bacteria from the portal vein and liver. In addition, translocation of gut bacteria through an intrahepatic portacaval shunt makes the liver more susceptible to various bacteria and fungi [63–65]. Uncontrollable infection may exacerbate liver injury, leading to severe hepatitis [66, 67]. 1.2.1.3 Changes in Host Transcriptional Factors and Severe Acute Exacerbation of CHB. Hepatic nuclear factors (HNFs) are hepatotropic determinants found to play important roles in the specific replication and expression of HBV in the liver. After binding to a specific site proximal to the HBV central promoter, HNF4 can increase the

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transcription and replication of HBV [68]. The expression of HNF4α was found to be significantly higher in patients with severe hepatitis B than in those with CHB and liver cirrhosis (LC) (P  0.05), indicating that HNF4 may be associated with severe acute exacerbation of CHB [69]. HNF3 has been found to inhibit nuclear hormone receptormediated viral replication, and HNF3 beta inhibition of HBV replication has been associated with a preferential reduction in the level of pregenomic compared with precore RNA [70]. HNF3 acts as a negative regulatory factor, which inhibits HBV transcription and replication by blocking the synthesis of pregenomic RNA and HBV DNA. HNF3 may play an antagonistic role in severe acute exacerbation of CHB by suppressing HBV replication, in contrast to other HNFs, like HNF4, which increase viral replication. Furthermore, HNF1 may be involved in the exacerbation of CHB by increasing the transcription and replication of HBVs with the A1764T and G1766A mutations. HBV-encoded proteins play significant roles in the severe acute exacerbation of CHB.  HBV x protein (HBx) is a multifunctional regulatory protein involved in many biologic processes, including gene transcription, signal transduction, protein degradation, cell cycle and cell apoptosis, as well as in HBV replication [71, 72]. HBV core protein (HBc) is also involved in a number of important functions, including host gene regulation. HBc and HBx initiate the transcription of hFGL2 through the c-Ets-2transcription factor, an initiation dependent on the activation of extracellular signal-regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK) signal pathways, respectively, leading to the over expression of inflammatory genes and the occurrence of inflammation [73]. HBx has been reported to recruit neutrophils and monocytes into the liver by activating interferon-gamma inducible protein 10 and monokine induced by interferon-gamma, enhancing the expression of the cytokines interleukin (IL)-6 and IL-32 in the liver and promoting inflammation [74] (Figs. 1.1, 1.2, and 1.3).

1.2.1.4 TNF-Related Apoptosis-Inducing Ligand (TRAIL) and Zinc-­ Finger Antiviral Protein (ZAP) TRAIL, a type II transmembrane protein belonging to the tumor necrosis factor (TNF) superfamily, has been found to induce tumor cell apoptosis by binding to its receptors expressed on the surface of target cells, leading to caspase activation [75]. TRAIL, however, has minimal adverse effects on normal cells. TRAIL was able to induce the death of virally infected, transformed hepatocytes and fatty acid-treated hepatocytes [75, 76]. More importantly, TRAIL has been reported to potentiate cytotoxic Fas signaling in the liver and enhance Fasmediated hepatotoxicity by inducing sustained activation of JNK, a member of the mitogen activated protein kinase family that contributes to many forms of liver injury [77, 78]. ZAP is a type of host antiviral factor originally obtained from a mouse cDNA library through high throughput screening of a functional genome. ZAP is a protein consisting of 776 amino acid, with 4 CCCH zip fingers in the N-terminal and no functional domains at the C-terminal. ZAP has been found to significantly

1  Introduction to Acute Exacerbation of Chronic Hepatitis B (AECHB) Sex Age Race/Ethnicity HLA T-cell receptors Cytokines Chemokines

Host Factors

Androgen level Xenobiotic metabolism enzymes DNA repair enzymes Hormone receptors Oncogenes Tumor suppressor genes

Acute or subacute insult SIRS, Sepsis, Alcohol Bleeding, Surgery

Chronic Hepatitis B

Acute exacerbation of CHB

Viral load Genotypes Mutants HDV HEV HIV

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Survival

Acute on chronic liver failure

Antiviral, Antibiotics Artifical liver system Liver protection drugs Liver transplantation

Death

MODS/MOF

Jaundice, Ascites Coagulopathy Hemorrhage Secondary Infection Endotoxemia Hepatorenal syndrome Hepatic encephalopathy Hepatopulmonary syndrome

Alcohol drinking Cigarette smoking Carcinogen exposure Antioxidant vitamins Selenium Environment Factors

Virus Factors

Fig. 1.1  The nature history of AECHB. In the cause of insult factors on patients, CHB severe acute exacerbated to ACLF. Pathology and pathophysiology mechanism of exacerbation is very complex, involving a wide range of host factors, viral factors and environment factors. ACLF presents in the form of jaundice, progressive hyperbilirubinemia; coagulopathy and hemorrhage; secondary infection; endotoxemia; hepatorenal syndrome; hepatic encephalopathy; hepatopulmonary syndrome. The common outcome is death from multiorgan failure Liver failure staging End Stage Medium Stage Early Stage Extreme fatigue Anorexia, vomit Abdominal distension TBil ≥ 171µmol/L increase of TBil >17.1µmol/L/d PTA ≤ 40% With or without HE and ascites

Obvious ascites Blutene chloaide, ecchymosis ≥ HE grade II 20%< PTA ≤ 30%

Hepatorenal syndrome Massive Variceal bleeding Sepsis or SIRS Refractory electrolyte disorder Severe bleeding tendency ≥ HE grade III PTA ≤ 20%

Acute on chronic liver failure

Fig. 1.2  Clinical progression grading of AECHB.  AECHB is a complicated multi-organ syndrome. According to the severity of liver dysfunction, coagulopathy, and occurrence of subsequential complications, ACLF can be staged into early, medium, and the end stages

