Tumor Necrosis Factor

CELL BIOLOGY RESEARCH PROGRESS SERIES

TUMOR NECROSIS FACTOR No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

CELL BIOLOGY RESEARCH PROGRESS SERIES Handbook of Cell Proliferation Andre P. Briggs and Jacob A. Coburn (Editors) 2009. ISBN: 978-1-60741-105-5 Cell Determination During Hematopoiesis Geoffrey Brown and Rhodri Ceredig) (Editors) 2009. ISBN: 978-1-60741-733-0 Tumor Necrosis Factor Toma P. Rossard (Editor) 2009. ISBN: 978-1-60741-708-8

CELL BIOLOGY RESEARCH PROGRESS SERIES

TUMOR NECROSIS FACTOR

TOMA P. ROSSARD EDITOR

Nova Biomedical Books New York

Copyright © 2009 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Library of Congress Cataloging-in-Publication Data Tumor necrosis factor / [edited by] Toma P. Rossard. p. ; cm. Includes bibliographic references and index. ISBN 978-1-61668-276-7 (E-Book) 1. Tumor necrosis factor. 2. Tumor necrosis factor--Therapeutic use. I. Rossard, Toma P. [DNLM: 1. Tumor Necrosis Factors--physiology. 2. Receptors, Tumor Necrosis Factor-therapeutic use. 3. Tumor Necrosis Factors--antagonists & inhibitors. 4. Tumor Necrosis Factors-genetics. QW 630 T9241 2009] QR185.8.T84T85 2009 616.07'9--dc22 2009024632 Published by Nova Science Publishers, Inc.  New York

Contents

Preface

vii

Chapter I

Tumor Necrosis Factor (TNF)–From Bench to Bed Side Indrajit Chowdhury and Ganapathy K. Bhat

Chapter II

Tumour Necrosis Factor Alpha Neutralization in the Medical Management of Crohn’s Disease Neil Gerard Docherty and P. Ronan O’Connell

Chapter III

Tumor Necrosis Factor-α and Biliary Tract Diseases Hiroko Ikeda, Kenichi Harada, Motoko Sasaki, Yasunori Sato and Yasuni Nakanuma

Chapter IV

Tumoricidal Effect of Tumor Necrosis Factor-Alpha in Isolated Limb Perfusion Treatment of Human Cancers Chandrakala Menon and Douglas L. Fraker

Chapter V

Tumor Necrosis Factor Antagonist Induced Psoriatic Skin Lesions Angelique N. Collamer and Daniel F. Battafarano

Chapter VI

A New Promising Role of Melatonin in Promoting Tumor Necrosis Factor Toxicity in Cancer Cells Rosa M. Sainz, Juan C. Mayo, Dun-Xian Tan and Russel J. Reiter

Chapter VII

Circulating TNF- α and Oral Health Condition in Elderly Japanese Hideaki Hayashida, Toshiyuki Saito, Reiko Furugen, Noboru Yamaguchi, Akihiro Yoshihara, Hiroshi Ogawa and Hideo Miyazaki

Chapter VIII

Tumor Necrotic Factor in T Cell Disorder: Hypothesis and Proof of Idea Viroj Wiwanitkit

Chapter IX

Tumor Necrotic Factor in Malaria Viroj Wiwanitkit

1

49 79

95 133

143 151

159 165

vi Chapter X

Chapter XI

Index

Contents Tumor Necrosis with Special Reference to Autophagic Cell Death (Self-Cannibalism) and Xeno- Cannibalism in Gastric Cancer: Our Experience and Review of the Literature Rosario A. Caruso Tumor Necrosis Factor and Carcinoma by Hepatitis B and C Virus Infection Kazuya Shirato and Tetsuya Mizutani

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205 221

Preface Tumor Necrosis Factor (TNF) is a member of a superfamily of proteins, each with 157 amino acids, which induce necrosis (death) of tumor cells and possess a wide range of proinflammatory actions. Tumor necrosis factor is a multifunctional cytokine with effects on lipid metabolism, coagulation, insulin resistance, and the function of endothelial cells lining blood vessels. Blocking the action of TNF has been shown to be beneficial in reducing the inflammation in inflammatory diseases such as Crohn's disease and rheumatoid arthritis. Inappropriate production of TNF or sustained activation of TNF signaling has been implicated in the pathogenesis of a wide spectrum of human diseases that include cancer, osteoporosis, sepsis, diabetes, and autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. Extensive research within the last two decades has shown that TNF can be a potential therapuetic agent in various diseases. This new important book gathers the latest research from around the globe in this field. Chapter 1 - The tumor necrosis factor (TNF) ligand is a family member of cytokines which transduces signal through two specific receptors, TNF-receptor type I (TNF-R1, CD120a, p55/60) and TNF-receptor type II (TNF-R2, CD120b, p75/80). Signaling through TNF-R1 is extremely complex, leading to both cell death and survival signals by the phosphorylation of number of protein kinases and by the activation of transcription factors. Role of TNF-R2 phosphorylation with signaling properties are less understood than TNF-R1. Activation of TNF signaling pathway controls cell survival, death, proliferation and differentiation that orchestrate the development, organization and homeostasis of lymphoid, mammary, neuronal and ectodermal tissues. Inappropriate production of TNF or sustained activation of TNF signaling has been implicated in the pathogenesis of a wide spectrum of human diseases that include cancer, osteoporosis, sepsis, diabetes, and autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. Extensive research within the last two decades has shown that TNF can be a potential therapeutic agent in various diseases. The recent continued expansion of the novel anti-TNF therapeutic agents has seen major improvements in the treatment of some inflammation-based human diseases including rheumatoid arthritis and Crohn’s disease, with other conditions currently being trialed using anti-TNF agents. In this review, author highlight author current knowledge of TNF and its signaling which plays a major role in cellular survival and death, with relation to therapeutic use of anti-TNF.

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Chapter 2 - Crohn’s disease (CD) is a chronic and debilitating inflammatory condition affecting principally the small intestine and colon. Tumour necrosis factor alpha (TNF-α) plays a key role in the pathophysiology of CD, most prominently via its role in intestinal macrophage and T-lymphocyte activation and through its effects on intestinal permeability and fibroblast mediated extracellular matrix remodeling. Polymorphisms in TNF-α receptor genes have also been implicated as disease modifiers in inflammatory bowel disease. In 1998, the anti-TNF-α monoclonal antibody infliximab was approved by the U.S Federal Drugs Administration (F.D.A) for use in the medical treatment of fistulae in CD. The drug is now licensed for induction and maintenance of remission of disease. More recently the humanized anti-TNF-α antibody adalimumab and the pegylated human antiTNF-α antibody fragment certolizumab have been licensed for use in CD. Trials of other anti-TNF-α agents in the treatment of CD, notably the CDP571 antibody, and the soluble type I and type II TNF-α receptors, etanercept and onercept, have failed to show sufficient efficacy to warrant FDA approval. In this chapter a background to the pathophysiology of CD is provided, the role of TNF-α as a key mediator of inflammation in CD is described and the process by which TNF-α neutralization has come to represent an important therapeutic tool in the medical management of CD is discussed. Finally, some of the concerns regarding the safety of TNF-α neutralizing therapy are reviewed. Chapter 3 - Tumor necrosis factor (TNF)-α is a pleiotropic cytokine involved in the pathophysiology of a variety of human diseases. This study reviews the roles of TNF-α in the pathogenesis of inflammatory biliary diseases such as primary biliary cirrhosis (PBC) and hepatolithiasis and also of cholangiocaricnoma. In PBC characterized by chronic destructive cholangitis of small intrahepatic bile ducts with their progressive loss, TNF-α which is produced by biliary epithelial cells and periductal inflammatory cells, induces the secretion of cytokines from biliary epithelial cells. TNF-α is also involved in the progressive bile duct loss via apoptosis and cellular senescence. Pathogen-associated molecular patterns which might originate from the gastrointestinal tract are involved in the secretion of TNF-α via interaction with TLR4. In hepatolithiasis, which is characterized by long-standing inflammation of intrahepatic large bile ducts with calculi formation, TNF-α is involved in the pathogenesis of intestinal metaplasia which is frequently seen in this disease. The expression of MUC2, an intestinal type mucin, and CDX-2, an intestine-specific transcription factor, characterize the intestinal metaplasia of the biliary mucosa. Human and cultural studies of biliary epithelial cells from polycystic kidney (PCK) rats which show chronic cholangitis with intestinal metaplasia, suggest that TNF-α is involved in the induction of CDX2 followed by an aberrant expression of MUC2, thereby playing a role in the pathogenesis of bile duct lesions of hepatolithiasis. TNF-α may also play an important role in the development and progression of cholangiocarcinoma. Matrix metalloproteinase-9 (MMP-9), an important enzyme in tumor invasion and metastasis, and cyclooxygenase-2 (COX-2) are frequently expressed in parallel in cholangiocarcinoma. In in vitro studies using cholangiocarcinoma cell line, a TNF-α/TNF-receptor 1 (TNF-R1) interaction induces COX-2 overexpression, MMP-9 production and activation, and increases the migration of cholangiocarcinoma cells. In conclusion, TNF-α plays a central role in the pathophysiology of inflammatory and

Preface

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neoplastic biliary diseases, and could be a target molecule in novel therapeutic strategic approaches for these biliary diseases. Chapter 4 - Tumor necrosis factor-alpha (TNF-alpha), first named for its anti-tumor property, is a pleiotropic cytokine implicated in many physiological and pathological reactions including cell death, cell survival, immune response, and inflammation. The human TNF-alpha gene is located on the short arm of chromosome 6. The gene was cloned in 1984 and recombinant human TNF-alpha (rhTNF-alpha) was successfully produced in Escherichia coli but could not be used as an effective systemic anticancer agent because of its dose-limiting toxicity. It is currently effectively used in regional therapy for melanomas and soft-tissue sarcomas using isolated limb perfusion (ILP), a surgical procedure which circulates high-dose therapeutics within an extremity (arms and legs), thus limiting systemic toxicity while avoiding the organs of metabolism. Objective response rates of 90-100% with 65-90% complete response rates have been reported in patients with in-transit metastases from melanoma. Overall response rates with soft-tissue sarcoma range from 58%-82% with 15-30% complete responses. These results, achieved with a single 90-minute ILP using rhTNF-alpha and melphalan, are significantly better than the best results achieved with combination systemic chemotherapy of 30% overall response rates and 0-5% complete responses for melanoma and 15-20% overall response rates with essentially no complete responses for sarcoma. The precise mechanism underlying the remarkable anti-tumor effect of rhTNF-alpha in conjunction with melphalan against solid tumors using ILP has not been fully elucidated. During ILP treatment, all of the normal tissues immediately surrounding the tumor i.e. skin, subcutaneous fat, muscle, bone, and peripheral nerve are subject to the same drug concentration as the tumors present in the extremity. Remarkably however, the combination of rhTNF-alpha and melphalan affects only tumor and not normal tissue and is known to work synergistically. ILP with melphalan-alone has activity for melanoma when the tumor burden (size and number of nodules) is limited. For large tumors or extensive disease, the response to melphalan-alone ILP is limited, but is significantly improved by adding rhTNF-alpha to the treatment regimen. It is widely accepted that melphalan, a nitrogen-mustard, forms damaging DNA adducts within dividing tumor cells. This chapter critically reviews recent data that provide insights into plausible mechanisms by which rhTNF-alpha asserts its anti-tumor activity. Chapter 5 - As experience with TNF antagonist therapy increases so does the recognition and reporting of unexpected and unusual adverse reactions. Other reported paradoxical autoimmune reactions observed with TNF antagonist therapies include the development of antinuclear and anti-double-stranded DNA antibodies, as well as drug induced lupus, cutaneous and systemic vasculitis, and the induction of demyelinating diseases and inflammatory bowel disease. The perplexing evidence that a medication class used to treat a disease such as psoriasis may exacerbate the condition, or induce psoriasis in patients with other autoimmune conditions, underscores that the pathophysiology of autoimmune diseases and their treatments is incompletely understood. Further observations and investigations are necessary to better elucidate this phenomenon. Chapter 6 - Melatonin, a neuroindole mainly produced by the pineal gland, has antioxidant, anti-proliferative and anti-inflammatory properties which can be responsible to

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its beneficial effects on human health. It is well known that melatonin serum levels are reduced in cancer patients since late 80’ies and its role on the growth of cancer cells has been clearly demonstrated. Melatonin directly inhibits the growth of several cell types from diverse embryological origins including breast, prostate, melanoma, lung, kidney or brain; additionally, melatonin is a modulator of the immune function. Physiologically, melatonin administration results in a functional enhancement of immune cells which might play a key role against cancer cells in vivo. In fact, there is a direct crosstalk between the pineal gland and the immune system in several ways. Previous data suggest that the increase in circulating tumor necrosis factor-alpha (TNFα), after a defense response transiently blocks nocturnal melatonin production. In fact, the transcription of arylalkylamine-N-acetyltransferase, the rate-limiting enzyme in melatonin biosynthesis, together with the synthesis of the melatonin precursor N-acetylserotonin, was inhibited by TNFα. It is also clear that cytokine production caused by infection or inflammation, including TNFα, is reduced by melatonin pretreatment. The remarkable ability of TNFα to inhibit the growth of malignant tumor cells is unfortunately limited by its systemic toxicity. New strategies are being tested in order to reduce TNFα toxicity without losing its antitumor efficiency. On the other hand TNFα, induced by a wide range of pathogenic stimuli induces other inflammatory mediators and proteases that act as tumor promoters. The role of TNF in cancer has been linked to all steps of carcinogenesis including carcinogenesis, cellular transformation, promotion, survival, proliferation, angiogenesis and metastasis and how the cytokine works in this intricate link is actually a matter of debate. Since melatonin has been claimed to prevent the toxicity of several anticancer drugs and more recently, to enhance the toxicity of TNFα in cancer cells, author will discuss here the innovative idea of the employment of melatonin in combination with TNFα in cancer treatments. The possible use of melatonin in preventing the toxicity of TNFα without losing its antitumor properties as well as its capacity to promote ability to kill cancer cells especially resistant to TNFα treatment is an idea which needs to be deeply explored. Chapter 7 - The oral cavity, especially area around teeth, is a hotbed for bacteria, which compose a biofilm that becomes a significant source of continuous subclinical infection. Tumor necrosis factor-α (TNF-α), which is secreted from adipose tissue and developing type 2 diabetes, is also known to be secreted in periodontal inflammation. Moreover, there is a two-way relationship between diabetes and periodontal disease. The author hypothesized that the circulating level of TNF-α is associated with the oral health condition, including periodontal disease. The purpose of this study was to assess the relationship between serum levels of TNF-α and the oral health condition in elderly Japanese people. Chapter 8 - Tumor necrotic factor is a specific cytokine resulting from the cellular immunity. In the process of cellular immunity, lymphocytes, especially the T lymphocyte, acts mainly in an immune response. The author hereby uses the systomics approach to formulate a hypothesis on tumor necrotic factor expression in important T cell disorders. In addition, further proof of the idea was done by matching with previously published reports on the corresponding proposed items. The models of testing include three important T cell disorders, human immunodeficiency virus (HIV) infection, T cell leukemia and Hodgkin’s lymphoma.