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Chronic Hepatitis B

Immune cells

Impaired CTL & DC Tregs

Immune moleculars

TLRs

Cytokines

TGF-β

PD-1

IL-10

Acute excerbation of CHB

Acute on chronic liver failure

Liver CD4/8+ T KCs , NK pDC , Th17

Death

Peripheral pDC

HLA-DR

TLRs

Liver TFN-α Serum IL-10

MODS / MOF

, IFN-γ , IFN-α

Serum IFN-α

Fig. 1.3  Immune mechanisms for the development of HBV related ACLF. In the immune tolerant chronic hepatitis B patients, the function of CTLs and dendritic cells are impaired, the number of regulatory T cells increases, with high levels of TGF-β,IL-10 and PD-1 but low expression of Toll-­ like receptors. In this stage, persistent HBV replication can be found in liver with no severe immune-mediated liver damage. Due to the hits from certain acute events, a large number of immune cells recruited into the liver, including CD4/8+ T cells, NK cells, monocytes, pDCs and Th17 cells, with subsequent increase of TNF-α, IFN-γ and reduction of IL-10 and IFN-α. These changes lead to an acute exacerbation of liver inflammation and ultimately cause liver failure. Comparing to that in patients who survive in ACLF, the peripheral pDC as well as HLA-DR and IFN- α remarkably decrease in patients who tend not to survive, and the mechanisms need to be further investigated

suppress HBV replication, suggesting that it may be involved in severe acute exacerbation of CHB by antagonizing transcriptional factors that increase HBV replication [79].

1.2.2 HBV Mutation and Severe Acute Exacerbation of CHB As a highly mutated genome, HBV mutations contribute significantly to the severe acute exacerbation of CHB. The precore mutation at nt 1896 (G to A) and core promoter mutations at nt 1762 (A to T) and nt 1764 (G to A) were found to be related to acute exacerbation of liver disease even fulminant hepatitis (FH), a rare condition in which rapid destruction of the liver parenchyma lead to coagulopathy, altered mental status and subsequently multiorgan failure [80, 81]. Replication activity was greater in these mutant HBVs than in wild-type, as well as to affect pre-C and C antigens. The duration of infection may be altered because a decrease in HBeAg expression may affect host immune responses. The presence of the double mutation C1766T and T1768A within the core promoter may also be related to severe acute exacerbation of CHB, since increased production of binding domain may enhance the transcription of pre-genome mRNA, increasing the synthesis and release of

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HBV DNA [82]. Nucleoside/nucleotide analogs targeting particular sites of viral polymerase, including lamivudine, entecavir, telbivudine, and tenofovir, have greatly improved the treatment of patients with hepatitis B. However, the emergence of drug-resistance mutations usually results in attenuated viral suppression and disease progression, and may lead to significant clinical deterioration [83].

1.2.3 Environmental Factors Liver is the main organ that metabolizes drugs, eliminate most through. biotransformation. Any physiopathological change in the liver will influence its metabolism of drugs, altering the therapeutic effects, toxicity and site effects of drugs, and making the liver more vulnerable. Drug-induced liver injury (DILI) is the second most frequent cause of acute liver failure, especially in developed countries [84]. There is an increasing trend of drug induced AECHB [43, 53, 85, 86]. Among the agents reported to induce liver injury are antibiotics, antituberculosis drugs as rifampicin and isoniazid, anticarcinogen, antineoplastic agents, antiparasitic agents, analgesic-antipyretic drugs, drugs that treat nervous-mental system disorders, anesthetics, antirheumatic agents, antiarthritic drugs, glucocorticoids, traditional Chinese medicines, and herbal medicines [86–91]. The exact mechanism of DILI is remain unknown. DILI occurs due to combination of host, drug and environmental factors often acting in concert [86, 92]. Alcohol may exacerbate the occurrence and development of liver disease in HBV infected patients, leading to severe hepatitis [41]. Many mechanisms may be involved, including the toxic effects of alcohol and its metabolites, oxidative stress, lipid peroxidation, toxicity to mitochondria, cytokines and inflammatory mediators, lack of oxygen, and secondary malnutrition caused by long-term alcohol consumption [93–95]. Furthermore, the general environment contains various harmful or toxic substances, including chemicals substances, radioactive materials, Selenium, Carcinogen, additives and antiseptics in foods, as well as insecticides and herbicides sprayed onto crops [84, 91]. Most of these harmful substances are metabolized by the liver, thereby inducing hepatic injuries. These agents are especially harmful in patients with chronic liver diseases, causing pathophysiologic changes in the liver, reducing the ability of the liver to metabolize these harmful or toxic materials, and lowering tolerance to drugs, all of which lead to greater deterioration in liver function and exacerbation of liver diseases [96].

1.2.4 Clinical Manifestations Severe acute exacerbation of CHB is a clinical and histologic manifestation of disease deterioration, characterized by increasing hepatic inflammation-necrosis [41, 97, 98]. Clinical manifestation of AECHB is determined by the chronic and acute insult and the possibility of the syndrome subsequently develops. The injuries

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involve the whole body, representing as systemic inflammatory response and high energy expenditure and catabolism. ALT was found to be the most sensitive marker of acute liver injury, with its activity related to the degree of hepatic injury. Serum bilirubin (SB) may dissociate from ALT under conditions of extensive hepatocyte necrosis, resulting in a rapid decrease in ALT activity and a significant increase in SB concentration. Increased ALT, above five times the upper limit of normal (ULN), is associated with a poor prognosis. SB is not a sensitive marker of hepatic damage, but its significant elevation (usually ≥10 ULN) is characteristic of severe hepatitis and liver failure, and may be important role in diagnosis of liver failure [83, 99, 100]. Histologic assessment of necroinflammatory activity, based on the examination of liver biopsy samples, is regarded as the golden standard for diagnosis of liver disease. The histologic characteristics of AECHB include ballooning degeneration, focal necrosis, confluent necrosis, bridging necrosis, extensive interface hepatitis, massive or submassive necrosis, neutrophil infiltration into hepatic lobules and portal area, and more than moderate intrahepatic cholestasis [43, 101, 102]. A marked degree of hepatocyte necrosis is regarded as a pathological indicator of severe acute exacerbation of CHB, manifesting as extensive focal necrosis, confluent necrosis, bridging necrosis, and massive or submassive necrosis, leading to an extreme form of exacerbation such as liver failure or severe hepatitis [73, 103]. The lack of liver detoxification, metabolic and regulatory functions and an altered immune response lead to life-threatening complications [85]. The subsequent complications include hepatic encephalopathy (HE), dysfunction of kidney, lung and other systems. HE is the most severe complication of AECHB, the mortality is especially higher in patients who progress to grade III/IV encephalopathy, for cerebral edema commonly occurs. Renal dysfunction, either due to the original insult or hyperdynamic circulation, is also common. Coagulopathy is another cardinal feature of AECHB, which results in a prolongation in prothrombin time [41, 104, 105].

1.2.5 Disease Classification and Progression Grading HBV ACLF combines an acute deterioration in liver function in an patient with pre-­ existing chronic liver disease and hepatic and extrahepatic organ failures. To recognize ACLF as probably a syndrome with several causes rather than a single disease, a classification was proposed on the basis of which divide ACLF to three types [106]. Type A disease is non-cirrhotic ACLF, which can be distinguished from acute liver failure only by histopathological evidence of substantial hepatic fibrosis. Type B disease is proposed to occur in patients with compensated cirrhosis with hepatic deterioration after an acute insult such as infection, surgery, or acute alcoholic hepatitis. Similar precipitants in patients with a previous or contemporaneous episode of cirrhotic decompensation has been called Type C disease. Severe acute exacerbation of CHB is a devastating multi-organ syndrome, involving both hepatic and extrahepatic organ failure, which characterized by severe liver cell dysfunction and resulting in a progressive elevation of jaundice, reduced liver capacity, coagulopathy, hemorrhage, secondary infection, hepatorenal

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syndrome, and hepatic encephalopathy [41, 43, 84, 107]. It is graded into three levels. This, however, needs to be prospectively evaluated. Fatigue and asthenia are common symptoms during the early stage of l, followed by progressively increasing malaise and anorexia and loss of appetite [108]. Sudden early stage manifestations include progressive deepening of jaundice (mainly hepatocellular jaundice), total bilirubin >10 ULN, a daily increase in SB > 17.1 μmol/L (1 mg/dL), dissociation of SB and ALT, and prothrombin activity (PTA) of 30–40%, without complications such as ascites and hepatic encephalopathy (HE) [107]. Immediate control of precipitating factors and immune reactions during the early stages of severe hepatitis can reduce digestive symptoms, such as nausea, vomiting, and abdominal distension, as well as increasing appetite. Moreover, jaundice may slowly subside, coagulation function may improve, and PTA may recover to above 40% [40, 101, 103]. A lack of timely control can result in progression to the middle stage of severe hepatitis. This stage is characterized by the aggravation of symptoms, including poor appetite, increases in vomiting, and hiccups. Patients may also manifest grade I/II HE, and (or) ascites, obvious hemorrhagic tendency (bleeding point or petechia), with 20% 1.5), complicated within 4 weeks by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease”. It is the main type of liver failure in China and Southeast

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Table 2.1  Pros and cons, using animals of main animal models Animal model Surgical Hepatectomy

Species

Pig, dog, rabbit, rat, mouse Devascularization Pig, dog, rabbit, mouse Chemical Acetaminophen Pig, dog, rabbit, rat, mouse Azoxymethane Mouse

Advantages

Disadvantages

Hepatic encephalopathy; reproducible

No reversibility; no long-term survival

Same as above

Same as above

Hepatic encephalopathy; no hazard

Non-reproducible; variable interval between damage and death Small size; hazard

Hepatic encephalopathy; reproducible Hepatic encephalopathy

CCl4

Pig, rabbit, rat, mouse

Concanavalin A D-Gal

Rat, mouse Pig, dog, rabbit, rat, mouse

Hepatic encephalopathy Hepatic encephalopathy

LPS

Rat, mouse

Hepatic encephalopathy

TAA

Rabbit, rat, mouse

Hepatic encephalopathy; reproducible; long time window before death

Sensitive mouse

Reproducible; no hazard

Rabbit

Hepatic encephalopathy; reproducible; no hazard

Viral WHV-3

RHDV

Non-reproducible; extrahepatic toxicity; small time window before death Small size Non-reproducible; hazard; variable interval between damage and death; species differences Non-reproducible; small size; hazard; small time window before death Hazard

Non-hepatic encephalopathy; only suitable for sensitive mouse Only suitable for rabbits

Asia, so it’s popular to study the animal models of ACLF in recent years. The research on the animal models of ACLF are based on the “two-hit” theory. Procedure one: The rats are immunized with human serum albumin to make immunological cirrhosis, then given different acute hits, such as intraperitoneal injection of D-Gal (1.2 g/kg), intravenous injection of LPS (30 mg/kg) or intraperitoneal injection of D-Gal (400 mg/kg) combined with LPS (100 μg/kg). The study showed that the cirrhotic rats challenged by the combined injection of D-Gal and LPS died within 13–19 h. Their liver pathogenic examination displayed regenerative nodules, massive or sub-massive necrosis, obvious hepatocyte apoptosis and the hyperplasia of the fibrous septa. Above features indicated that it can be used as successful animal model of ACLF. While on the basis of immunological cirrhosis, large dose of D-Gal or LPS was given alone, only marked macrovesicular or

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microvesicular steatosis, small patch necrosis or no obvious necrotic lesions could be observed in the liver, and it could not cause the massive or sub-massive necrosis in cirrhotic rats. Procedure two: SD rats are intraperitoneally inoculated with vegetable oil containing 50% CCl4 every 3 days for 10–12 weeks to cause cirrhosis. On this basis, ACLF could be induced with the hit of D-Gal or LPS combined D-Gal, manifested as patchy, massive or sub-massive necrosis in the liver. ACLF animal models can also be induced by the intraperitoneal injection of CCl4 on the basis of cirrhosis.