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Chapter 9 - Malaria is an important tropical-mosquito-borne blood infection. Malaria has a complex pathogenesis. Tumor necrotic factor, a cytokine, is widely mentioned for its correlation to malaria in various aspects. In this article, the author will briefly review the important reports on tumor necrotic factor in malaria. Chapter 10 - Cell death is a field that has attracted much attention in recent years, leading to several new and important insights in cell biology, development, and pathology, but the recruitment of many new researchers to the field has led to some confusion in terms. According to the Recommendations of the Nomenclature Committee on Cell Death, the definition of cell death must be based on precise terms of the parameters that describe the presumed cell death pathway involved. Since the precise characterization of biochemical checkpoints controlling cell death is still awaiting, different cell death types are defined by morphological criteria. Four morphological entities, without a clear reference to precise biochemical mechanism, have been described: apoptosis, autophagy, necrosis and mitotic catastrophe. The most common and well-defined form of programmed cell death is apoptosis, which is a physiological “cell-suicide” programme that is essential for embryonic development, immune system function and the maintenance of tissue homeostasis in multicellular organisms. Apoptosis is characterized by the activation of a specific family of cysteine proteases, the caspases, followed by a series of morphological changes including cellular and nuclear shrinkage (pyknosis), chromatin condensation and nuclear fragmentation (karyorhexis) with formation of apoptotic bodies. Tumor growth involves two essential deviations from the normal state including the induction of proliferative stimuli, and simultaneous suppression of potentially compensatory cell death. It is well recognized that apoptosis is impaired in many cancers by mutations in genes such as p53, but nonapoptotic mechanisms have been largely overlooked in studies of cancer causation, progression and therapy. It has recently suggested that the development of an invasive cancer involves a progressive switch from predominantly apoptotic to necrotic tumor cell death. This disordered cell death is supported by the frequent observation in a large number of common tumors that the presence of microscopic necrosis predicts a poor prognosis. This review summarizes the recent discoveries on cell death and its role in neoplasms. In particular, author experience on the ultrastructural features of autophagic cell death and xenocannibalism in gastric carcinomas is reported. Profound knowledge of the morphology of cell death may be useful for inform and drive the development of more effective biologic therapies for patients with cancer. Chapter 11 - Hepatitis B virus (HBV) and hepatitis C virus (HCV) are global public health problems. The clinical courses of both HBV and HCV infection vary from acute hepatitis to chronic persistent infection that may progress to cirrhosis and carcinoma. Acute inflammation is a defense response and chronic inflammation can lead to cancer. Several proinflammatory gene products have been identified in both HBV- and HCV-infected patients. The expression of these genes is mainly regulated by the transcription factor NFB, which is constitutively active in most tumors and is induced by tumor promoters and carcinogenic viral proteins. Anti-inflammatory agents that suppress NF- B may be useful in both the prevention and treatment of cancer. Tumor necrosis factor alpha (TNF ) is thought to be an important factor underlying the mechanisms of action of these types of viral hepatitis and carcinoma because TNF plays an important role in the host immune response to HBV

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and HCV infection. Recent studies indicated that TNF promoter polymorphisms are significantly associated with viral clearance. Neutralization of TNF (anti-TNF therapy) has been shown to be associated with activation of HBV infection, but not HCV infection. However, TNF has been reported to induce clearance of HBV. One of the inhibitory mechanisms of anti-TNF therapy is that TNF blocks HBV replication by promoting destabilization of viral nucleocapsids. HCV proteins are able to trigger production of TNF and modulate nuclear factor kappa B activation and apoptosis stimulated by this cytokine. This review highlights the importance of TNF production for hepatitis and carcinoma by HBV and HCV infection.

In: Tumor Necrosis Factor Editor: Toma P. Rossard

ISBN: 978-1-60741-708-8 © 2009 Nova Science Publishers, Inc.

Chapter I

Tumor Necrosis Factor (TNF)–From Bench to Bed Side Indrajit Chowdhury and *Ganapathy K. Bhat Department of Obstetrics and Gynecology, Morehouse School of Medicine, Atlanta, Georgia 30310, USA

Abstract The tumor necrosis factor (TNF) ligand is a family member of cytokines which transduces signal through two specific receptors, TNF-receptor type I (TNF-R1, CD120a, p55/60) and TNF-receptor type II (TNF-R2, CD120b, p75/80). Signaling through TNFR1 is extremely complex, leading to both cell death and survival signals by the phosphorylation of number of protein kinases and by the activation of transcription factors. Role of TNF-R2 phosphorylation with signaling properties are less understood than TNF-R1. Activation of TNF signaling pathway controls cell survival, death, proliferation and differentiation that orchestrate the development, organization and homeostasis of lymphoid, mammary, neuronal and ectodermal tissues. Inappropriate production of TNF or sustained activation of TNF signaling has been implicated in the pathogenesis of a wide spectrum of human diseases that include cancer, osteoporosis, sepsis, diabetes, and autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. Extensive research within the last two decades has shown that TNF can be a potential therapeutic agent in various diseases. The recent continued expansion of the novel anti-TNF therapeutic agents has seen major improvements in the treatment of some inflammation-based human diseases including rheumatoid arthritis and Crohn’s disease, with other conditions currently being trialed using anti-TNF agents. In this review, we highlight our current knowledge of TNF and its signaling which plays a major role in cellular survival and death, with relation to therapeutic use of anti-TNF.

Keywords: tumor necrosis factor, TNF receptor, lymphotoxin, apoptosis. *

Correspondence: [email protected]

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Indrajit Chowdhury and Ganapathy K. Bhat

Abbreviations ARE, adenine-uracil–rich elements; CCL, chemokine (C-C motif) ligand; CRP, Creactive protein; CXCL, chemokine (C-X-C motif) ligand; ICAM-1, intercellular adhesion molecule; IL, interleukin; LPS, lipopolysaccharide; LT, lymphotoxin (LTα, LTβ); mAbs, monoclonal antibodies; MCP-1, macrophage chemoattractant protein-1; MMP, matrix metalloproteinase; NF-κB, nuclear factor kappa-B; NK, natural killer; RA, rheumatoid arthritis; PI, package insert; RANKL, receptor activator of nuclear factor kappa-B ligand; RANTES, regulated on activation, normal T cell expressed and secreted; SPPL, signal peptide peptidase-like proteases; THD, TNF homology domain; sTNF, soluble tumor necrosis factor; TACE, tumor necrosis factor-alpha–converting enzyme; THP-1, human acute monocytic leukemia cell line; TLR, toll-like receptor; TNF, tumor necrosis factor; TNFβ, lymphotoxin; tmTNF, transmembrane tumor necrosis factor; TNFR, tumor necrosis factor receptor; TNF-R1, TNF-receptor type I ; TNF-R2, TNF-receptor type 2 ; TNFSF, TNF ligand superfamily; ECD, N-terminal extracellular domain; TMD, transmembrane domain; ICD, Cterminus intracellular domain; CRD- cysteine-rich domain; DD, death domain; DR, death receptor; TNFRSF, TNF-receptor super family; PKC, protein kinase C; PKA, protein kinase A; TSF, thyroid stimulating factor; sTNFR, soluble TNF receptor; PLAD, extracellular Nterminal pre-ligand association domain; SODD, silencer of death domain; TRADD, TNFRassociated death domain protein; RIP-1, serine/threonine kinase receptor interacting protein1; MAPK, mitogen activated protein kinase; IKK, inhibitor of κB; FADD, Fas-associated DD protein; TIM, TRAF-interacting motifs; ASK-1, apoptosis-signaling kinase-1; ERK, extracellular signal-related kinase; PI3K, phosphotidyl-inositol-3 kinase; JNK, c-Jun-Nterminal kinase; CRH, corticotrophin releasing hormone; IRS-1, insulin receptor substrate-1; INF-γ, interferon-γ; XIAP, X-linked inhibitor of apoptosis; XAF1, XIAP associated factor 1; PCD, program cell death; CAD, caspases-activated DNase; FLIP, FLICE inhibitory protein 9IFLICE); Bcl2, B-cell lymphoma 2; SNP, single-nucleotide polymorphisms; PCR, polymerase chain reaction; MHC, major histocompatibility complex; TRAF, tumor necrosis factor receptor-associated factor; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling; UTR, untranslated region; VCAM-1, vascular cell adhesion molecule; VEGF, vascular endothelial growth factor.

Introduction Tumor necrosis factor (TNF) is a pleiotropic cytokine, formally known as tumor necrosis factor-alpha, cachexin or cachectin. It has the ability to induce tumor cell necrosis, hence the name. In 1968, Dr. Gale A Granger from the University of California discovered this family member and reported it as a cytotoxic factor produced by lymphocytes and named it as lymphotoxin (LT) (Allcock et al., 2004). Credit for this discovery is also shared by Dr. Nancy H. Ruddle from Yale University, who reported the same activity in a series of published work in the same year. In 1975, Dr. Lloyd Old and his colleagues from Memorial SloanKettering Cancer Center reported another cytotoxic factor produced by macrophages, and named it tumor necrosis factor (TNF) (Haranaka et al., 1986; Aoki et al., 2006). Both (LT

Tumor Necrosis Factor (TNF)–From Bench to Bed Side

3

and TNF) factors are described based on their ability to kill mouse fibrosarcoma L-929 cells. Later, TNF was isolated and identified as TNF-alpha (TNFα) from the serum of mice treated with bacterial endotoxin, which was shown to replicate the ability of endotoxin to induce hemorrhagic necrosis and methylcholanthrene-induced sarcomas (Carswell et al., 1975; Pfeffer, 2003). Subsequently, TNFα has been identified as an active component of “Coley’s toxin” from a crude bacterial filtrate of Coley’s mixture (Wiemann and Starnes, 1994). The discovery and initial characterization of TNFα did not provide any evidence to the role of TNF in cell physiology. It was only cloning of the TNFα gene in 1984 led to the era of clinical experimentation for cancer therapy (Gray et al., 1984; Shirai et al., 1985). In 1985, Bruce A. Beutler and Anthony Cerami renamed cachectin as TNFα, a hormone that induces cachexia (Balpsso et al., 2005). The cDNAs encoding LT and TNFα were revealed to be similar and binding of TNFα to its receptor by displacement of LT confirmed the functional homology between these two factors (Balding et al., 2003). The sequential and functional homology of TNFα and LT led to their renaming as TNF and LTα, respectively. TNF is a general term that includes transmembrane TNF (tmTNF) and soluble TNF (sTNF) in the context of tissues/cells and fluids respectively. LT is a general term for the family of lymphotoxins. Today, a pubmed search of the word “TNF” reveals several thousand (>71,300) publications related to TNF in various functional roles. Based on the current research in TNF family, we reviewed the structure and properties of TNF and activation of the TNF-cascade signaling system with special emphasis on physiological role in relation to cell survival, death and different diseases.

TNF The human TNF gene (TNFα) was cloned in 1984 and maps within the major histocompatibility complex to chromosome 6p21.3 adjacent to the genes encoding LTα and LTβ (Pennica et al., 1984; Gray et al., 1984; Hajeer et al., 2000), (Figure 1). Together these comprise the TNF locus. At least five microsatellite markers have been identified in the TNF locus with multiple alleles allowing the precise definition of haplotypes (Udalova et al., 1993). It spans about 3 kb with 4 exons. The last exon encodes for more than 80% of the secreted TNF protein (Nedwin et al., 1985; Beutler et al., 1986). The 3' UTR of TNFα contains AU-rich element (ARE). TNFα is mainly produced in the activated macrophages, monocytes, T-cell and natural killer (NK) cell lymphocytes although a lower expression is known in other cells including mast cells, endothelial cells, cardiac myocytes, adipose tissue, fibroblasts, smooth muscle cells, astrocytes, Kupffer cells, keratinocytes and in various tumor cells (Fiers et al., 1996; Oppenheim and Feldmann, 2000; Thomson and Lotze, 2003). A large amount of TNFα is released in response to lipopolysaccharide, bacterial products, and interleukin-1 (IL-1). The TNF protein is synthesized as a 26 kDa (212 amino acids) long type II transmembrane (membrane-bound) pro-peptide (pro-TNF) as stable homotrimers (Kriegler et al., 1988; Tang and Hung, 1996). The structure of TNF is characterized by an intracellular N-terminus and extracellular C terminus with conserved C-terminal domain named the “TNF homology domain” (THD). The THD is a 150 amino acid long sequence containing a

Indrajit Chowdhury and Ganapathy K. Bhat

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conserved framework of aromatic and hydrophobic residues. This trimeric domain is responsible for receptor binding.

Figure 1. A. Schematic representation of the location of tumor necrosis factor (TNF) cluster in the major histocompatibility complex on chromosome 6. B. Schematic representation of gene structure of TNFα, LTβ and LTα with direction of transcription followed by single nucleotide polymorphism.

Table 1. Human TNF ligand superfamily (TNFSF) Nomenclature (ligand symbol) TNFSF1A/ TNFSF2

TNFSF1B TNFSF1 TNFSF3

TNFSF4

TNFSF5

TNFSF6

Gene names

Gene location

mRNA accession number

TNF TNF-α Cachectin Necrosin Cytotoxin DIF LTα TNF-β LT LTβ TNFC LTB OX40-L gp34 TXGP-1 CD252 CD40-L CD154 Gp39 TRAP Fas-L CD95L CD178 ApoI L

6p21.3

NM_000594

6p21.3

NM_000595

6p21.3

NM_002341

1q25

NM_003326

Xq26

NM_000074

1q23

NM_000639

Tumor Necrosis Factor (TNF)–From Bench to Bed Side TNFSF7

TNFSF8 TNFSF9 TNFSF10

TNFSF11

TNFSF12

TNFSF12-13 TNFSF13

TNFSF13B

TNFSF14

TNFSF15

TNFSF18

CD27-L CD70 Ki-24 CD30-L CD153 4-IBB-L CDw137L TRAIL Apo-2L TL2 CD253 RANK-L TRANCE OPGL ODF CD254 TWEAK Apo-3L CD255 TWE-PRIL APRIL TALL2 TRDL-1 ZTNF2 TNFSF13A CD256 BAFF BLYS TALL1 zTNF4 THANK DTL CD257 LIGHT LTg TR2 HVEM-L CD258 TL1A TL1 VEGI GITRL AITRL TL6 EDA1 EDA2

19p13

NM_001252

9q33

NM_001244

19p13.3

NM_003811

3q26

NM_003810

13q14

NM_003701

17p13

NM_003809

17p13.1 17p13.1

NM_003808

13q32-q34

NM-006573

19p13.3

NM_003807

9q33

NM_005118

1q23

NM_005092

Xq12-q13.1 Xq12-q13.1

NM_001399 AF061189

5

Since its initial discovery, researchers have shown the existence of 18 TNF ligand family genes encoding 20 type II transmembrane proteins, which are classified under TNF ligand superfamily (TNFSF). These different members of TNFSF are located to different chromosomes (Zhang et al., 2004). TNFSF includes TNFα, lymphotoxin-α (LT-α, TNF-β), fibroblast associated surface ligand (FasL, CD95L, CD178), CD30L, CD40L and LIGHT (Locksley et al., 2001) (Table 1). The comparative amino acid alignment studies demonstrate ~15-20% identity among the TNF family members. To date, atomic-level structures are available for the THD of TNF, LTα, CD40L and TRAIL (Eck and Sprang, 1989; Jones et al., 1989; Banner et al., 1993; Karpusas et al., 1995; Hymowitz et al., 1999; Mongkolsapaya et

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Indrajit Chowdhury and Ganapathy K. Bhat

al., 1999; Cha et al., 2000). THD is a compact bell-shaped truncated homotrimer pyramid, formed by three identical monomers which assemble around a 3-fold axis with ~60 0A in height. This β-sandwich structure is formed by two stacks of β-pleated sheets, each containing five anti-parallel β-strands that adopt a classical ‘jelly-roll’ topology, a typical structure for the TNF family, but shows structural homology with several viral coat proteins (Eck and Sprang, 1989). This truncated pyramid structure has also variable loops protruding out of the compact core of conserved anti-parallel β-strand. The inner sheet (strands designated as A, A’, H, C and F) is involved in trimer contacts, and the outer sheet (strands designated as B, B’, D, E and G) is exposed at the surface. TNF contains a single disulphide bridge linking the CD and EF loops (Bodmer et al., 2002). A single cysteine residue in the EF loop is involved in the coordination of a Zn (II) ion, with each monomer contributing to one coordination position while the fourth coordination position is occupied by an internal solvent molecule or a chloride counter-ion. This metal-binding site is unique in the TNF family, and affects its stability and bioactivity (Bodmer et al., 2002). The incomplete Zn coordination causes the formation of partial oxidized disulfide-links which results in cellular toxicity (Lawrence et al., 2001). Structural analysis revealed that TNFα contains one disulphide bond (Aggarwal et al., 1987). TNF can act in its membrane-bound form through cell-to-cell contact. However, soluble homotrimer TNF (sTNF) form is more effective for distant action. From the membraneintegrated form, the soluble 51 kDa homotrimeric cytokine (sTNF) with a subunit molecular mass of 17 kDa (157 amino acids) is released by limited proteolysis using the metalloproteinase (Jones et al., 1989; Eck and Sprang, 1989; Eck et al., 1992; Black et al., 1997). In the cleavage of different TNF ligands, distinct metalloproteases are involved such as the ADAM (a disintegrin and metalloproteinase domain or TNF-converting enzyme, TACE, also called ADAM17) which act on TNF and RANKL ligands (Black et al., 1997; Lum et al., 1999; Bouwmeester et al., 2004), matrilysin acting on Fas ligand (FasL) (Powell et al., 1999), and members of the subtilisin-like furin family acting on BAFF, EDA, TWEAK and APRIL-members of the TNF family (Schneider et al., 1999; Chen et al., 2001). TNF expressions and its biological activities are tightly regulated at the level of gene transcription and protein processing (Han et al., 1990). TNF is barely detectable in quiescent cells. The production of TNF in macrophages can be induced by a wide variety of stimuli, including bacteria, viruses, immune complexes, cytokines (e.g., IL-1, IL-17, granulocyte macrophage colony-stimulating factor, interferon-γ), complement factors, tumor cells, irradiation, ischemia/hypoxia and trauma. Many stimuli induce TNF mRNA within 30 min, but most of the regulation of TNF expression occurs post-transcriptionally. Adenine-Uracil rich elements (ARE) and flanking sequences in the 3-untranslated region regulate the translation and degradation of TNF mRNA (Han et al., 1990). Translation of TNF mRNA results in the intracellular production of trimeric pro-TNF protein, which lacks a signal peptide and is inserted into the plasma membrane as tmTNF. The production of TNF by cells is regulated by positive and negative feedback loops initiated by TNF-induced factors. TNFα can directly induce the expression and release of several humoral factors including interleukin (IL)-10, IL-1, IL-2, interferon-γ, corticosteroids and prostanoids which in turn regulate TNFα production through negative feedback loops (Butler et al., 1989; Scales et al., 1989; Platzer et al., 1995; Caughey et al., 1997). TNF also induces some negative feedback regulators, such as IL-10, prostaglandins

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and corticosteroids that inhibit transcription of TNF mRNA. TNFα is also down regulated by the expression of TNF-receptor (TNFR) (van der Poll et al., 1995). In vitro site mutagenesis of the cysteine residues demonstrated that the disulfide bond is important for the biological function of TNFα (Narachi et al., 1987). The soluble TNF (sTNF) is an essential form for the negative regulation of TNF biological activity (Bemelmans et al., 1996). The sTNF is unstable but biologically active. The 51 kDa homotrimeric sTNF dissociates at concentrations below the nanomolar range, thereby losing its bioactivity.

TNF Receptor TNF exhibits its biological properties upon binding to its cognate membrane receptors. The high affinity cell surface receptor for TNFα was first discovered in 1985 (Aggarwal et al., 1985). By crosslinking the ligand with the receptor through reversible and irreversible crosslinkers and by immunoaffinity chromatography, the TNF receptor was isolated with an approximate molecular mass of 70 kDa (Stauber et al., 1988). In 1990, two different TNF receptor genes were cloned with a predicted molecular mass of 55-60 kDa and 75-80kDa which are referred as p60 (or p55) and p80 (or p75) respectively (Loetscher et al., 1990; Schall et al., 1990; Smith et al., 1990). The smaller (p60/p55) TNF receptor is named as TNFR1 (also known as TNFRSF1, CD120a, p55TNFR, p60) and the larger form (p80/p75) as TNFR2 (also known as TNFRSF1b, CD120b, p75TNFR, p80). Each p60 and p80 TNF receptors are encoded by a single gene. The gene for the human p60 receptor is located on chromosome 12p13 and the p80 receptor gene is on chromosome 1p36. The p60 receptor is encoded by three exons. The gene structure for human p80 receptor spans approximately 43 kbp, consists of 10 exons (ranging in length from 34 bp to 2.5 kbp) and nine introns (ranging from 343 bp to 19 kbp) (Santee and Owen-Schaub, 1996). Consensus elements for transcription factors of TNFR are present in the 5’-flanking region of the promoter which include T cell factor 1 (TCF-1), Ikaros, AP-1, CK-2, IL-6 receptor E (IL-6RE), ISRE, GAS, NFκB, and SP-1. The unusual (GATA)n and (GAA)(GGA) repeats are found within intron 1. Both TNF receptors are type I transmembrane glycoproteins consisting of an N-terminal extracellular domain (ECD), a transmembrane domain (TMD) and a C-terminal intracellular domain (ICD). The ECDs of both the receptors (TNFR1 and TNFR2) contain four wellconserved cysteine-rich domains (CRDs) (Naismith and Sprang, 1998). The amino acid sequences of ICD of the two receptors are quite dissimilar and lack any intrinsic enzymatic activity. The p60 receptor has 426 amino acid residues consisting of an ECD of 182 amino acids, a TMD of 21 amino acids and an ICD of 221 amino acids. From this, the predicted molecular mass of p60 receptor is about 47.5 kDa. Since the apparent molecular mass of the p60 receptor is between 55 and 60 kDa, the difference is due to the presence of three potential N-linked glycosylation sites in the ECD of the receptor. The ECD of p60 receptor has a net charge opposite to that of the TNF, suggesting electrostatic interaction. The p80 receptor is a 75-80 kDa glycosylated protein, and it consists of 439 amino acid residues with an ECD of 235 amino acids, a TMD of 30 amino acid residues and an ICD of 174 amino acids (Tartaglia and Goeddel, 1992; Gruss and Dower, 1995).

Indrajit Chowdhury and Ganapathy K. Bhat

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The ECDs of both p60 and p80 receptors contain four cysteine-rich domain repeats, each consisting of six cysteine residues. The ICDs of both the receptors are completely distinct. The most striking feature of ICD of the p60 receptor is a region of approximately 80 amino acid recidues near the C-terminus called the death domain (DD) (Tartaglia et al., 1993b). This region is homologous to Fas, death receptor (DR)-3, DR4, DR5 and DR6 that are implicated in cell death (Ashkenazi and Dixit, 1998). In contrast, the p80 receptor lacks a DD but contains a serine-rich region that undergoes phosphorylation in a ligand-independent manner (Pennica et al., 1992; Darnay et al., 1994a; Beyaert et al., 1995). Table 2. Human TNF receptor superfamily (TNFRSF) Nomenclature (symbol) TNFRSF1A

TNFRSF1B

TNFRSF3

TNFRSF4

TNFRSF5 TNFRSF6

TNFRSF6B

TNFRSF7

Gene names

Gene location

Adaptor proteins

Ligands

12p13.2-p13.31

mRNA accession # NM_001065

TNFR-1 TNFR type 1 CD120a TNFAR P55TNFR TNFR60 P55-60 TNFR2 TNFR type 2 CD120b TNFR80 P75TNFR TNFBR P75-80 TNFR III LTβR TNFCR LTBR TNFR-RP TNFR2-RP CD18 OX40 ACT35 TXGP1L CD134 CD40 Fas CD95 Apo-1 APT1 TNFRSF6A DcR3 TR6 M68 CD27 S152 Tp55 T14

TRADD

TNFSF1 TNFSF2

1p36.3-p36.2

NM_001066

TRAFF1,2,5

TNFSF1 TNFSF2

12p13

NM_002342

TRAFF3,4,5

TNFSF3

1p36

NM_003327

TRAFF1,2,3,5

TNFSF4

20q12-q13.2 10q24.1

NM_001250 NM_000043

TRAFF1,2,3,5,6 FADD

TNFSF5 TNFSF6

20q13

NM_003823

12p13

NM_001242

TRAF2 SIVA

TNFSF14 TNFSF15 TNFSF6 TNFSF7

Tumor Necrosis Factor (TNF)–From Bench to Bed Side TNFRSF8 TNFRSF9

TNFRSF10A

TNFRSF10B

TNFRSF10C

TNFRSF10D

TNFRSF11A

TNFRSF11B

TNFRSF12

TNFRSF12A

TNFRSF12L TNFRSF13B TNFRSF13C

CD30 Ki-1 4-IBB CDw137 ILA TRAIL-R1 DR4 Apo-2 CD261 DR5 TRAIL-R2 KILLER CD262 TRICK2A TRICKB DcR1 TRAIL-R3 LIT TRID CD263 DcR2 TRAIL-R4 TRUNDD CD264

RANK ODFR TRANCE-R CD265 OPG TR1 OCIF APO-3 DR3 TRAMP TRS WSL-1 LARD DDR3 WSL-LR TWEAK-R Fn14 FGF-inducible 14 CD266 DR3L TACI CD267 BAFF-R CD268 BR3

9

1p36

NM_001243

TRAF1,2,3,5

TNFSF8

1p36

NM_001561

TRAF1,2,3

TNFSF9

8p21

NM_003844

FADD

TNFSF10A

8p22-p21

NM_003842

FADD

TNFSF10B

8p22-p21

NM_003841

TNFSF10C

8p21

NM_003840

TNFSF10D

18q22.1

NM_003839

8q24

NM_002546

1p36.3

NM_003790

TRADD

TNFSF15 TNFSF12

16p13.3

NM_016639

TRAF1,2,3,5

TNFSF12

1p36.2 17p11.2

NM_012452

TRAF2,5,6

TNFSF13B

22q13.1-q13.31

NM_052945

TRAF3

TNFSF13B

TRAF1,2,3,5,6

TNFSF11

TNFSF11

Indrajit Chowdhury and Ganapathy K. Bhat

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Table 2. (Continued) TNFRSF14

LIGHT-R TR2 HVEM HVEA ATAR

1p36.3-p36.2

NM_003820

TRAF1,2,3,5

TNFSF14 TNFSF1 BTLA

TNFRSF16

p75NGFR NGF-R NTR CD271 P75NTR BCMA BCM TNFRSF13 TNFRSF13a CD269 GITR AITR TROY TAJ TAJ-α TRADE RELT EDAR DR6 Death Receptor 6 SOBa Tnfrh2 Tnfrsf1a12 mDcTrailr2 mSOB Tnfrh1 mDcTrailr1 XEDAR mTNFRH3

17q21-q22

NM_002507

TRAF2,4,6

NGF BDNF NT-3 NT-4

16p13.1

NM_001192

TRAF1,2,3,5,6

TNFRSF17

TNFRSF18 TNFRSF19

TNFRSF19L TNFRSF21

TNFRSF22

TNFRSF23

TNFRSF24 -pending

TNFSF13

1p36.3

NM_004195

TRAF1,2,3

13q12.11-q12.3

NM_018647

TRAF1,2,3,5

11q13.4 2q11-q13 6p12.2-p21.1

NM_152222 NM_022336 NM_014452

TRAF1

Xq11.1 Unknown

NM_021783 Unknown

TNFSF13B TNFSF18

TRADD

At present, 29 transmembrane glycoprotein TNF receptors are known in human, including TNFR1, TNFR2, Fas, CD40, the low affinity nerve growth factor receptor (p75NGFR), TRAIL receptors, death receptors (DR), receptor activator of NFκB (RANK) and osteoprotegrin (OPG), which are grouped together under TNF receptor superfamily (TNFRSF) (Table 2) (Locksley et al., 2001). A hallmark of the TNFR superfamily is the presence of extracellular N-terminus domain characterized by one to six cysteine-rich motifs/domains (CRDs), which are pseudo-repeats typically containing six highly conserved cysteine residues engaged in the formation of three disulphide bonds (Smith et al., 1994; Ashkenazi, 2002). Among all the receptors, number of CRDs varies from one to four, except in the case of CD30, where the three CRDs have been partially duplicated in the human but

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not in the mouse sequence. The repeated and regular arrangement of CRDs confers an elongated shape to the receptors, which is stabilized by a short twisted ladder of disulphide bridges. Sequence alignment of TNF receptor family members in the absence of structural information is difficult because the spacing of cysteine residues is not always conserved between receptors. TNF receptors share about 20-30% homology among the TNFR members. The cDNA of human TNF receptor p60 and p80 showed 64% and 62% sequence homologies, respectively, to the corresponding mouse receptor. However, the p60 receptor is most conserved in the ECD (70%), whereas the p80 is conserved in the ICD (73%). Due to this reason p60 receptor binds both human and murine TNF ligands whereas human p80 receptor binds only human and not mouse TNF ligand (Lewis et al., 1991). The ECD of murine p60 and p80 are 28% identical to each other. The p60 and p80 forms of the TNF receptors are also homologous to several other members of the TNF receptor super-family which include Fas, DR3, DR4, DR5, DR6, NGF (31%), RANK, CD40 (40%), CD27, CD30, Ox40, and 41BB characterized by the presence of cysteine-rich domains in their ECDs. (Gruss and Dower, 1995; Ashkenazi and Dixit, 1998). The major area of homology between these receptors is in their ECD, which may range from 25 to 30%. In addition, several viral open reading frames (ORF) have been found to encode for soluble TNF receptor-like molecules. This includes SFV-T2 in Shope fibrosarcoma virus and Va53 or SsIF19R in vaccinia virus, MYX-T2, G4R, CrmB, and CrmD.

Structural Classification of TNFRSF Naismith and Sprang (1998) have introduced a classification based on distinct structural modules that greatly facilitates sequence comparison between TNF receptors. Each module type is designated by a letter (A, B, C and N for crystallized modules, and X for modules of unknown structure), and by a numeral indicating the number of disulfide bridges it contains (Bodmer et al., 2002). A typical CRD is usually composed of an A1–B2 or A2–B1 module or, less frequently, a different pair of modules. A1 modules are 12–27 amino acids long, consist of three short β strands linked by turns, and contain a single disulfide bridge connecting strands 1 and 3, yielding a characteristic C-shaped structure. A2 modules contain a second disulfide bridge linking the second and third strands without affecting the overall structure. B modules are 21–24 amino acids long and comprise three anti-parallel strands adopting an S-shaped fold reminiscent of a paper clip. In this case, the fold is constrained by two entangled disulfide bridges linking strands 1 and 3 in B2 modules. The first disulfide bridge is replaced by a hydrogen bond in B1 modules (Naismith and Sprang, 1998). The structure of A and B modules is also reflected at the level of the primary sequence by the conservation of a few non-cysteine residues. Other modules are less frequently present in TNFRSF. So far, the N-terminal N1 modules have been found only in the TRAIL receptors, in which they precede the first A1–B2 CRD. Structurally, the N1 module resembles the second half of a B module (Hymowitz et al., 1999; Mongkolsapaya et al., 1999; Cha et al., 2000). The fourth CRD of TNF-R1 contains an A1–C2 module pair, in which the cysteine connectivity of C2 is distinct from that of a B2 module. TACI, BCMA and Fn14 also contain putative A1–C2 CRDs, but these remain to be demonstrated at the structural level. The

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recently described BAFF receptor (BAFFR) contains a single X2 module whose sequence resembles an A module entangled with the beginning of a B module (Thompson et al., 2001). TNF receptors are often viewed as monomers, principally because they appear in this form in crystal structures of ligand–receptor complexes. However, TNF-R1 has also been crystallized as both head-to-head and head-to-tail dimers (Naismith et al., 1996), and there is genetic and experimental evidence that Fas, TNF-R1 and CD40 exist as oligomers within the plasma membrane (Siegel et al., 2000).