2.4.4 S  everal Points Should Be Noted in Building the Animal Models of Liver Failure Terblanche proposed the six requirements, which were reversibility, reproducibility, death from liver failure, a therapeutic window, a large animal model, and minimal hazard to personnel [55] respectively, for a satisfactory animal model of hepatic failure. Reversibility: In the ALF model, the mortality rate of untreated animals should be high, and the damaged liver could be restored reversibly with effective therapy. Repeatability: Repeatability and stability are needed for ideal animal models. Died of AFL: The experimental animals should ultimately die of liver failure rather than other causes, it’s important to determine the efficacy. A therapeutic window: The time interval between applying damaging process to animals and the death should not be too short, so there is enough time to make intervention and assess the efficacy. Large animal model: The large animal model is convenient to collect the blood sample or tissue continuously for a series of study, and the data derived from large animal perhaps has more reference value for the clinical practice. Minimal harm to the experimental personnel: all of the chemicals used in experiments should be safe to the experimental personnel and harmless to the environment.

2.4.4.1 Animal Species Currently the animals used in the models of liver injury include the pigs, dogs, rats and rabbits. Large animal (pigs and dogs) models of ALF are mainly made by surgical procedures and surgical plus drug-toxic procedures. If toxic drugs are used in large animal alone, it’s difficult to control the dosage, the terminal of the experiment is unclear and the repeatability is poor. On the contrary, small animal (rats and rabbits) models of ALF are more suitable to be established by hepatotoxic substance, such as CCl4, thioacetamide and D-Gal, etc. Rats have been widely used in animal experiments due to their strong reproductive capacity, great vitality, wide availability and cheap price. The rat liver failure model has been served as common tools to study the mechanism and therapy of liver failure. The hepatic anatomy and physiological indexes of the pigs are similar to those of humans, and the experiments are easy to operate, so the pig is the first choice to build large animal models of liver failure.

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The liver cells of different animals have various sensitivities to different hepatotoxic drugs. For example, the murid is most sensitive to acetaminophen, followed by the dogs, rabbits and monkeys.

2.4.4.2 Observation Items The general observatory items include the diet, movement, hair color and urine color of experimental animals. The biochemical parameters (serum ALT, total bilirubin, blood ammonia), the liver pathological changes under hematoxylin-eosin staining and the molecular biological indicator associated with the study shall also be detected and recorded. And on this basis, other observation items can be set based on the experimental design. 2.4.4.3 Anesthetic Sometimes the anesthesia is needed for the large animals before administration of hepatotoxic drugs. The thiopental, pentobarbital, ketamine and halothane are usually used. The anesthetic toxicity to animals must be taken into account, especially the co-toxicity of anesthetic and experimental drugs. The thiopental, pentobarbital and other barbiturates and halothane can aggravate the hepatotoxicity induced by acetaminophen. The aggravation of the hepatotoxicity has been confirmed both by pathological study and the surviving statistics. Intramuscular injection with thiopental (25 mg/kg) once a day for 4 days, followed by injecting 400 mg/kg of acetaminophen intravenously, as high as 80% of the dogs died within 48 h. While the dogs only received the injection of acetaminophen (700  mg/kg), all animals survived. Halothane also can aggravate the liver injury, the success rate of animal models anesthetized by halothane is significantly increased, while the success rate reduced obviously as anesthetized by other drugs. In terms of safety, thiopental has a higher frequent to induce anesthetic accident, it can obviously inhibit the respiratory center and induce throat and bronchial spasm. Overall, compound ketamine is relatively safe to be used, there is no deaths as it is intramuscularly injected with a dose of 0.05 mL/kg. 2.4.4.4 Extrahepatic Toxicity of the Drug In the aforementioned common chemical models, the extrahepatic toxicity of acetaminophen is most obvious, mainly shown as hypoglycemia, methemoglobinemia and pulmonary edema, animals died without exception when the latter two occurred simultaneously. In addition, acetaminophen can cause hemolytic anemia. 2.4.4.5 Requirements of the Projects The miniature pigs are usually chosen to build the surgical liver failure animal model which requires rich operative experience. And only large animals are eligible to the study of artificial liver support system. In view of being easy to operate and repeatability, rats are frequently used to investigate the internal therapeutic strategies of liver failure and its model is often built by poisoning method. Until now, D-Gal and thioacetamide are most frequently reported and widely used in spite of their advantages and disadvantages.

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The animal model of acetaminophen is suitable for the study of drugs and chemical liver failure, but the model of D-Gal is also used in the study of drug-induced liver failure. In numerous chemical models of liver failure, the model of D-Gal has been verified to able to simulate the pathophysiological process and clinical pictures of human ALF. The large animal model developed by D-Gal is very similar to clinical viral liver failure and has been widely used to study the bioartificial liver support system and the pathogenesis of ALF. For viral liver failure, the viral model is more appropriate. Unfortunately, the use of infective agents to develop animal model of ALF has in general unsuccessful. MHV-3 only infects susceptible mice and RHDV only infects the rabbit. The large animal models imitating the human infection are absent still. Although a large quantities of liver failure models has been report, no one experimental model can fully and accurately reflect the nature of the specific liver damage to meet all the needs of scientific research so far because the human liver function is complex, the factors leading to liver failure are various and the clinical manifestations and complications of ALF are diverse. Researchers should establish an appropriate animal model according to different research purpose, technical proficiency and economic conditions. The key to the success of the model is to control the biological characteristics of experimental animals strictly. It should be the focus of research to find more satisfied hepatotoxic substances or combine the simple surgical procedures with drugs to establish a large animal model of ALF.

2.5

Hepatic Encephalopathy

Qiong-Fang Zhang and Da-Zhi Zhang Hepatic encephalopathy (HE) due to metabolic disturbance is a complex neuropsychiatric syndrome caused by severe liver dysfunction or disorder and is one of the common complications and causes of death in severe liver diseases. Patients with HE mainly present with neuronal or mental abnormalities and disturbance of consciousness, even coma and death. The clinical manifestations and the severity of the disease vary because of its complex pathogenesis.

2.5.1 T  he Concept and Clinical Classification of Hepatic Encephalopathy 2.5.1.1 The Concept Hepatic encephalopathy is the result of acute and chronic hepatic failure caused by cirrhosis or various kinds of portosystemic shunt (PSS) created. A diagnosis of HE can be made after excluding encephalon diseases. The syndrome is caused by metabolic disorders and is potentially reversible. HE clinical features differ due to the wide degree and range of neuropsychiatric symptoms that vary from subtle abnormalities detected only by intelligence tests or electrophysiological methods geared for detecting

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personality changes to abnormal behavior, intellectual impairment, and even different degrees of consciousness disorders. HE was previously known as hepatic coma, but that is only one of the worst severe signs of HE and does not represent all types of HE.

2.5.1.2 HE Classifications In 2003, the World Congress of Gastroenterology (WCOG) suggested that based on the cause HE can be divided into three types (A, B, and C) [56, 57]. Type A: Type A is acute liver failure-related HE and the symptoms occur within 2  weeks. In subacute liver failure-related HE, the symptoms of HE occur within 2–12 weeks with or without predisposing factors. Type B: Patients with type B HE have obvious PSS and normal liver histology without associated intrinsic liver disease. These clinical manifestations are similar to those in patients with HE and cirrhosis. The PSS may be spontaneous or caused by surgical or interventional procedures [58]. Common causes of PSS include congenital vascular malformation, intrahepatic or extrahepatic portal vein obstruction (including trauma, carcinoid, and bone marrow hyperplastic disease caused by a high coagulation state due to portal vein branch embolization and thrombosis) and generation of portal hypertension by oppression of lymphoma, metastatic tumors, and bile duct carcinoma. Type C: Type C HE is related to chronic liver diseases, with cirrhosis being the most common type, and is generally accompanied by portal hypertension and PSS.  Type C HE is mainly caused by liver function failure, rather than by PSS. According to the clinical manifestations, duration and characteristics, type C can be divided into three types: episodic HE, persistent HE, and minimal HE [56]. Episodic HE Episodic HE, related to chronic hepatic disease, is defined as a disturbance of consciousness and cognitive change in a short time and can be alleviated by spontaneous remission or drug treatment in the short term, which cannot be explained by a relevant preexisting mental disorder. Episodic HE can be divided into three types according to the presence of known risk factors: (1) incentive type: there is a clear history of predisposing factors; (2) spontaneous type: there is no history of predisposing factors; (3) recurrent type: HE attacks more than two times within a year. Persistent HE Persistent HE related to chronic hepatic disease is defined as an occurrence of continuous neural mental abnormality, including cognitive decline, disturbance of consciousness, coma and even death. Persistent HE can be further divided into three types according to the severity of the disturbance in the patient’s self-control and self-discipline: (1) mildest type, namely West Haven level 1; (2) severe type: namely West Haven level 2–4; and (3) therapeutic resistance type: medication can alleviate HE quickly, but withdrawal can aggravate HE rapidly. Minimal HE Patients with minimal HE, with normal clinical manifestations and routine biochemical tests, have mild cognitive and psychomotor deficits detected by

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neuropsychology and neural physiology tests, and these patients usually have a history of chronic hepatic disease [59]. The prevalence of minimal HE in patients with cirrhosis is 30–80%. Patients with minimal HE with reduced physical and mental ability have gained more and more attention recently because they have a high risk of accidents when engaged in occupations involving mechanical, or driving work.

2.5.2 Pathogenesis The pathogenesis of HE has not been fully elucidated so far, and many theories have been put forward. It is generally believed that HE is caused by acute and chronic liver failure and/or PSS. When toxic substances absorbed by the intestines cannot be detoxified and cleared by (or through) the liver, they directly enter into the systemic circulation and pass through the blood-brain barrier to reach the brain tissue and cause central nervous system dysfunction. A variety of the risk factors mentioned above can result in HE.  Hyperammonemia is still recognized as one of the most important factors, especially in HE related to chronic liver disease, liver cirrhosis and/or PSS. According to the ammonia intoxication theory several factors including false neurotransmitters, such as γ-aminobutyric acid/benzodiazepine (GABA/Bz) receptor complex, an imbalance in the ratio of branched chain amino acids to aromatic amino acids, brain cell edema, astrocyte dysfunction, mercaptan, short chain fatty acid toxicity and manganese deposition are all involved in the occurrence of HE [60].

2.5.2.1 Ammonia Intoxication Theory Ammonia intoxication caused by an ammonia metabolism disorder is the most important factor in the pathogenesis of HE [61]. Ammonia comes mainly from the gut and the generation and absorption of ammonia increase in a serious liver disease when excess ammonia cannot be cleared sufficiently by ornithine cycle due to serious damage to liver parenchyma. When PSS occurs, intestinal ammonia directly enters the systemic circulation without liver detoxification, resulting in increased blood ammonia. High levels of blood ammonia can enter the brain through the blood-brain barrier and generate central nervous system toxicity by interfering with cerebral energy metabolism, neurotransmitter and nerve cell membrane ion transport; increasing cerebral edema; and changing gene expression (such as stellate cell glutamate carrier, stellate cell structural protein, glial fibrillary acidic protein, peripheral benzodiazepine receptor and aquaporin-4) and inducing the mitochondrial permeability transition (MPT). The main way of removing ammonia from the brain is through urea cycle. During glutamine synthesis, glutamic acid is formed from ammonia and α-ketoglutaric acid and the glutamic acid combines with ammonia to generate glutamine. This process requires ATP and consumes a large amount of α-ketoglutaric acid, which interferes with the brain energy metabolism and causes an energy supply shortage in brain cells. Glutamate is an important excitatory neurotransmitter in the brain, and lack of glutamate increases inhibition in the brain. Glutamine synthetase is present in astrocytes, where glutamic acid is detoxified to glutamine. Glutamine is a strong