TNFR Expression, Regulation and Release TNFR1 (p60) is constitutively expressed in most cell types/tissues and seems to be the key mediator for both the membrane-bound and soluble trimeric forms of TNF signaling. In contrast, TNFR2 (p80) is predominantly expressed in immune system/cells and hematopoietic cells such as macrophages, neutrophils, lymphocytes (B and T cells), thymocytes and mast cells, and plays a major role in the lymphoid system (Grell et al., 1995). Recent studies suggest that TNFR2 (p80) is also expressed in endothelial cells, cardiac myocytes and prostate cells. Most cells exhibit a receptor density of around 1000 sites/cell but in some it is as high as 5000 sites/cell. There is very little known about the regulation of TNF receptors at the transcription, translation, or post-translation levels. Interestingly, TNF can both up-regulate and downregulate its own receptor in a cell type-specific manner. Among the regulators are protein kinase C activators (PKC) (phorbol esters), PKC inhibitors (Staurosporine), PKA activators (Dibutyryl cAMP, Forskolin), cytokines (IFNαβγ, IL-2, IL-4, IL-6, IL-8), TNF, thyroid stimulating factor (TSF), microtubule depolymerizing agents (Vincristine, Vinblastine, Cholchicine) and other factors (hydrogen peroxide, retinal, glucocorticoids, taxol, calcium ionophore, platelet activating factor, okadaic acid, iodoacetic acid) (Aggarwal and Natarajan, 1996). Similar to sTNF, in response to different stimuli, the extracellular domains of both the TNF receptors are cleaved from the membrane resulting in soluble form of TNF receptors (sTNFR) released in extra-cellular space, fluid or in cell culture media. TNFR2 is cleaved by TACE (Solomon et al., 1999). The release of sTNFR1 through proteolytic enzyme is still unknown. The soluble form of both the receptors have been detected in vivo, in serum, synovial fluids, cerebro spinal fluids, ovarian fluids and secreted in urine (Seckinger et al., 1989; Engelmann et al., 1989, 1990). The concentration of soluble receptors in serum of normal healthy individuals is high. There are higher levels of soluble p80 receptors (2-4 ng/ml) than the soluble p60 receptors (1 ng/ml) in normal sera. Even though their binding affinities are much lower than those of the membrane receptors, the secreted sTNFRs eventually neutralize TNF (Wallach et al., 1991). Shedding of sTNFR1 seems to be physiologically important since mutations leading to cleavage resistance are related to dominantly inherited auto-inflammatory syndromes (TNFR1-associated periodic syndromes) (McDemott et al., 1999). A major function of sTNFR is the clearance of TNF from the serum (Van Zee et al., 1992; Bemelmans et al., 1993). Moreover, the sTNFR can bind TNF and act as a competitor to the cell surface receptor (Engelmann et al., 1989; Seckinger et al., 1989; Olsson et al., 1989). The level of membrane bound TNFRs are elevated during pathological condition (Aderka et al., 1993; Deloron et al.,

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1994). In inflammatory diseases, the levels of sTNFRs are several folds higher than in the normal serum (Cope, 1992; Roux-Lombard, 1993). Finally, the density of TNFR is an important factor in facilitating TNF signaling (Chan and Aggarwal, 1994).

TNF Ligand-Receptor Interactions Both types of TNF receptors have a high affinity for TNF in the range of 0.1-1nM. Both sTNF and tmTNF ligands can bind to both TNFR1 and TNFR2, but certain pairings are favored over others; namely, sTNF binding to TNFR1 and tmTNF binding to TNFR2. Although sTNF binds to both receptors on human cells with high affinity, it preferentially binds to TNFR1 (dissociation constant [Kd]~20 pM) versus TNFR2 (Kd~400 pM ), with a 30-fold faster dissociation rate from TNFR2 than from TNFR1 (Grell et al., 1998). Other data from in vivo studies corroborate the conclusion that most of the biologic activities of sTNF are mediated through TNFR1 (Ksontini et al., 1998). In contrast, tmTNF preferentially binds to TNFR2 and is thought to exert most of its inflammatory and proapoptotic activities through TNFR2 (Grell et al., 1995). In 1993, Banner and colleagues published a seminal study unraveling the first structure of a TNF ligand (LTα) bound to its cognate receptor (TNF-R1). Both TNFR1 and TNFR2 contain an extracellular N-terminal pre-ligand association domain (PLAD), a distinct region from the ligand binding region that precomplexes with receptors to form a trimerized complex, particularly upon activation by ligand (MacEwan, 2002). PLAD keeps TNFR1 and TNFR2 in a pre-assembled oligomeric status to avoid auto-activation (Chan et al., 2000). This asymmetric unit contains three receptors and three ligands assembled as a hexameric complex in which a single TNF trimer binds to three receptor molecules. Receptor p80 binds at the C-terminal of the cytokine whereas p60 binds more towards the N-terminus (Loetscher et al., 1993; van Ostade et al., 1994; Haridas et al., 1998). Upon ligand binding, the receptor undergoes a conformational change towards a higherorder receptor complex to achieve signal competence and a three fold geometry is conserved for TNFR complexes (Wajant et al., 2003). The regions of contact between TNF and TNFRs are very diverse among the family members and contribute to the specific interaction of TNFTNFRs pairs. TNFR binds in the groove formed by the trimeric ligand at the interface of each pair of monomers with their long parallel axis to the C3 symmetry. A conformational change occurs upon complex formation that substantially affects the TNF-TNFR loop (CD and AA′) (Banner et al., 1993; Hymowitz et al., 1999). There are mainly two contact regions between the TNFR and the TNF. The first contact area involves TNFR residues corresponding to the second CRD of the receptor (A1 plus half of B2) and loops DE and AA′ of two adjacent ligand subunits. This area is based on a central hydrophobic interaction containing a relatively conserved tyrosine residue (present in loop DE of TNF, LTα, FasL, TRAIL, LIGHT and VEGI) that is crucial for receptor binding in TNF, LTα, FasL and TRAIL. In the second, more polar interaction region, the remainder of the second CRD (second half of B2) and the A1 module of the third CRD of TNF-R1 make contacts with the CD and EF loops of two adjacent ligand subunits. Indeed, the geometry of the receptor–ligand complex matches that of TRAF-2, a trimeric intracellular adaptor molecule mediating TNFR2 and CD40 signals (Park et al., 1999).

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More recently, highly similar crystal structures have been reported for complexes between TRAIL and TRAIL-R2, confirming that the 3:3 stoichometry is the likely basis of the signaling unit or a key event for initiation of signal transduction (Hymowitz et al., 1999; Mongkolsapaya et al., 1999; Cha et al., 2000; Locksley et al., 2001). In addition, a third central interaction region is present in TRAIL–TRAILR2 structure. The central region involves residues 131–135 of the AA′ loop that penetrates into the central interaction region upon binding, forming several specific polar interactions. This additional interaction patch is specific to TRAIL because of its long AA′ loop (Cha et al., 2000). TNFR1 contains a DD motif region in its cytoplasmic tail of approximately 80 amino acids, which is absent in TNFR2. Thus, TNFR1 is known as the archetypal death receptor (DR) because of its ability to bind DD-containing cytoplasmic proteins that activate pro-apoptotic signaling in many cells.

TNF-Dependent Signaling Pathways TNF transduces its signal through two distinct receptors TNFR1 (p55/60) and TNFR2 (p75/80) (Table 3). The signaling of TNFR2 is likely underestimated, even though the affinity of TNFR2 for TNF is five times higher than the TNFR1 (Grell et al., 1998; MacEwan, 2002). Most of the research on TNFR signaling is based on sTNF. TNFR1 is activated equally well by both the soluble and membrane-bound TNF forms. In contrast, TNFR2 is poorly activated by sTNF ligands. TNFR2 is efficaciously stimulated by the membrane-bound form of TNF (Grell et al., 1995). Most of the clues for TNFR2 signaling have been gained from the transgenic knock-out studies in mice (Lewis et al., 1991; MacEwan, 2002). Although both TNF receptors contain a highly homologous, cysteine-rich extracellular domain, their intracellular regions do not show sequence homology The ICD of both the receptors lack homology to the catalytic domain of either Tyr or Ser/Thr-specific protein kinases or to nucleotide-binding proteins. Further, both the receptors possess no enzymatic activity. In fact, the signals they transduce are transmitted through the recruitment of more than a dozen of different signaling proteins, which together initiate signaling cascades leading to the activation of effector proteins like caspases, protein kinases etc. Thus, the cytoplasmic domains of both the receptors have been shown to bind to distinct serine/threonine kinases and cause the phosphorylation of distinct transcription factors and different protein factors leading to distinct functional paths (Darnay et al., 1994a, b; 1995; Beyaert et al., 1995; Bodmer et al., 2002; Bertazza and Mocellin, 2008).

TNFR1-Dependent Signaling A series of proteins have been identified that bind to TNFR1 (Table 3), leading to various cellular responses (Tewari and Dixit, 1996; Drnay and Aggarwal, 1997; Singh and Aggarwal, 1998). Based on the presence of specific signaling motifs or domains in TNFR1 cytoplasmic tail (Figure 2), TNFR1 signaling is divided into three major subgroups which activate NFκB, JNK and apoptosis.

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Table 3. Interacting proteins at the cytoplasmic domain of the TNF receptors. p60 receptor (TNFR1) 55.11 protein BRE (brain and reproductive organ expression) Factor-associated with N-Smase activation (FAN) MAP kinase-activated death domain protein (MADD) p60 TNF receptor associated kinase 60-TRAK Sentrin Silencer of death domains (SODD) TNF receptor-associated death domain protein (TRADD) TNF receptor-associated protein (TRAP1) TNF receptor-associated protein (TRAP2) Fas-associated death domain protein (FADD/Mort 1) TNF receptor-associated factor (TRAF2) A20 zinc finger protein Apoptosis signal-regulating kinase 1 (ASK1) Cellular inhibitor of apoptosis (cIAP1) Cellular inhibitor of apoptosis (cIAP2) Germinal center kinase (GCK) NKκB-inducing kinase (NIK) Receptor-interacting protein (RIP) TRAF2-interacting protein (I-TRAF/TANK) FADD-like ICE (FLICE/MACH) FLICE-interacting protein (I-FLICE/CASH/FLIP/MRIT) P80 receptor (TNFR2) Cellular inhibitor of apoptosis (cIAP1) Cellular inhibitor of apoptosis (cIAP2) TNF receptor-associated factor (TRAF2) TNF receptor-associated factor (TRAF1) p80-TRAK

The first group of signaling is based on DD. Initial step in TNFR1 signaling involves the binding of the TNF homotrimer to the extracellular domain of the receptor at the plasma membrane, which induces TNFR1 trimerization (their tips fitting into the grooves formed between TNF monomers). This binding causes a conformational change in the receptor, leading to the dissociation of the inhibitory protein silencer of death domains (SODD) from the intracellular death domain. The cytoplasmic domain of the unstimulated receptor is bound by SODD preventing constitutive signaling of TNFR1 through blocking the binding of adaptor proteins to the DD of TNFR1 (Bodmer et al., 2002). Upon TNF stimulation, SODD is released serving as a platform for subsequent protein binding. Subsequently, TNFRassociated death domain protein (TRADD) binds to TRAF-2 with the recruitment of intracellular domain with the serine/threonine kinase receptor interacting protein-1 (RIP-1) (Hsu et al., 1996). This adaptor complex recruits other key proteins and forms a complex, termed complex I, which is responsible for intracellular signaling events. This protein complex I, is believed to activate the NFκB pathway via mitogen activated protein (MAP) kinase kinase-3 (MEKK-3) leading to phosphorylation of the inhibitor of κB kinase (IKK), which in turn phosphorylates the inhibitor of κB (IκB) (Yang et al., 2001). Inhibitory protein, IκBα normally binds to NFκB and inhibits its translocation.

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Figure 2. Schematic representation of the proximal components of the tumor necrosis factor (TNF) receptor type 1 (TNFR1) and TNF receptor type 2 (TNFR2) signal- transduction pathways and their relationships to the activation and inhibition of programmed cell death and inflammation. TNFR1 transduces apoptotic and anti-inflammatory signals through the recruitment of Fas-associated death domain protein (FADD) and subsequent recruitment and activation of caspase 8. TNFR1 also mediates anti-apoptotic and inflammatory responses through the recruitment of TNF-receptor-associated factor 2 (TRAF2) and receptor-interacting protein 1 (RIP1). As also shown in the figure, TNFR2 recruits TRAF1 and TRAF2 to transmit its anti-apoptotic and inflammatory signals.

The IKK dependent phosphorylation of IκBα leads to the ubiquitinated-dependent degradation of IκB and allows NFκB to enter the nucleus to initiate gene transcription. NFκB is a heterodimeric transcription factor that translocates to the nucleus and mediates the transcription of a vast array of proteins such as transcription factors leading to the activation of stress response genes, cytokines, chemokines, regulators of apoptosis, immune receptors, cell cycle proteins and adhesion molecules. Subsequently, these factors are involved in cell survival, proliferation and inflammatory responses, development, oncogenesis or cellular stress (Shishodia and Aggarwal, 2002). The NFκB pathway can also be activated through TRAF-2 (Devin et al., 2001). The second signaling pathway elicits cell death through TRAF signaling proteins. In this, the receptor complex I is internalized and a complex consisting of TRADD, RIP-1 and TRAF-2 is released/dissociated from TNFR1 (Schutze et al., 1999). Then, Fas-associated DD protein (FADD) binds to TRADD and recruits FLICE. Subsequently, it activates a family of aspartate-specific cysteine protease, mainly pro-caspase-8 as a complex II or as a cytoplasmic complex. This signaling step results in activation of caspase-8 through two distinct pathways which finally activates caspase-3 and initiates apoptosis (Wang et al., 2008). In a third signaling pathway, receptors act through the cytoplasmic tail, which contains one or more TRAF-interacting motifs (TIM). Activation of TIM-containing TNF receptors leads to the direct recruitment of TRAF family members that are recruited to the TRADD-

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RIP-TRAF-2 complex which interacts with the apoptosis-signalling kinase-1 (ASK-1). ASK1 is a MAPKKK, a member of the MEKK family. This protein complex activates the MAP kinase kinases MEK-4 and MEK-6 (Ichijo et al., 1997). MEK-4 and MEK-6 phosphorylate and activate c-Jun N-terminal kinases (JNKs), p38 MAPKs, extracellular signal-related kinase (ERK), inhibitor of nuclear factor kappa-B (NFκB) kinase (IκB kinase, IKK), phosphatidyl-inositol-3 kinase (PI3K) and AKT (Rivas et al., 2008). JNKs phosphorylate cJun, a subunit of the transcription factor activating protein-1 (AP-1). Similar to NFκB, AP-1 is considered to promote inflammation and cell survival. However, TNF signaling through ASK-1 has been shown to provoke cell death as over expression of dominant-negative ASK1 or knock-down inhibits TNF-induced apoptosis (Ichijo et al., 1997; Tobiume et al., 2001).

TNFR2-Dependent Signaling Compared to TNFR1-dependent signaling, TNFR2-mediated pathways are less well understood. TNFR2 only responds to the membrane-bound form of the TNF homotrimer (Chen and Goeddel, 2002; Pfeffer, 2003; Aggarwal, 2003). Pro-TNF binds to TNFR2 through direct cell-to-cell contact and presents a higher affinity for this receptor than the sTNF form. Since TNFR2 lacks a DD, it cannot stimulate the apoptotic process directly. However, TNFR2 mediates signaling via TRAFs (Table 3, Figure 2) and shares signaling effects with TNFR1 which activates NFκB and JNKs. The intracellular domain of TNFR2 can directly interact with TRAF-2 resulting in recruitment of RIP and FADD and activation of caspases (Aggarwal, 2003; Wajant et al., 2003). Thus, depending on the cell type, TNFR2 can promote proliferation and apoptosis (MacEwan, 2002). TNFR2 was also shown to activate the endothelial/epithelial tyrosine kinase (Etk) which in turn results in activation of the PI3K-Akt pathway via vascular endothelial growth factor (VEGF), thereby modulating cell adhesion, proliferation, migration and survival (Chen and Goeddel, 2002; Gaur and Aggarwal, 2003; Zhang et al., 2003). Since, TNFR family members do not contain functional intracellular signaling domains and motifs, these “decoy” receptors cannot provide intracellular signaling unless they can effectively compete with other receptor groups for their corresponding ligands, which adds a further level of regulation to the TNF family ligands (Bertazza and Mocellin, 2008). Interestingly, few soluble forms of TNFRSF receptors such as CD27, CD30, CD40, 4-1BB, CD95, TNFR1 and TNFR2 act as circulating decoy receptors (DcR). Due to lack of transmembrane and cytoplasmic domains in circulating decoy receptors, they bind ligand without inducing cell signaling activation. However, these receptors add an extra level of regulation to the activity of respective ligands (Bertazza and Mocellin, 2008). Thus, TNF through TNFR1 and TNFR2 receptor induces numerous signaling pathways which provoke a variety of cellular effects (Figure 3). The complexity and crosstalk of TNF signaling pathways still have to be elucidated.

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Figure 3. Schematic representation of TNF-induced cell survival and cell death signaling pathways mediated through membrane bound TNFR1 and TNFR2 receptors (for details see the text).

Lymphotoxin LT was identified in tumor cells as a cytotoxic factor and homologous partner of TNFα, and named as lymphotoxin (LT/TNFβ) (Pennica et al., 1985). LT is mainly produced in lymphoid cells, but binds to the same surface receptor as TNFα (Old, 1985). LTs are trimeric molecules composed of various combinations of α and/or β monomers, including LTα3, LTα1β2 and LTα2β1. LTα is the official name for LTα3 but is sometimes used in the literature to denote any LT molecule containing an LTα chain. LTα is a glycoprotein with no cysteine residues (Aggarwal et al., 1987). As members of the TNF superfamily (TNFSF), LTs have many similarities to TNF, but with some distinct molecular and biological differences (Gommerman & Browning, 2003; Ware, 2005). First, there are several distinct ligands in the LT family. LTα3, formerly called TNFβ, is structurally similar to sTNF in that it is a soluble homotrimer composed of 17-kDa monomers and it binds specifically to TNFR1 and TNFR2 to exert its biologic activities. The affinities of LTα3 for TNFR1 and TNFR2 are comparable to those of TNF; but, unlike TNF, LTα3 does not rapidly dissociate from TNFR2 (Medvedev et al., 1996), suggesting that ligand passing of LTα3 from TNFR2 to TNFR1 is unlikely to occur. LTαβ is structurally distinct from LTα3 and comprises 2 membrane-anchored heterotrimers, the predominant LTα1β2 form and a minor LTα2β1 form. Both LTαβ forms interact with the LTβ receptor (LTβR), but the LTα2β1 form also binds less avidly to TNFR1 and TNFR2 than to the LTβR (Crowe et al., 1994;

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Williams-Abbott et al., 1997; Ware, 2005). Unique among TNF superfamily members, the LTα monomer contains a traditional signal peptide which is secreted in soluble form. The LTα monomer can only be membrane anchored when co-expressed and associated with LTβ monomers to form LTαβ heterotrimers. Follicular B cells and CD4 T cells in the spleen constitutively express LTαβ, but expression of LTαβ can be induced on splenic T cells by the cytokines IL-4 and IL-7 and the chemokine (C-C motif) ligands (CCL) 19 and 21 (Luther et al., 2002), and on a human T-cell line by TNF (Voon et al., 2004). The LTβR that interacts with LTαβ-bearing lymphocytes is not expressed on T-cells, B cells or NK cells but is constitutively expressed on stromal fibroblasts, epithelial cells and myeloid cells, such as monocytes/macrophages, dendritic cells and mast cells (Murphy et al., 1998; Ware, 2005). The cellular distribution of LTαβ ligands on lymphoid cells and LTβR on stromal and parenchymal cells, coupled with the requirement for cell–cell contact to initiate LTβR signaling, suggests a functional role of LTαβ in the interaction of lymphoid cells with surrounding stromal cells. Signaling via the LTβR is similar to that of TNFR1. The intracellular domains of LTβR couple with the adapter protein TRAF2 or TRAF3, which activate both the conventional and alternative NF-κB1 pathways, leading to the induction of inflammatory genes and genes involved in lymphoid tissue neogenesis (Ware, 2005). The LTβR does not contain a death domain, therefore, apoptosis pathways are not activated by LTαβ.

Functional Role of TNF Members of the TNF-superfamily play important roles in cellular functions. TNF displays a functional duality being involved in tissue regeneration and destruction (Table 4). Due to several motifs in TNF receptors, it has a wide spectrum of bioactivities and most cells show at least response sensitivity to TNF. TNF has been called a sentinel cytokine or “the body's fire alarm” (Feldmann & Steinman, 2005), as it initiates the defense response to local injury. The activation occurs within 5 minutes and with as little as 1pM TNF concentration (Chaturvedi et al., 1994). TNF concentrations seem to determine whether the cytokine exerts beneficial or harmful effects. At low concentrations in tissues, TNF is thought to have beneficial effects, such as the augmentation of host defense mechanisms against infections. Low concentrations over a long period of TNFs are often associated with cachexia. At high concentrations, TNF can lead to excess inflammation and organ injury. In disease states, TNF is generally considered to be a proinflammatory cytokine, along with IL-1, IL-17, and other cytokines. Also high doses of sTNF in response to lipopolysaccharides and other bacterial toxins play a key role in the development of septic shock (Mannel and Echtenacher, 2000). A simplified view of the role of TNF in inflammation and some immune-mediated inflammatory diseases is that expression of TNF is increased in the affected tissues as a result of innate and adaptive immune responses. TNF then mediates a variety of direct pathogenic effects and induces the production of other mediators of inflammation and tissue destruction, placing it at the head of an inflammatory cascade within an inflammatory network. TNF may also be considered as one particularly important proinflammatory cytokine in an intricate network rather than in an inflammatory cascade. Much less is known about the roles of the LT family in diseases, but at least some of its member’s functions are similar to those of TNF.

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Indrajit Chowdhury and Ganapathy K. Bhat Table 4. Functional role of TNF mediated through TNFR-1 and TNFR-2. p60 receptor (TNFR1) Antiproliferation Antiviral activities Apoptosis Cytotoxicity Endothelial cell adhesion molecules Generation of lymphocyte-activated killer (LAK) cells Growth stimulation HLA class I and II Ag expression Induction of c-fos Induction of IL-2 receptor Induction of IL-6 Induction of Mn superoxide dismutase mRNA Induction of NFκB activation Production of ceramide Production of diacylglycerol Proliferation of natural killer (NK) cells Prostaglandin E2 synthesis Stimulation of phospholipase A2 Stimulation of protein kinase C Stimulation of spingomyelinase p80 receptor (TNFR2) Antiproliferation Apoptosis Cytotoxicity DNA fragmentation Generation of NK and LAK cells Induction of IL-6 Induction of NFκB activation Prolifeartion of thymocytes

TNF has a number of actions on various organ systems, together with Interleukin-1 (IL1) and Interleukin-6 (IL-6). In the hypothalamus, TNF stimulates the hypothalamic-pituitaryadrenal axis which in turn stimulates the release of corticotropin releasing hormone (CRH), suppressing appetite and fever. In the liver, TNF stimulates the acute phase response leading to an increase in C-reactive protein and a number of other mediators. TNF also induces insulin resistance by promoting serine-phosphorylation of insulin receptor substrate-1 (IRS1), which impairs insulin signaling. TNF is a potent chemoattractant for neutrophils, it helps them to stick to the endothelial cells for migration; on macrophages TNF stimulates phagocytosis, production of IL-1 oxidants and the inflammatory lipid prostaglandin E2 (PGE2) A locally increasing concentration of TNF causes the cardinal signs of inflammation such as heat, swelling, redness and pain; high concentrations of TNF induce shock-like symptoms. The prolonged exposure to low concentrations of TNF causes cachexia (weakness, loss of weight and muscle atrophy), a wasting syndrome which can be found in tumor patients (Beutler et al., 1985). The TNF is also involved in the progression of many autoimmune diseases (Taylor et al., 2000; Blam et al., 2001). Due to its inflammatory response, TNF in turn causes many of the clinical problems associated with autoimmune

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disorders such as rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, psoriasis and refractory asthma. Thus, under physiological conditions, TNF is involved in immune surveillance and defense, cellular homeostasis, protection against certain neurological insults as well as in the control of cell survival, proliferation, migration, and differentiation. Also, TNF has protective functions of pathogen-reactive cells through activation or inactivation of transcription factors or gene induction (Wajant et al., 2003; Sriram et al., 2007). Perhaps most important of these are NFκB, which is responsible for many of the inflammatory effects of the TNF and another transcription factor, AP-1 which mediates through JNK (Brenner et al., 1989). TNF is the most potent activator of different transcription factors described to date. TNF receptor mediates the activation of promoters of various genes containing NFκB, AP-1, SP-1, or cmyc binding sites. All the functional roles of TNF indicate that there must be a complex interaction pattern between TNF concentration, tissue and cell type, TNF receptor distribution and duration of TNF stimulation leading to a specific physiological or pathological reaction.

TNF Induced Apoptosis/Programmed Cell Death (PCD) Numerous studies have investigated the role of TNF in programmed cell death/apoptosis. In summary, the local TNF productions are critically involved in the physiological balance of cellular turnover and renewal. Cytotoxic effects of TNF have been demonstrated alone or in combination with interferon γ (INFγ) in in-vitro culture system. Utilization of either TNFR1 or TNFR2-specific TNF proteins suggested that apoptosis is predominantly induced trough TNFR1 (Yui et al., 1996). The TNF/TNFR1 complex is internalized into endocytic vesicles in which various adapter proteins assemble and initiate the signaling cascades, leading to apoptosis (Higuchi & Aggarwal, 1994; Micheau & Tschopp, 2003; Schneider-Brachert et al., 2004). Association of the TNF/TNFR1 complex with lipid rafts, but not internalization, is required for the pathway leading to NF-κB1 activation (Legler et al., 2003; SchneiderBrachert et al., 2004; D'Alessio et al., 2005). Alternatively, TNF may also induce cell death by antagonizing the caspase-inhibitory action of XIAP (X-linked inhibitor of apoptosis) through elevation of the pro-apoptotic protein XAF1 (XIAP associated factor 1) (Straszewski-Chavez et al., 2007). The TNF-TNFR complex mediates two distinct forms of “extrinsic” (or death receptor mediated) PCD. The first is classical apoptosis, which is characterized by caspase-dependent chromatin condensation and fragmentation, membrane blebbing and regeneration of apoptotic bodies. The second is necrosis-like caspase-independent PCD, characterized by absent or marginal chromatin condensation, lack of nuclear fragmentation and disruption of membrane integrity (Chowdhury et al., 2006). The former is considered to be the best characterized type of TNF-driven PCD. Activation of caspase-8 by FADD starts the caspase cascade. Caspases are synthesized as pro-enzymes and are activated by the cleavage at specific aspartic acid residues (Chowdhury et al., 2008). Beside causing DNA fragmentation through the activation of caspase-activated DNase (CAD), caspases can also initiate the mitochondrial apoptotic

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pathway (or “intrinsic” death pathway) leading to the release of several mediators such as cytochrome c that further amplify the caspase cascade (Chowdhury et al., 2006). TNF also activates the evolutionary conserved lysosomal death pathway, which is mainly mediated by the cathepsin protease family (Foghsgaard et al., 2001). In normal circumstances, TNFR1 induces the transcription and activation of inflammatory genes (Locksley et al., 2001). TNFR1 signaling also provides a mechanism to suppress the apoptotic stimulus. TNFR1 recruits TRAF2 and RIP to activate the NFκB and JNK pathways, which can initiate inflammatory, proliferative and apoptotic responses. NFκB activation promotes the synthesis of IκB and anti-apoptotic factors, such as FLICE inhibitory protein (FLIP or IFLICE and FLAME) and IAP family members (IAP-1/2, survivin), which are potent inhibitors of PCD. RIP is a key mediator of the NFκB activation. Its cleavage by caspase-8 separates the N-terminal kinase domain (RIPn) from the C-terminal DD (RIPc) and results in ablation of the RIP-mediated activation of NFκB (Kim et al., 2000). JNK has the ultimate role in controlling TNF-driven PCD, which is less clear at present (Varfolomeev and Ashkenazi, 2004). In an apparent dichotomy, TNFR1 assembles a signaling complex that can promote both cell death and survival. Physiological or disease-related alteration or virtually any intracellular apoptosis-related factor can tip the balance towards PCD or cell survival. Cell can be refractory to TNF-driven PCD owing to the over-expression of anti-apoptotic proteins, such as NFκB, FLIP, mitochondrial apoptotic pathway-related factors (Bcl2, BclxL) or the down regulation of pro-apoptotic factors (caspases) (Chowdhury et al., 2006, 2008; Bertazza and Mocellin, 2008). Additionally, TNF signaling network intersects several other metabolic pathways such as generation of ceramide which plays an important role in the induction of apoptosis (Pandey et al., 2006). Ceramide exerts its pro-apoptotic effect by inhibiting the activity of anti-apoptotic factors (protein kinase B, PKB/Akt), and by stimulating TRADD recruitment to TNFR1 with subsequent increased activation of caspase-8 (De Nadai et al., 2000). Impairment of the activity of spingomyelinases, which generate ceramide following TNF stimulation, reduces the ability of the cytokines to induce apoptosis (Liu et al., 1998). Additionally, short chain ceramide analogues increase cell sensitivity to TNF-induced PCD (De Nadai et al., 2000). The above studies represent a complex network of interrelations with TNF and pro-/anti-apoptotic molecules, making the prediction of the final outcome particularly very complex.

Genetic Diversity of TNF and Human Abnormalities TNF is a pro-inflammatory cytokine that provides a rapid form of host defense against infection and employed against a variety of pathogens. Overproduction of TNF has been implicated in a variety of human diseases, including sepsis, psoriasis, multiple sclerosis, cerebral malaria, diabetes, ankylosing spondylitis, tuberculosis, alopecia areata, asthma, inflammatory bowel disease, hepatitis b, cystic fibrosis, Crohn disease, cancer and autoimmune diseases such as autosomal dominant polycystic kidney disease, rheumatoid arthritis and systemic lupus erythematosus.. Susceptibility to many of these diseases have a

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genetic basis. Thus, TNF gene is considered to be a candidate predisposing gene. However, unraveling the importance of genetic variation in the TNF gene to disease susceptibility or severity is complicated due to its presence within the MHC location, a highly polymorphic region that encodes numerous genes involved in immunologic responses. Most of the genetic studies are restricted to TNFα polymorphism. Recent studies have demonstrated that some of these familial disorders are due to the multiple bi-allelic single polymorphisms in the up-stream of the proximal promoter of TNFA (Hajeer and Hutchinson, 2000; Khalil et al., 2006). Single-nucleotide polymorphisms (SNPs) in regulatory regions of TNFA genes have been also associated with susceptibility to a number of complex disorders with inflammatory response. Using PCR and sequencing technology, the entire coding region and 1,053 bp upstream of the transcription start site of the TNFA gene have been screened for polymorphisms. There are five polymorphisms identified: 4 are located in the upstream region at positions -857, -851, -308; and -238 from the first transcribed nucleotide, and 1 is found in a non-translated region at position +691 (Herrmann et al., 1998). Three SNPs located at nucleotides -238, -308, and -376 with respect to the TNF transcriptional start site are all substitutions of adenine for guanine. Knight et al. (1999) referred to the allelic types as -238G/-238A, -308G/-308A, and -376G/-376A. The variation in the TNFA promoter region has been found to be associated with susceptibility to cerebral malaria (McGuire et al., 1994), mucocutaneous leishmaniasis (Cabrera et al., 1995), death from meningococcal disease (Nadel et al., 1996), lepromatous leprosy (Roy et al., 1997), scarring trachoma (Conway et al., 1997) and asthma (Moffatt and Cookson, 1997). The best studied is substitution at position -308 relative to the transcription start site. The rare alleles at position -308 (denoted TNFA-308*2) are associated with high production of TNFα (Braun et al., 1996; Kroeger et al., 1997; Huizinga et al., 1997). The effect of TNFA-308 was first shown by comparing TNFα responses of donors with and without TNFA-308*2. However, most Caucasians with TNFA-308*2 carry a conserved ancestral MHC haplotype defined as HLA-A1, B8, LTA+250*2, TNFA-308*2, DR3 and DQ2. Carriers of all or part of this haplotype have been associated with many diseases with immunopathological aetiology. It has been impossible to ascertain as to which of the genes and alleles are directly responsible, TNFA-308 remains a candidate because it can affect transcription in luciferase reporter constructs (Kroeger et al., 1997). However, its importance in vivo is unclear (Bayley et al., 2004).