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intracellular osmotic agent, and increases in glutamine can lead to brain cell swelling. Reports have identified a strong correlation between the content of glutamine in cerebrospinal fluid (CSF) and the degree of HE [62]. During HE, excess ammonia under the effect of glutamine synthetase, not only reduces the formation of active glutamate but also consumes a lot of energy, leading to the accumulation of glutamine, which increases intracellular osmotic pressure and causes brain cell swelling. Swollen astrocytes with impaired function further affect ammonia metabolism, reduce the ability of neurons to efficiently uptake or release extracellular ions and neurotransmitters, and stimulate glial cell synthesis of neurosteroids by upregulating their expression of the peripheral-type Bz receptor (translocator protein, 18 kDa). Neurosteroid is an endogenous Bz that can enhance GABA nerve tension and cause symptoms in patients with HE [63] (Fig. 2.3). Recent studies have shown Cerebral edema

Glutamine synthase Glutamine

Glutamate NH3

Inflammation

Endothelial dysfunction

NH3 Muscle glutamine synthase

Glutamine

NH3

NH3

NH3

Enterohepatic circulation

Urea cycle

NH3

NH3

Portacaval shunt

NH3

Cirrhosis

Glutaminase

Urea Glutamine

Urea

NH3

Urinary excretion

Brush border mucosal enzyme Diet Glutaminase Ammonia(NH3) Glutamate

Urea Bacteria

Fig. 2.3  The pathogenesis of hepatic encephalopathy schematic diagram

Glutamine

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that the metabolic rate of cerebral ammonia in HE patients is increased. Increased levels of blood ammonia enter the brain through the blood-brain barrier. Brain dysfunction also occurs even if blood ammonia levels appear normal; this partially explains the occurrence of HE in the case of normal blood ammonia and invalidates HE treatment by simply reducing blood ammonia. In addition, increasing evidence suggests a synergistic effect between blood ammonia and its metabolic disorders with systemic inflammation, nerve steroids, oxidative stress, nitrification stress, manganese poisoning, and GABA/Bz [63].

2.5.2.2 The GABA/Bz Receptor Theory The main inhibitory neurotransmitter in the mammalian brain is GABA.  Plasma GABA is derived from the conversion of glutamic acid by glutamate decarboxylase in intestinal bacterial. Notably, GABA has dual role. On one hand, during liver function failure and PSS, the removal of GABA in liver is significantly decreased; on the other hand, GABA can directly enter the systemic circulation bypassing the liver, resulting in increased concentration of GABA in blood. The concentration of GABA in CSF and brain tissue increases as more GABA crosses the abnormal blood-brain barrier. In addition, endogenous Bz was found in the blood and CSF, and the GABA receptor on the membrane surface of the brain’s postsynaptic neurons increased significantly in some patients with HE and in animal models. This receptor not only combines with GABA but also binds to barbiturates (BARB) and Bz on different parts of the receptor surface; thus, it has been named the GABA/Bz complex receptor or the super receptor complex. When liver function is severely impaired, the binding affinity of this complex receptor to its three ligands is also increased. Binding of GABA, BARB, or Bz with the complex receptor can promote entry of chloride ions from neuronal membrane ion channels into the cytoplasm of postsynaptic neurons, causing membrane hyperpolarization and nerve conduction inhibition. HE symptoms were relieved in about 30% of patients treated with a GABA receptor antagonist or Bz receptor antagonist, and GABA/Bz and ammonia were reported to act synergistically in HE.  Recently, some studies focused on peripheral type Bz receptors, which are different from central GABA [64, 65]. Some questions, including the source of endogenous Bz, and the correlation between the increased degree of GABA or Bz and the disease, remain to be answered. Therefore, therapy targeted at reducing the blood ammonia concentration in patients with HE and significantly reducing the increased GABA nerve tension seems reasonable [66], but may not be completely effective. Treatment effects of reducing ammonia vary, because of the different levels of ammonia in HE patients that can be produced by the interaction between various known or unknown factors and the different effects of Bz receptor antagonists. 2.5.2.3 False Neurotransmitters and Imbalance of Amino Acid Metabolism Theory This theory is related to the metabolism of aromatic amino acids (AAA), the precursors of true neurotransmitters, including norepinephrine and dopamine. Due to the reduction in the liver’s detoxification function or formation of PSS, the amines

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(phenylethylamine and tyramine) produced in the intestine cannot be cleared completely, resulting in elevated concentrations of these amines in the systemic circulation and increased levels in the brain through the blood-brain barrier. Under the effect of β-hydroxylase, phenethanolamine and β-hydroxytyramine (β-dopamine) are generated from phenylethylamine and tyramine, respectively and are similar to norepinephrine and dopamine in chemical structure. These amines can be taken up, stored and released by adrenergic neurons in the brainstem reticular structure. Phenethanolamine and β-hydroxytyramine are called false neurotransmitters because of their low physiological effects on the postsynaptic membrane, which is about 1/10 of norepinephrine. When these false neurotransmitters accumulate in the nerve synapse, they can outcompete or replace normal neurotransmitters, resulting in a disorder of nerve conduction. It was reported that plasma AAA (such as phenylalanine, tyrosine, and tryptophan) increased and branched-chain amino acids (BCAA, such as valine, leucine, isoleucine) decreased in patients with decompensated liver cirrhosis, leading to an imbalance of amino acid metabolism. AAA are decomposed and metabolized in the liver, and liver failure decreases AAA decomposition resulting in an elevated concentration of AAA in the plasma. Insulin can promote BCAAs entering muscle, which is then broken down and metabolized in the skeletal muscle instead of the liver. Insulin inactivation is decreased in patients with liver failure, promoting a large number of BCAAs entering the muscle tissue and decreasing the concentration of BCAAs in plasma. Finally, the BCAA/AAA ratio is reduced from a normal 3–3.5:1 to 1:1 or lower. The above process reduces the BCAA concentration, but increases the AAA concentration, leading to an increase in synthesis of false neurotransmitters and reduction of the normal neurotransmitter [67–69].

2.5.2.4 Manganese Poisoning Theory The epidemiological data suggests that manganese poisoning and HE extrapyramidal have common clinical symptoms. The liver is an important organ for manganese excretion. The concentration of blood manganese can be increased when liver function is affected, during PSS, or when excretion of bile is reduced. Manganese content in plasma was sharply increased in more than 80% of patients with acute hepatitis and liver cirrhosis and the density of globus pallidus increased in the brain basal ganglia of HE patients (partially two to seven times higher by MRI). Based on histological results, the above changes were caused by manganese deposition, which disappears after liver transplantation. It has been suggested that manganese deposition may cause dopamine dysfunction. Deposition of manganese not only cause direct brain injury, it can influence the function of 5-hydroxytryptamine (5-HT), norepinephrine and GABA neurotransmitters; impair astrocyte function; and have a synergistic effect with ammonia. However, there is no reliable correlation between the concentration of serum manganese and HE severity, which may be due to the chronic deposition of manganese [70]. The characteristic change in MRI imaging as the deposition of manganese remains to be verified. The effectiveness of manganese removal to improve the symptoms and neurological signs of patients with HE needs further validation.

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2.5.2.5 Additional Theories The synergistic toxic effects between toxins (ammonia and mercaptan) and short chain fatty acids [71], the 5-HT hypothesis, the effect of Helicobacter pylori urease, opioids, endotoxin, tumor necrosis factor, melatonin, and hepatitis B virus termed additional theories of HE syndrome. This theory also suggests the same hypothesis mentioned in the above theories.

2.5.3 Common Predisposing Factors of HE Due to the extensive amount of liver cell damage caused by acute liver failure in type A HE, the residual liver cells cannot effectively remove toxins leading to central nervous system dysfunction. Type A HE, known as non-ammonia encephalopathy, is endogenous HE without clear causative agents. Simple type B HE is rare in mainland china; the liver can clear limited metabolic toxins in patients with chronic liver failure or PSS, but once these toxins exceed the compensatory capacity of the liver, type C HE occurs. The occurrence of type C HE is largely related to the following risk factors, which are the most important factors in the prevention and treatment of HE.

2.5.3.1 Excessive Intake of Nitrogen Patients with chronic liver failure or PSS are less tolerant to the protein found in food, especially animal protein. A large amount of ammonia and AAA are produced by the decomposition of intestinal bacteria, which can induce HE. Oral ammonium salts, urea, and methionine can induce HE by increasing the absorption of nitrogenous substances and elevating blood ammonia. 2.5.3.2 Massive Hemorrhage in the Digestive Tract Intestinal production of ammonia can be increased by hemorrhage in the intestine (100 mL of blood contains 15–20 g protein). At the same time, because of the lack of isoleucine in the blood, after digestion and absorption of a hemorrhage, extra blood leucine and valine increase BCAA decomposition by enhancing the activity of BCAA dehydrogenase, thereby exacerbating the imbalance in the BCAA/AAA ratio. Loss of blood volume, cerebral ischemia and hypoxia also increase the sensitivity of the central nervous system to ammonia and other toxic substances [60]. 2.5.3.3 Infection and Sepsis/Systemic Inflammatory Response Syndrome, SIRS Infections such as spontaneous peritonitis, pneumonia, and urinary tract infection can increase tissue decomposition and production of ammonia. Secondary sepsis or SIRS induce HE through TNF-α, IL-1, IL-6 and other inflammatory factors, exacerbates oxidative stress, and increases the blood-brain barrier permeability of ammonia and other toxic molecules to liver and brain [72]. Studies have shown that SIRS is directly related to the deterioration of HE in patients with liver cirrhosis, and its extent and mortality increase with the deterioration of SIRS [73]. Similarly, SIRS is

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a common factor in triggering chronic liver failure characterized by HE and renal failure. In a study of patients with liver cirrhosis, artificially-induced hyperammonemia by oral administration of glutamine may have worsened the results of psycho-­ mental testing in ten cases of sepsis patients; while brain toxicity was not obvious after the inflammation was relieved, the observation of decreased cytokine levels indicated that infection and induced inflammatory mediators enhanced brain toxicity of hyperammonemia. Accordingly, some researchers suggested that SIRS could be an independent pathogenesis of HE rather than a risk factor [73].

2.5.3.4 Water and Electrolyte Disturbance Hyponatremia can affect the intracellular osmotic pressure and lead to brain edema, which induces HE. Hypokalemia is often associated with metabolic alkalosis [74]. Mass use of diuretics or extraction of ascites can also cause alkalosis. Ammonia is easily absorbed by the intestinal tract or through the blood-brain barrier inducing HE [75]. 2.5.3.5 Azotemia A variety of reasons can cause pre-renal azotemia such as hypovolemia, anorexia, diarrhea, limiting the amount of liquid, mass use of diuretics, or extraction of ascites. Hepatorenal syndrome or other causes can result in renal azotemia. Pre-renal azotemia and renal azotemia caused by hepatorenal syndrome or other causes can increase the concentration of ammonia in the blood. 2.5.3.6 Other Theories Several other predisposing factors can contribute to HE such as constipation, hypoglycemia, the use of sedatives and proton pump inhibitors, and epilepsy. After the occurrence of constipation and intestinal obstruction, the patient’s intestinal mucosa is exposed to ammonia longer thus increasing the absorption of ammonia. Hypoglycemia can reduce brain deamination. The binding of sedatives, hypnotics and the brain GABA/Bz receptor produce an inhibitory effect on the brain. It was reported that proton pump inhibitors increase the risk of HE in patients with cirrhosis in a population study [76]. Another study also suggested that epilepsy was associated with an increased risk of HE in patients with cirrhosis [77].

2.5.4 Pathological Changes in HE Patients with type A HE often have no obvious anatomical abnormalities in their brains, but 38–50% of patients have brain edema, which may be a secondary change of the disease. Hypertrophy and hyperplasia of the original plasma astrocytes in gray matter and subcortical tissue can be found in patients with type C HE. Patients with longer course of the disease will exhibit brain atrophy (especially in patients with alcoholic cirrhosis) of different degrees, thinning of the cerebral cortex, loss of neurons and nerve fibers, and deep cortical sheet necrosis, even the cerebellum and the base may also be involved.