Sepsis Studies have shown a higher incidence of septicemic shock and type 1 respiratory failure following a community-acquired pneumonia-associated carriage of TNFA-308*2 (Waterer et al., 2001). Similarly, neonates with TNFA-308*2 are three times more likely to die following sepsis (Hedberg et al., 2004). Further, anti-TNF treatment to these sepsis patients is reported to have partial survival benefit (Reinhart and Karzai, 2001). However, few studies have shown no correlation between TNFA-308 alleles and sepsis (Calvano et al., 2003).

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Burn Injury Carriage of TNFA-308*2 is also associated with a higher risk of severe sepsis following burns (Barber et al., 2004). Burn patients are very susceptible to bacterial infection, which induces higher TNFα release. Thus, burned patients with sepsis have a higher level of circulating TNFα (69%) compared with those without sepsis (33%). High patient mortality (71%) has been observed when high circulating TNFα was detected (Marano et al., 1990). Overwhelming tissue damage secondary to burns evokes intense systemic inflammatory responses leading to spillage of inflammatory mediators from local to systemic circulation and affecting remote organs such as lungs. The treatment of burns is traditionally focused on correcting the fluid and electrolyte imbalance, pain relief, nutritional support, along with wound care. Some studies suggested that soluble TNFR1 and TNFR2 may be prognostic markers in burn patients (Barber et al., 2004). Early intervention with surgical excision of infected burn wounds and grafting can reduce circulating TNFα levels (Chai et al., 2000). In superficial and partial thickness burn injuries, surface local cooling may inhibit TNFαinduced capillary perfusion failure and leucocyte responses (Westermann et al., 1999). AntiTNF antibody may reduce susceptibility to infection, but the dose and time of administration are critical.

Acute Pancreatitis Acute pancreatitis induces production of TNFα. Inflammation of the pancreas evokes local and systemic responses resulting in the release of high inflammatory mediators such as IL-6, IL-8 and TNFα in to the circulation. The over-production of TNFα causes a significant influence on remote organs such as lung and kidney (Pooran et al., 2003). The patients with high risk of severe acute pancreatitis have been shown to be associated with TNFA-308*2 (Balog et al., 2005). The continuous high-volume renal replacement therapy in acute renal failure patients following acute pancreatitis has a beneficial role in removing excess inflammatory mediators and reducing the high circulating TNF load (Li et al., 2003; Morgera et al., 2003).

Ischemia Reperfusion Injury After cardiaopulmonary bypass surgery, TNFA-308*2 has been associated with pulmonary complications similar to TNFα mediated ischemia reperfusion injury (Yende et al., 2004). TNFα has also been implicated in remote organ injury (Gilmont et al., 1996; Welborn et al., 1996). Immunotherapy has been used to treat reperfusion injury in many animal models and can reduce injury to local and remote organs following ischemia and reperfusion (Gurevitch et al., 1997; Yang et al., 1998; Tsuruma et al., 1998; Gasines et al., 1999; Souza et al., 2001; Pascher et al., 2005). The preliminary clinical data have shown the benefit of anti-TNF therapy in patients with ischemic reperfusion injury. However, these studies require further evaluation (Pascher et al., 2005).

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Trauma Surgical trauma is associated with tissue damage and production of high TNFα. Earlier studies have demonstrated a correlation between the severity of injury and level of soluble TNFR1 and TNFR2, but the method used was not sensitive enough to detect TNFα (Ciant et al., 1994). In contrast, a later study showed that TNF and IL-8 are significantly increased in the first 4 hours post-trauma (Ferguson et al., 1997). Individuals with alleles TNFA-308*2 and LTA+250*2 which are commonly carried together as a haplotype have a high risk of post-trauma severe sepsis and death (O’Keefe et al., 2002; Majetschak et al., 2002).

Cancer TNFα is a major mediator of the cachexia of cancer (Mantovani et al., 1998). Patients with solid tumours have high circulating TNFα and are associated with poor prognosis (Michalaki et al., 2004). Carriage of TNFA-308*2 is related to higher clinical tumour stage of prostate cancer (Oh et al., 2000) and it affects initial susceptibility to hepatocellular carcinoma (Ho et al., 2004), gastric carcinoma (Machado et al., 2003) and breast carcinoma (Mestiri et al., 2001). Furthermore, alleles TNFA+488 and TNFA-859 can affect risk of bladder cancer (Mestiri et al., 2001), TNFA-238 and TNFA+488 affect renal cell carcinoma (Nakajima et al., 2001) and TNFA+488 affects risk of prostate cancer (Oh et al., 2000).

Anti-TNF Therapeutics TNF through its receptors mediates the induction of a wide variety of genes that are involved in autoimmune diseases, inflammation, tumorigenesis, sepsis and tumor metastasis. TNFα was the first cytokine to be employed for cancer biotherapy as a pleiotropic protein. Due to serious side effects (i.e. shock-like syndrome) of high dose of systemic TNF administration, researchers investigated the delivery of TNF through the loco-regional route (i.e. isolated limb perfusion for the treatment of patients with locally advanced melanoma and soft tissue sarcomas) which culminated in a license from the European Medicine Evaluation Agency (EMEA) for the TNF-based treatment of limb-threatening soft tissue sarcomas (Spriggs et al., 1988; Lejeune et al., 1998; Eggermont et al., 2003; Rossi et al., 2003). The development of TNFα antagonists is the newest and most successful in clinical applications of cytokine biology till date. Usage of sTNF receptors and TNF-neutralising antibodies became important therapeutic strategies for aforementioned disorders (Feldmann et al., 2003). Today, there are 3 registered TNF antagonists in the United States and the European Union: infliximab, etanercept and adalimumab (Table 5); each is indicated for several immunemediated inflammatory diseases. The current status of the registered TNF antagonists and the clinical trials of two other antagonists can be accessed at http://clinicaltrials.gov/, http://www.who.int/, or http://www.actr.org.au/. Although different immune-mediated inflammatory diseases involve distinct target organs or tissues, they appear to share some common underlying mechanisms involving TNF. Infliximab and adalimumab are monoclonal

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antibodies (mAbs) that specifically bind TNF while etanercept is a TNF-receptor Fc-fusion protein that binds TNF and lymphotoxin (LT) family members. In addition, two other TNFantagonists are in development, namely certolizumabpegol (refered to as certolizumab) and golimumab, although relatively little information is publicly available on these molecules (Tracey et al., 2008). All agents except etanercept are anti-TNF mAbs or fragments thereof. Natural mAbs are derived from single B cells that clonally express copies of a unique heavy (H) chain and a unique light (L) chain that are covalently linked to form an antibody molecule of unique specificity. Engineered mAbs can be structurally identical to natural mAbs but are created by gene splicing and mutation procedures, mimicking natural gene rearrangement and somatic mutation events in B cells (Salfeld et al., 1998). Infliximab, adalimumab and golimumab are full-length, bivalent IgG mAbs, whereas certolizumab is a monovalent Fab1 antibody fragment covalently linked to polyethylene glycol. IgG antibody molecules are composed of two-H and two-L polypeptide chains, each of which contains 3 complementarity determining regions in the N-terminal (VH and VL) domains. An IgG molecule is composed of 2 antigen-binding Fab arms, linked to a glycosylated Fc region via a flexible hinge region. The antigen-binding site on each Fab portion of a mAb is generally composed of amino acids from the 6 complementarity determining regions in each H:L chain pair. Infliximab is a chimeric protein containing ~25% mouse-derived amino acids comprising the VH and VL domains and ~75% human-derived amino acids comprising the CH1 and Fc constant regions. Certolizumab is a humanized protein containing amino acid sequences in the complementarity-determining regions derived from a mouse anti-TNF mAb and inserted into human VH and VL domain frameworks. Adalimumab and golimumab are fully human mAbs. The TNF–antagonist mAbs also differ in their IgG isotypes, the Fc regions of which govern effector functions, like complement fixation and Fc receptor–mediated biological activities. Infliximab, adalimumab and golimumab are IgG1 antibodies, which are capable of complement fixation and Fc receptor binding. Certolizumab is a Fab1 fragment of an IgG1 mAb and lacks effector functions because it has no Fc region. The hinge region of certolizumab is modified and covalently linked to 2 cross linked chains of 20 kDa of polyethylene glycol to enhance solubility and half-life in vivo (Weir et al., 2006; Tracey et al., 2008). Etanercept is a genetically engineered fusion protein composed of a dimer of the extracellular portions of human TNFR2 fused to the Fc portion of human IgG1. The TNFR2 portion contains 4 domains, and the C-terminal domain includes a 57-residue region that contains 13 O-glycosylated residues and 11 proline residues (Kohno et al., 2005a). In endothelial cells, the plasma half-lives of antibodies appear to be largely governed by the binding of their Fc regions to the neonatal Fc receptor (FcRn) (Lobo et al., 2004). Although the amino acid sequences of the Fc regions are identical, the markedly shorter plasma half-life of etanercept versus IgG1 mAbs or other Fc fusion proteins (Lobo et al., 2004) suggests that the conformation or steric accessibility of the Fc region of etanercept may be different from those of the Fc regions of the IgG1 antibodies infliximab and adalimumab. The effect of the glycosylated C-terminal domain of TNFR2 on the structure and function of the adjacent Fc region of etanercept is unclear. No data have been reported on the binding affinities of etanercept for FcRn or other Fc receptors. In comparison, the long plasma half-lives of infliximab, adalimumab and golimumab suggest that they bind to FcRn like natural IgG1 molecules.

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Ligand-binding studies using BIA core surface plasmon resonance technology measures the on rate and off-rate of an agent binding to a ligand. The ratio of these rates determines the binding affinity of the agent for the ligand, usually expressed as a dissociation constant, Kd. All of these mAbs/agents bind sTNF with high affinity, with Kd values in the sub-nM range. However, there are some important differences between agents in their kinetic parameters of binding. Infliximab and adalimumab have been reported to have slower on-rates and off rates than etanercept (Santora et al., 2001; Scallon et al., 2002). Recent studies with current BIA core methodology found that the on-rate for etanercept was about twice that of infliximab or adalimumab while the off-rates of the 3 agents were comparable (Kaymakcalan et al., 2006a). Infliximab binds to both the 17-kDa monomer and the 51-kDa trimer forms of sTNF, whereas etanercept binds only to the trimer form with each receptor arm contacting comparable epitopes on different faces of the trimer (Scallon et al., 2002). Thus, infliximab and etanercept probably bind to different epitopes on sTNF (Scallon et al., 2002). Similar studies comparing the binding of adalimumab to sTNF monomer and trimer have not been reported. Differences have been reported on the size, composition and stability of complexes formed between sTNF and the different agents. As bivalent mAbs, infliximab and adalimumab can bind two sTNF trimers simultaneously, allowing multimeric complexes to form under permissive stochiometric conditions (Santora et al., 2001; Scallon et al., 2002; Tracey et al., 2008). In contrast, each molecule of etanercept appears to bind to sTNF by interacting with a single sTNF trimer, generally resulting in small 1:1 complexes (Scallon et al., 2002). The cellular and biochemical consequences of binding to tmTNF by TNF antagonists may depend on tmTNF cross linking and may be influenced by several factors. In contrast to the anti-TNF mAbs, which have the potential to crosslink two tmTNF trimers, it appears that etanercept preferentially binds with both receptor arms to a single tmTNF trimer with little or no potential to crosslink one tmTNF trimer to another (Scallon et al., 2002; Tracey et al., 2008). Variations in cell–surface density of tmTNF may underly some of the apparent discrepancies between cellular tmTNF-binding studies. Low-density expression of tmTNF might favor binding of infliximab, adalimumab and etanercept to a single tmTNF, without crosslinking; whereas high-density tmTNF expression might favor crosslinking and greater-avidity binding to tmTNF by infliximab or adalimumab, but probably not etanercept. Interestingly, certolizumab is a monovalent PEGylated Fab1 molecule that should not be able to crosslink tmTNF, yet it has been found to induce reverse signaling in cells (Nesbitt et al., 2006 Tracey et al., 2008). Current evidence suggests that the above described drugs have dual functions and can act as antagonists by blocking tmTNF interactions with TNFR1/2, or as agonists by initiating reverse signaling leading to apoptosis, cell activation or cytokine suppression. With regard to their tmTNF-antagonist activities, measured as inhibition of TNFR-mediated endothelial cell activation by tmTNF-transfected cells, infliximab, adalimumab and certolizumab had comparable activity (Gramlick et al., 2006). Binding of TNF antagonists to tmTNF initiates reverse signaling pathways that intersect with those induced by LPS, zymosan or other stimuli. Simultaneous engagement of these signaling pathways results in suppression of cytokine production, possibly by exhaustion of common signaling components (Eissner et al., 2004). The novel intramembrane proteases SPPL2a and SPPL2b were recently identified and shown to be necessary for tmTNF-mediated reverse signaling in IL-12 production by human

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dendritic cells (Friedmann et al., 2006). It is possible that some reverse-signaling pathways initiated by TNF antagonists involve the activation of these proteases. Table 5. Biochemical characteristics of TNF antagonists. Infliximab REMICADE

Etanercept ENBREL

Adalimumab HUMIRA

Certolizumab NA

Golimumab NA

Synonyms /historical Class

cA2

p75TNFRFc Fc-fusion protein

D2E7

CDP870

CNTO-148

Monoclonal antibody

Monoclonal antibody fragment

Monoclona l antibody

Structure

Mo/Hu chimeric IgG1κ

Hu sTNFR2Fcγ1

Hu IgG1κ

PEG-Hu IgG1κ Fab1

Hu IgG1κ

Molecular weight (kDa)

150

150

150

Specificit y to TNF ligands

sTNF, tmTNF

sTNF, tmTNF,

sTNF, tmTNF

sTNF, tmTNF

sTNF, tmTNF

Specificit y to LT ligands

–

LTα3, LTα2β1

–

–

–

Dosages

3–10 mg/kg q4–8w

25 mg biw; 50 mg qw

40 mg eow; 40 mg qw

100, 200 or 400 mg q4w

50 or 100 mg q2w or q4w

Half-life (t ½)

8–10 days

4 days

10–20 days

Brand name

Monoclonal antibody

95

14 days

150

7–20 days

References Remicade PI (2006), Enbrel PI (2007), Humira PI (2007)

Kay et al.(2006), Remicade PI (2006), Weir et al.(2006), Enbrel PI (2007), Humira PI (2007) Kay et al.(2006), Remicade PI (2006), Weir et al.(2006), Enbrel PI (2007), Humira PI (2007) Kay et al.(2006), Remicade PI (2006), Weir et al.(2006), Enbrel PI (2007), Humira PI (2007) Kay et al.(2006), Remicade PI (2006), Weir et al.(2006), Enbrel PI (2007), Humira PI (2007) Browning et al.(1995), Crowe et al.(1994), WilliamsAbbott et al.(1997), Scallon et al. (2002), Ware (2005) Furst et al. (2003), Haraoui (2005), Kay et al.(2006), Remicade PI (2006), Weir et al.(2006), Enbrel PI (2007), Humira PI (2007) Remicade PI (2006), Weir et al.(2006), Enbrel PI (2007), Humira PI (2007), Zhou et al.(2007)