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2.5.5 Clinical Epidemiology The majority of patients with cirrhosis may have different degrees of HE at some stage in the course of the disease. The incidence of HE in patients with liver cirrhosis is at least 10–50% in mainland China while the incidence of Post-TIPS (transjugular intrahepatic portosystemic shunt) HE is 25–45%. If patients with chronic liver disease have HE, the outcome is poor; the 1 year survival rate is lower than 50% and the 3 year survival rate is less than 25% [77, 78]. The incidence of mild HE is 39.9% in mainland China in patients with liver cirrhosis, 24.8% in patients with Child-Pugh A, 39.4% in patients with Child-Pugh B, and 56.1% in patients with Child-Pugh C. The incidence of mild HE is not significantly associated with cirrhosis; however, with the increased degree of decompensated liver cirrhosis, the incidence of mild HE increase. Several studies have found that the incidence of depression and anxiety in patients also increased, With the increase of liver function damage, the incidence also increased, and the outcome is poor [79, 80].

2.5.6 Clinical Manifestation and Stages of HE The clinical manifestations of HE vary, because of the difference in the nature of underlying disease, the degree of liver cell damage, the speed of injury and incentives. They are not specific to HE compared with other metabolic encephalopathies. Early pathological changes of HE are mild HE. The neuropsychological and intelligence tests detect mild form of HE, which exhibit no clear clinical symptoms and often develop symptomatic HE. The main clinical manifestations seen in acute liver failure induced by type A He are rapid- onset jaundice, bleeding, decrease in prothrombin, and eventually, change in mental status that can start as mild confusion but progress to coma and even death. Type C HE is characterized by chronic recurrent episodes of changes in personality and behavior [81], stupor and coma, which is often accompanied by increased muscle tone, hyperreflexia, hepatic flap, ankle clonus or positive Babinski sign and nervous system abnormalities. Most patients in the early stages relapse, but then their symptoms become persistent. HE often has a variety of risk factors such as consuming a high-protein diet or discontinuing treatment of HE. Patients with type C HE not only have the clinical manifestations of encephalopathy, they also have chronic liver injury, cirrhosis and other clinical manifestations [56]. Observation of encephalopathy dynamic changes is beneficial for early diagnosis, treatment and analysis of treatment efficacy. HE can be graded and quantified according to the degree of disturbance of consciousness, nervous system performance and EEG changes. According to the 2009 edition of the “Consensus on the Diagnosis and Treatment of Hepatic Encephalopathy” in China, HE is divided into 0–4 periods, but each period can be overlapping or distinct but each period can be overlapping (Table  2.2). At present, scholars have stressed that the occurrence of HE is a

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Table 2.2  Clinical staging of hepatic encephalopathy Staging Grade 0 (MHE) Grade 1

Grade 2

Grade 3

Grade 4

The degree of cognitive dysfunction, personality and abnormal behavior No change in behavior and personality, but minor abnormalities on psychological or intelligence tests Patients with mild personality changes or behavioral disorders, such as euphoria or depressed state; urinary and fecal incontinence accurate response, but articulation is slow and unclear; impaired concentration or sleep cycle inversion Patients with sleep disorders and mental disorders, slow response, disorientation, decreased computing power and understanding, unclear speech, writing disorders, behavioral Abnormalities, obvious sleep time inversion, even hallucinations, fear and manic. Involuntary movement or movement disorders Patients with lethargy and insanity and can be awakened and answer questions, but often be unconscious or illusory Patients with loss of consciousness and cannot be awakened; Shallow coma patients with pain response; Deep coma

Signs of the nervous system No

Changes in EEG Normal α rhythm

Asterixis

α and θ rhythms

Tendon hyperreflexia, increased muscle tone, positive sign of ankle clonus, Babinski and asterixis

Persistent θ, occasional δ

Tendon hyperreflexia, increased muscle tone, positive sign of ankle clonus, and asterixis Shallow coma patients with positive sign of ankle clonus, tendon hyperreflexia and increased muscle tone Deep coma patients with no response to stimulation

Transient complex waves with spike and slow waves Persistent δ, many complex wave with spike and slow waves

continuous progression of the disease and should be viewed as a continuum of a wide range of neuropsychiatric abnormalities, rather than isolated clinical stages. According to the traditional West Haven criteria diagnosing grade 1 HE is based on clinical signs and physician assessments, resulting in diagnostic criteria confusion [82]. To facilitate international communication and guide clinical practice, in 2011 the International Society of Hepatic Encephalopathy and Nitrogen Metabolism (ISHEN) proposed a new five-grade method for HE: HE (West Haven Level 0), covert HE (West Haven Level 1 HE), mild overt HE (West Haven Level 2 HE), severe overt HE (West Haven Level 3 HE) and coma overt HE (West Haven Level 4 HE) [58] (Fig. 2.4). Covert HE is diagnosed by a variety of neuropsychological and intelligence tests; the evaluation of overt HE widely uses the modified West Haven semi-quantitative grading table for the analysis of patients with neuropsychiatric state (Table  2.3), the Glasgow coma scale for the analysis of the degree of

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Fig. 2.4  HE classification of ammonia metabolism association and West Haven

Mild HE

Normal West Haven Grade 0

HE Grade 1

Covert HE West Haven Grade 1

HE Grade 2

Mild overt HE West Haven Grade 2

HE Grade 3

Severe overt HE West Haven Grade 3

HE Grade 4

Coma overt HE West Haven Grade 4

Table 2.3  West Haven semi-quantitative classification Grade 0 Minimal HE

1

2

3

4

Symptom No abnormality seen Abnormal results on neuropsychological tests Normal examination Abnormal results on psychometric tests Trivial lack of awareness Euphoria or anxiety Shortened attention span Impaired performance of addition Lethargy or apathy Minimal disorientation in time or place Subtle personality change Inappropriate behavior Impaired performance of subtraction Somnolence to semi-stupor Gross disorientation

Coma (does not respond to verbal or noxious stimuli)

Operation definition Two or more tests PHES > 2SD ICT > 5 CF ≤ 39 Hz Respond seven animal names within 120 s Normally oriented in time and space

Disoriented for time (at least three of the following are wrong: day, week, month, season, or year) Normally oriented in space Disoriented for space (at least three of the following wrongly reported: country, state/ region, city or place) Disoriented for time GCS 8–14 GCS