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Furthermore, several studies have shown clear concentration dependence for the induction of apoptosis by TNF antagonists. A recent study demonstrated concentration dependence of infliximab- and adalimumabinduced apoptosis of normal blood T cells (Chaudhary et al., 2006). Etanercept also induced apoptosis at a concentration of 10 μg/mL, but not at 1.0 or 0.1 μg/mL (Catrina et al., 2005). Further studies are needed to investigate the incidence and role of apoptosis in vivo in TNF antagonist therapy and the relationship to in vivo drug concentrations and to know the methodological differences among the many in vitro studies. Most importantly, the relevance of apoptosis with regard to the efficacy and safety of TNF antagonists in disease state is still an open question. Infliximab, etanercept, adalimumab, golimumab and complexes of these agents with TNF are all likely to bind to FcγR's and FcRn and to modulate a variety of cellular functions in vivo, but further research is needed to precisely define these interactions and possible differences among these agents. TNF antagonists may induce cytotoxicity of tmTNF-bearing cells by Fc-dependent mechanisms, including complement–dependent cytotoxicity (CDC) and ADCC. Complement activation by the classical pathway can be initiated by the binding of C1q to the CH2 domain in the Fc region of cell-bound antibodies or Fc-fusion proteins. Cross-linking of cell-bound Fc-containing molecules by C1q can initiate the complement cascade, leading to formation of the membrane attack complex, pore formation and cell lysis. Macrophages and NK cells mediate ADCC by binding their FcγRs to the CH2 domains of Fc-containing molecules of the target cell, thereby crosslinking the FcRs and inducing enzyme-mediated lysis of the target cell. Both CDC and ADCC require a threshold level of density of target cell-bound Fc-containing molecules to trigger cell lysis. Despite a vast amount of data supporting a role for TNF in lymphoid organization, innate immunity and adaptive immunity, there is relatively little direct evidence that TNF antagonists are immunosuppressive in clinical use. Many of the hallmarks of chronic inflammation such as leukocyte recruitment, activation, proliferation, and production of inflammatory mediators are reduced by TNF antagonist therapy and thus have their mechanistic link to TNF empirically confirmed. As more than 100 cytokines and chemokines have been identified, many of them studied in TNF antagonist–treated patients, a concept has emerged that TNF is at the top of the proinflammatory cytokine cascade (Feldmann, 2002). In fact, the overall effect of TNF antagonism on the immune system of patients appears to be one of normalizing immune homeostasis, with some evidence for immune enhancement (Maurice et al., 1999). Normalization of immune function by TNF antagonists involves down-regulation of the inflammation and immune reactions that drive RA, Crohn's disease, psoriasis and other diseases. It appears that TNF antagonism can reverse some disease-related immune suppression and, in some cases, it enhances the immune response to foreign antigens. One exception to this generalization is the class effect of TNF antagonists whereby they appear to impair host defense against microbial infections, particularly reactivation of intracellular bacterial infections, which have been observed in a small percentage of treated patients. These TNF-antagonists are now licensed for use in wide variety of diseases such as rheumatoid arthritis, Crohn’s disease, patients with TRAPS, advanced heart failure, etc (Ito et al., 1987; Paya et al., 1988; Nokta et al., 1991; Schmitt et al., 1992; Franklin, 1999; Jones and

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Moreland, 1999; Bell and Kamm, 2000; Galon et al., 2000; Furst et al., 2003; Mian et al., 2005). In cancer patients, new approaches combining TNFα with other cytokines and tumoricidal agents have showed promising results (Pluzanska, 1994). The preclinical studies and early clinical trials have resulted in the evaluation of the safety of recombinant TNFα on humans with cancer (Saks and Rosenblum, 1992). However, anti-cancer treatment with recombinant TNFα has many adverse side effects with modest benefits even at low doses (Skillings et al., 1992). High doses of TNFα can induce tumour regression, but are associated with severe adverse effects. Conversely, low dose of TNFα can be tolerated, but was associated with cancer progression in some preclinical studies (Mocellin et al., 2005). Due to these reasons, the role of TNF in cancer therapy is debated in many countries (Ten Hagen et al., 2001; Mocellin et al., 2005; Balkwill, 2006; Lejeune et al., 2006; Cornett et al., 2006).

Conclusion TNF and its membrane-bound and soluble receptors play unique and pivotal roles as master conductors of immune function (innate immunity vs adaptive immunity) and cellular survival or death in different cells/tissues. Most of the studies suggest that TNF concentrations, receptor distribution and duration of TNF stimulation may determine whether the cytokine has beneficial or adverse effects on cell types. Although the picture is still very complex and far from being fully drawn, we can sense that there is a balance between immunostimulatory versus immunoregulatory functions and cellular survival versus death. The value of genotyping for TNFA polymorphisms as a prognostic marker warrants further development. Ligand-neutralizing strategies (such as anti-TNF therapies) have been pursued and successfully used in the clinic to treat various disorders, however, they failed to show any benefit in surgically related conditions. The TNF antagonists infliximab, etanercept, adalimumab, certolizumab and golimumab that differ in their molecular structures and pharmacokinetic properties are all effective therapeutic agents in different diseases. Their strong clinical efficacy and the potent neutralization of sTNF and tmTNF suggest that they achieve efficacy by preventing TNF from inducing TNFR mediated cellular functions. These functions include cell activation, cell proliferation, cytokine and chemokine production and a sequel of these functions such as cell recruitment, inflammation, immune regulation, angiogenesis and extracellular matrix degradation. Thus, TNF family is very rich in exciting possibilities for future therapeutic strategies tailored to specific functional arms of the immune system and cellular survival or death. TNFα is not the only mediator involved in cellular survival versus death, therefore using combination therapies may be useful. Furthermore, relatively little attention has been paid to the contribution of LT to the pathogenesis of immune-mediated inflammatory diseases or to whether the efficacy of etanercept depends on its ability to bind LT ligands (namely LTα3 and LTα2β1). New insights into the mechanisms of action of TNF antagonists and related distinctions between the agents will undoubtedly emerge as greater numbers of diseases are treated by TNF blockade, however, these speculations require further evaluations with animal experiments and clinical trial. Also continuous research and improvement of model systems are required

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to gain more insights into the complex functions of TNF in physiological and pathological conditions.

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In: Tumor Necrosis Factor Editor: Toma P. Rossard

ISBN: 978-1-60741-708-8 © 2009 Nova Science Publishers, Inc.

Chapter II

Tumour Necrosis Factor Alpha Neutralization in the Medical Management of Crohn’s Disease Neil Gerard Docherty1,2 and P. Ronan O’Connell1,2 1

School of Medicine and Medical Sciences, University College Dublin Belfield, Dublin 4, Ireland, and 2Surgical Professorial Unit, St Vincent’s University Hospital, Elm Park, Dublin 4 Ireland

Abstract Crohn’s disease (CD) is a chronic and debilitating inflammatory condition affecting principally the small intestine and colon. Tumour necrosis factor alpha (TNF-α) plays a key role in the pathophysiology of CD, most prominently via its role in intestinal macrophage and T-lymphocyte activation and through its effects on intestinal permeability and fibroblast mediated extracellular matrix remodeling. Polymorphisms in TNF-α receptor genes have also been implicated as disease modifiers in inflammatory bowel disease. In 1998, the anti-TNF-α monoclonal antibody infliximab was approved by the U.S Federal Drugs Administration (F.D.A) for use in the medical treatment of fistulae in CD. The drug is now licensed for induction and maintenance of remission of disease. More recently the humanized anti-TNF-α antibody adalimumab and the pegylated human antiTNF-α antibody fragment certolizumab have been licensed for use in CD. Trials of other anti-TNF-α agents in the treatment of CD, notably the CDP571 antibody, and the soluble type I and type II TNF-α receptors, etanercept and onercept, have failed to show sufficient efficacy to warrant FDA approval. In this chapter a background to the pathophysiology of CD is provided, the role of TNF-α as a key mediator of inflammation in CD is described and the process by which TNF-α neutralization has come to represent an important therapeutic tool in the medical management of CD is discussed. Finally, some of the concerns regarding the safety of TNF-α neutralizing therapy are reviewed (232).

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Introduction Crohn’s disease (CD) is a chronic relapsing inflammatory disease of the gastrointestinal tract most commonly occurring at the terminal ileum, but also frequently affecting the colon and anorectal area. In contrast, ulcerative colitis (UC) (the other major subtype of inflammatory bowel disease), is confined to the large bowel. The typical lesions of CD can occasionally be found elsewhere in the gastrointestinal tract, and indeed CD can occur anywhere from the mouth to the anus. The complexity of CD pathogenesis and the fact that its precise aetiology remains uncertain, in part explains why the disease continues to present such a significant challenge in terms of successful medical management. This is reflected in the continuing requirement for surgical intervention to treat complications of CD. Surgery is however rarely curative and itself can lead to complications such as short bowel syndrome. However, in recent years, improved understanding of the molecular and cellular basis of the inflammatory response has allowed for rational design of pharmacological interventions that target key steps in intestinal inflammation. Targeting of TNF-α activity in CD with biological based therapies represents a case in point.

CD-A Historical Perspective The definitive description of Crohn’s disease is attributed to Burril B Crohn, Leon Ginzberg and Gordon Oppenheimer and stems from clinical and pathological findings in a cohort of 14 patients operated on by A.A Berg at The Mount Sinai Hospital, New York who were found to have tuberculin negative, non-caseating, granulomatous terminal ileitis. These data were presented in 1932 to the American Gastroenterology and American Medical Associations and subsequently published in The Journal of The American Medical Association under the title of Regional Ileitis-A Pathologic and Clinical Entity.[1,2] One year later a further article was published in Surgery, Gynecology and Obstetrics to which the eponymous title of Chronic Cicatrizing Enteritis: Regional Ileitis (Crohn) was given.

Common Symptoms, Diagnosis and ExtraIntestinal Manifestations CD most frequently occurs in the ileum and colon and gives rise to a variety of symptoms dependent on severity, disease location and complicating factors. At diagnosis, a clinical picture indicative of intestinal inflammation frequently predominates and is characterized by fever, abdominal pain with palpable mass, diarrhoea with or without faecal blood, malnutrition and in paediatric patients, evidence of growth retardation. The presence of perianal disease provides for the provisional exclusion of ulcerative colitis as a cause. Endoscopic evidence of non-continuous aphthous ulceration and cobblestoning of the mucosa is typical of CD. Histological visualization of deep fissuring of the mucosa explains the cobblestoning effect observable on endoscopy. Histology typically demonstrates blunting and

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shortening of intestinal villi with transmural inflammation [3]. The presence of a non caseating, granulomatous reaction in the absence of clinical evidence of tuberculosis is considered pathognomic for CD. Over 50% patients with CD demonstrate seropositivity for anti-Saccharomyces Cervisae antibodies, however, in contrast to patients with ulcerative colitis, patients with CD have a low prevalence of anti-neutrophil cytoplasmic antibody positivity [4]. HLA-B27 linked extra-intestinal manifestations such as ankylosing spondylitis and primary sclerosing cholangitis occur less frequently in CD than in ulcerative colitis [4] . Standardized assessment of CD activity post-diagnosis is measured using a disease activity index which relies on a cumulative scoring system based on the combination of scores for eight differentially weighted clinical and biochemical observations [5].

Clinical Patterns and Progression (The Vienna Classification) A definitive classification system for the behaviour and location of CD was established in 1998 and is termed The Vienna Classification [6]. It consists of a triple coding system for age at diagnosis (A), location (L) and behaviour (B). The designation A1 or A2 refers to diagnosis having been made at under or over the age of 40 respectively. Grading for location (L) is made as L1 (terminal ileal), L2 (colonic), L3 (ileocolonic) or L4 (upper gastrointestinal). Behaviour (B) is classed as non-stricturing, non penetrating (B1), stricturing (B2) or penetrating (B3). While the purely inflammatory designation of B1 predominates at diagnosis, at least 50% of patients progress to a more complicated phenotype (B2 or B3) within 10 years [7]. Progression to B2 has been shown to be more common in isolated ileal disease while progression to B3 is more common in ileo-colonic disease. Changes in disease location occur with much less frequency [7]. Stricturing disease is characterized by transmural, fibrotic thickening of the intestinal wall associated with the accumulation of fibrofatty plaques which emerge from the mesentery to interpose themselves between the muscular and serosal layers of the bowel. Strictures frequently progress to the point of causing intestinal obstructions that necessitate surgical resection. Penetrating disease is characterized by entero-cutaneous, entero-vesicular and entero-enteric fistulae formation with the formation of abscesses, and also frequently requires surgical management.

Medical and Surgical Management In brief, the mainstay of medical therapy in active CD is a step-up, step-down usage of medications, dependent on disease activity. In mild disease, the use of more benign antiinflammatory agents [5-aminosalylic acid) predominates and progresses to the use of oral corticosteroids (prednisone, budesonide) in moderate disease. In moderate to severe disease the use of parenteral corticosteroids and immunomodulators/immunosuppressants (azathioprine, methotrexate and cyclosporine A) is used with the more recent addition of antiTNF-α based therapies (infliximab, adalimumab and certolizumab), these being of particular

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use in refractory disease. There is also a role for broad spectrum antibiotic use (e.g metronidazole and ciprofloxacin) across the varying grades of disease activity in CD and in disease complicated by fistulae [8]. The value of maintenance therapy in CD is questionable and contrasts with the beneficial results reported for use of maintanence aminosalicylates in ulcerative colitis. However, evolving evidence points to the judicious use of azathiorprine, methotrexate and anti-TNF-α therapies as being useful in providing for extended periods of remission following control of acutely active CD [8]. Surgical intervention in stricturing CD ranges from balloon dilatation and stenting to stricturoplasty (surgical widening of the stricuterd segment without resection to segmental resection of stricutered segments of intestine). However, the disease frequently recurs at, or proximal to, the site of anastomosis. Surgery is also required for the excision of fistulating bowel when conservative measures fail. The typical features of the presentation, progression and medical management of CD are summarized in Figure 1.

Figure 1. Typical continuum of CD diagnosis and management The flow diagram summarizes the typical continuum of disease progression, treatment and long-term issues involved in the management of CD as a chronic and debilitating disease.

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Development of Intestinal Inflammation in Crohn’s Disease Physiological versus Pathological Inflammation, Barrier Function and Chronic Intestinal Inflammatory Responses A distinguishing feature of inflammatory responses within the intestine is that under normal conditions, both the small intestine and colon manifest a state of controlled inflammation characterized by the presence of a mucosal immune system intimately linked to local lymphoid aggregates (e.g Peyers patches in the ileum) and local mesenteric lymph nodes. The presence and complexity of mucosal immunity parallels the graded increase in bacterial colonization of the intestinal lumen occurring aborally from the duodenum to the rectum. Normally, distinct separation of the luminal microflora from the wall of the intestine is maintained via the mucous gel layer and epithelial barrier function of the mucosa. Active sampling and antigen presenting activity of the luminal bacterial load by dendritic cells (DC) [9]and mucosal epithelial microfold cells (M cells) [10] allow maintenance of tolerance to normal gut flora and activation of localized innate and adaptive responses to the presence of pathogenic bacteria. Secretory IgA antibodies and anti-microbial defensin peptides, derived from Paneth cells, also act to ensure that an appropriate physiological immune response prevents chronic intestinal inflammation [11, 12]. Pathological intestinal inflammation occurs in response to perturbations either in the demarcation between host and microfloral cell populations, alterations in tolerance to normal luminal antigens, or the presence of pathogenic and /or invasive species that hyperactivate the mucosal immune system. It is therefore plausible to suggest that a mixture of genetic, environmental and infectious factors may combine to lead to pathological inflammation in CD. Agreement on a definitive initiating factor in the pathological inflammation observed in CD remains elusive, however, at some stage in the pathogenesis increased exposure of the deeper layers of the intestinal wall to normal gut microflora plays a key role in the progression of inflammation. This is underlined by the fact that in spontaneous and genetically modified mouse models of IBD (e.g. interleukin-10 (IL-10) knockout and SAM1/Yit), there is an absolute requirement for intestinal colonization, with no disease observed in these lines when born and maintained in a germ free environment [13, 14].

Molecular and Cellular Aspects of the Chronic Intestinal Inflammatory Response Immunologically, the pattern of inflammation observed in CD is considered to be a Tlymphocyte driven process [15]. The activation and persistence of T-lymphocytes in the intestinal lamina propria, lymphoid aggregates and mesenteric lymph nodes and their circulation and reinfiltration of the mucosal epithelium marks a crucial stage in the development of inflammation. The lymphocyte is consequently an important target of a

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variety of the commonly used medications in CD including calcineurin inhibitors and TNF-α neutralizing therapies. Engagement of the T-cell receptor by MHC class II restricted antigen on antigen presenting cells (APC) such as dendritic cells and macrophages is a pre-requisite for T helper (TH) lymphocyte activation. Evidence suggests that in CD, increased activation of the CD4 positive TH1 and TH17 lineages and a decrease in the prevalence of Fox p3 positive T regulatory (TREG) cells is key to disease progression, a profile that is reversed upon the induction of remission [16, 17]. The preponderance of a TH1 response is reliant on reciprocal exchange of pro-inflammatory cytokines between the T-lymphocytes and the APC. Macrophage derived TNF-α and interleukin-1 (IL-1) are important in this exchange and that reciprocal secretion of the macrophage activation factor, interferon gamma (IFN-γ) and the Tlymphocyte mitogen interleukin-2 represents an important axis operative in the amplification of local TH1 driven cell-mediated immunity and tissue destruction observed in CD [18] . Production of interleukin-12 (IL-12) in myeloid APC cell populations in response to IFN- γ acts to increase T-lymphocyte production of IFN-γ in an amplification loop which leads to selective polarization of the TH1 phenotype [19]. IFN-γ also mediates repression of interleukin-4 (IL-4) expression in uncommitted Tlymphocytes via signal transducer and activator of transcription 3 (STAT3) mediated activation of a 3’ silencer element on the IL-4 gene, thereby inhibiting TH2 maturation [20] (Figure 2.). This is likely to underpin the observation that in CD, cell mediated reactions are more prominent than humoral responses. Release of cytotoxic factors such as highly reactive oxygen species and pro-inflammatory arachadonic acid metabolites from T-lymphocyte primed innate cells is a key feature of the cellular nature of tissue injury in the TH1 response. However, a number of recent studies have questioned this paradigm given that a somewhat impaired innate immune response in terms of phagocytosis and the oxidative burst occurs in neutrophils and macrophages isolated from patients with CD [21, 22]. Recently activation of a subset of CD4 positive T-lymphocytes termed TH17 (due to secretion of interleukin-17 (IL-17)) has been highlighted in CD [23]. Differentiation of the TH17 subtype has been shown to be dependent on co-stimulatory effects of transforming growth factor beta-1 (TGF-β1) and acute phase proteins such as interleukin -6 (IL-6), which has been proven to be a TNF-α inducible gene [24, 25]. TH17 proliferation and proinflammatory cytokine production occur secondary to the effects of interleukin-23 (IL-23) stimulation due to preferential expression of the IL-23 receptor on cells of this lineage [26] (Figure 2). Auto-inhibition of T-lymphocyte responses can be mediated via anergic T-lymphocytes that lack the ability to transmit the IL-2 mediated activatory second signal and by TREG cells and T suppressor cells (TS), both of which can be distinguished on the basis of differences in cluster of differentiation markers and anti-inflammatory cytokine release. Tolerance induced by type 1 TREG cells occurs via the effects of TGF-β1 and IL-10 on the proliferation and activation state of innate immune cells and the other polarized T-lymphocyte phenotypes [27]. Loss of this subset in CD may reflect changes in TGF-β1 bioactivity in the presence of a pro-inflammatory milieu of other cytokines such as IL-6 [24]. T-lymphocyte activation in CD is accompanied by increased infiltration by leukocytes of both the innate and adaptive arms of the immune response. Infiltration from the mucosal

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microvascular bed occurs on the activated capillary endothelium via leukocyte docking through integrin α4β7 mediated interaction with adhesion molecules such as the gut homing, mucosal vascular addressin (MAdCAM-1) [28, 29] .

Figure 2. Cytokine Networks in TH1 and TH17 Polarization 1) Engagement of MHC class II restricted antigen by the T-cell receptor on CD4 positive TH lymphocytes leads to reciprocal exchange of IFN-γ, TNF-α and IL-12 between the T-lymphocyte and the APC, leading to the promotion of TH1 phenotype maturation. 2) Inhibition of IL-4 expression by IFN-γ aids in suppressing the development of the TH2 phenotype. 3) IL-1 linked expression of IL-2 and the subsequent autocrine/paracrine effects of IL-2 lead to activation of a default proliferation pathway in the T-lymphocyte. 4) TGF-β1 in combination with IL-6 and 5) via the proliferatory effects of IL-23, causes selective polarisation of the IL-17 secreting TH17 phenotype at the expense of the production of TREG cells.

Perpetuation of inflammation at the site of reaction is promoted by damage to the mucosal epithelium, allowing for mass penetration of bacterial antigenic load into the intestinal wall. Incorrect post-transcriptional modification of mucin genes such as MUC2 with subsequent thinning of the mucous gel layer, apoptosis of secretory and absorptive mucosal epithelial cells and the reported negative effect of cytokines such as TNF-α on epithelial junction patency are all implicated in this phenomenon [30, 31]. Inflammation leads to the manifestation of the classical symptoms of CD. Mucosal inflammation, loss of epithelial structure and subsequent ulceration leads to faecal blood loss, while associated changes in absorption and secretion rates contribute to diarrhoea and malabsorption. As inflammation spreads transmurally in CD, functional aspects of gut motility become disturbed secondary to inflammation of the muscularis mucosae and muscularis propria, and loss of the interstitial cells of Cajal [32]. A specific feature of CD inflammation is the formation of non-caseating epithelioid granulomas. Given the current view that CD is non-infectious in origin such features, more characteristic of a primary bacterial infection, remain perplexing. A prospective French study published in 2005 followed a cohort of 188 patients with CD diagnosed in 1994 and 1995, reported a five year incidence of granuloma formation of 37% [33] . This raises the

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possibility that the presence or absence of granulomatous disease may be of significance in disease classification and subsequent treatment strategies.

Genetic, Environmental, Infectious, or All of the Above? Theories in Crohn’s Disease Aetiology The role of genetics A role for genetic susceptibility in CD in populations of European/Middle Eastern descent is clear from studies demonstrating that monozygotic concordance rates in CD are high (58.3% in one Swedish study) and the incidence in first degree relatives is also elevated, particularly in Ashkenazi Jewish populations [34, 35]. However, CD is much less common in the Japanese population, and shows less evidence of familial linkage. When it occurs it preferentially affects males, essentially reversing observations in Western societies [36]. Genome wide studies across a large population of a total of 3,230 patients and 4,829 controls recently reported 33 candidate CD susceptibility loci on multiple chromosomes [37]. Short nucleotides polymorphisms in the NOD2/CARD15 gene (16q12), ATG16L1 (2q37) and IRGM (5q33) genes are of particular interest given that they are all specific for CD, and all code for proteins involved in autophagic processing of intracellular pathogens [37]. Among the most extensively studied changes in these regions are one frameshift and two missense single nucleotide polymorphisms (SNPs) in the caspase activation and recruitment 15 domain (CARD15) of the gene coding for the intracellular bacterial pattern recognition receptor, nucleotide-binding oligomerization domain containing 2 (NOD2) protein on chromosome 16q12. In 2002, a large multi-centre study involving patients in Belgium, Denmark, France, Germany, Ireland, Italy, Spain, and Sweden demonstrated the presence of at least one mutant allele in 50% of patients with CD, 17% of whom were homozygous for mutant alleles [38]. Homozygous patients showed an earlier age of onset and a significantly higher risk of developing fibrostenosing disease. Notably, these mutations are rare in African American and Asian patients with CD [39, 40]. NOD2 mutations are thought to be linked to an impaired tolerance to normal microflora occurring secondary to reduced NFkappaB activation in APCs during processing of luminal bacteria derived antigen [41]. Evidence from mice heterozygous for NOD2 mutations also shows reduced production of defensins by intestinal Paneth cells and reflects similar findings in CD patients [42, 43]. A caveat in the last observation is that Paneth cell depletion can occur as a result of inflammatory damage, independent of NOD2 mutation, and therefore whether a direct cause-effect relationship exists between NOD2 mutation and altered defensin production requires further clarification [44]. Other genes of particular interest in CD genetics are the IL-23 receptor and a number of its downstream signalling intermediates such as STAT3 [37, 45]. However, mutations in these genes are more likely to act as disease modifiers given that they are also present in ulcerative colitis. Not withstanding, they are still of great interest given recent identification of the TH 17 lineage as a key immunological marker of CD inflammation.

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Environmental theories A role for the hygiene hypothesis of autoimmune disease in the development of CD is supported by a number of lines of evidence. Firstly, the prevelance of CD in both the North American and European populations points to a role for increased sanitation and/or dietary factors as a factor in the first versus third world differences in incidence. A non-genetic component in this finding is supported by studies demonstrating that lack of access to tap water, lower birth rank and a large number of siblings is relatively protective in terms of CD [46, 47]. Interestingly, an increased incidence of CD has occurred in parallel with industrialization in Southern mainland China and Hong Kong [48]. Conversely, a lower incidence of CD has been noted within Irish traveller groups, a subgroup within a first world society in whom relatively poorer access to sanitation and cramped living conditions have traditionally been a notable feature [49]. These observations suggest that differences in sanitation and living conditions, as opposed to dietary changes, are instrumental in the differences in disease rates observed between societies. Smoking is a well established risk factor for CD, and for reactivation of quiescent disease. Additionally smoking cessation has been proven to be of value in preventing postoperative recurrence in CD [50]. How smoking habit interacts with the hygiene hypothesis represents an interesting question, i.e are smokers in less developed societies any more prone to develop CD relative to their non-smoker peers? Bacterial causes A role for enhanced bacterial translocation as a consequence of impaired barrier function is well established in CD. However a number of theories point to a primary role for bacteria in the aetiological basis of CD. Despite its original definition as being non tuberculoid in nature, both culture and DNA based techniques have shown mycobacterium avium paratuberculosis (MAP) positivity in CD tissue [51, 52], although findings have been inconsistent [53]. Initial optimisim about the efficacy of anti-mycobacterial triple therapy in CD has recently been tempered as a result of disappointing long term outcomes regarding disease reactivation [54]. Adherent and invasive strains of E.Coli, have also been shown to preferentially colonise CD mucosa [55]. The inability to determine whether these events are cause or effect in clinical samples limits conclusions regarding their role in aetiology per se. The “cold-chain” hypothesis of Crohn’s disease links specific lifestyle changes to microbiology and genetics in postulating that cold resistant bacteria such as yersinia enterocolitica might predominate in refrigerated food and lead to CD in genetically predisposed individuals [56]. The concept of intestinal dysbiosis represents a further theory implicating a role for bacteria in CD. Studies have shown profound changes in the number and variety of bacteria in active CD although some of these changes may be the result of diarrhoea and inflammation as opposed to inherent differences in the microbiota. However, an increased total bacterial count and a relative preponderance of subspecies of the phyla Bacteroidetes and Proteobacteria at the expense of Firmcutes is characteristic of the CD ileum independent of

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inflammation, suggesting that some disease specific differences in colonization may contribute to disease development [57]. To date, studies of the impact of probiotic therapy as a method of correcting intestinal dysbiosis and thereby attenuating disease activity in CD have been inconclusive.

Role of TNF-α in Crohn’s Disease Pathophysiology TNF-α is implicated in the pathophysiology of CD in both pro-apoptotic and antiapoptotic pathways in epithelial cells and T-lymphocytes, via its effects on epithelial junctional complexes in the mucosal epithelium, through its effects on endothelial activation and via modulation of the extracellular matrix remodelling response (Figure 3).

Figure 3. TNF-α in CD pathophysiology A low power transmural image of the resected terminal ileum of a patient with fibrostenosing CD highlighting the key histopathological features of the lesion observed in this phenotype and annotating the areas of CD pathophysiology in which TNF-α is proposed to play a key role. A) The activation and apoptotic resistance phenotype of T-lymphocytes B) Epithelial barrier dysfunction C) Endothelial activation and leukocyte diapedesis D) Extracellular matrix remodeling (H&E x4) Abbreviations:M-mucosa, MM-muscularis mucosae, SM-submucosa, CM-circular muscle, LMlongtidudinal muscle, S-serosa, Galyx-glycocalyx, IL-intestinal lumen, VT-villous tip LP- lamina propria, ME- mucosal epithelium, MV-microvasculature, FB-fibroblast, MFB-myofibroblast, V-vein, A-artery Cellular abbreviations: MØ-macrophage, m-microfold cell, D-dendritic cell, T0 –naive Tlymphocyte, TH1–T helper 1 lymphocyte TH17–T helper 17 lymphocyte, HCV-high capillary venule, MicroV-enterocyte microvilli. Molecular abbreviations: IFNγ-interferon gamma, IL-23-interleukin 23, IL-6-intereukin 6, TGF-β1-transforming growth factor beta 1, mTNF-α-membrane bound TNF alpha, MadCAM- mucosal addressin cellular adhesion molecule, VCAM-vascular cell adhesion molecule, ICAM-intercellular adhesion molecule, AAC-apical adhesion complex.

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Seminal Publications on TNF-α in CD The first report documenting increased expression of TNF-α in CD appeared in 1990 in which the relative increase in TNF-α secretion in CD versus ulcerative colitis was documented in biopsy specimens of children with inflammatory bowel disease [58]. The explanation for this finding was attributed to the relative increase in T-lymphocyte activity in CD. In this study, a reduction in TNF-α levels occurred in parallel with immunosuppressant induced remission of disease, implicating a key role for TNF-α in intestinal inflammation. A follow up immunohistochemical study demonstrated that TNF-α immunoreactivity in CD localized primarily to mononuclear cells within the lamina propria and furthermore extended to dense aggregates of macrophages within the submucosa [59]. In progressive disease in which fibrofatty expansion of the mesentery occurs, TNF-α has more recently been identified as a key cytokine released by the expanded adipocyte population [60]. In the intervening years, the repercussions of the findings of these original reports have been clarified as a result of the elucidation of the precise molecular mechanisms by which TNF-α acts as a mediator of inflammation in patients with CD (Figure 2). This combined knowledge underpins current day use of TNF-α neutralization in medical management of CD

TNF-α and T-lymphocyte activation A key feature of the activated T-lymphocyte is resistance to apoptosis which confers longevity and leads to accumulation at sites of inflammation. T-lymphocytes from inflamed mucosa of patients with CD are resistant to apoptosis partly through a reduced expression of the pro-apoptotic protein Bax and a coincident elevation in Bcl(XL)/Bax ratios [61]. A role for the TNF-α/NFkappaB survival pathway in this phenomenon is implied from studies in which TNF-α neutralization in stimulated Jurkat T lymphocyte cultures results in apoptosis, characterized by a decrease in Bcl-2/Bax ratios [62, 63]. In both studies these response were specific for activated T-lymphocytes. These studies also demonstrate that autocrine signalling via the membrane bound form of TNF-α (mTNF-α) predominates in T-lymphocyte resistance to apoptosis.

Macrophages and TNF-α in CD Development of a TH1 or TH2 response is determined not only by the nature of the antigenic stimulus, but at least in part by the type of APC involved in activation of the naïve T-lymphocyte. Macrophages have been demonstrated to selectively induce a TH1 response characterized by TNF-α and IL-12 release following toll like receptor mediated antigenic detection, a process amplified by reciprocal release of IFNγ on the part of the T-lymphocyte [64]. Early identification of TNF-α expression in myeloid cells in CD tissue demonstrates that macrophages constitute an important source of TNF-α in CD. Release of soluble TNF-α by the macrophage is then able to mediate a number of the injury processes in CD inflammation as listed below.

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TNF-α and Endothelial Activation in CD TNF-α has been demonstrated to induce the expression of various adhesion molecules involved in the rolling and diapedesis of leukocytes in the inflamed intestine. Studies in human intestinal microvascular endothelial cells demonstrate NFkappaB mediated induction of vascular cell adhesion molecule-1 (VCAM-1) and the mucosal addressin cell adhesion molecule (MadCAM) by soluble TNF-α [28] . MadCAM induction by TNF-α has also been demonstrated in cultured murine endothelial cells [29].

Evidence That TNF-α Affects Epithelial Barrier Function in CD Epithelial barrier dysfunction can become manifest in response to various alterations, notably in response to reduced mucous gel patency, epithelial apoptosis, downregulation of junctional adhesion complexes and via increases in transcellular transport of antigen. TNF-α has been implicated in all of the above in CD pathogenesis Treatment of T84 human colonic epithelial cells with TNF-α and IFNγ leads to a synergistic increase in apoptosis, internalization of the junctional complex proteins, junctional adhesion molecule-1 (JAM1), occludin and claudin and cytoplasmic redistribution of E-cadherin [31] Interestingly the effects on barrier function were not prevented in these studies when apoptosis was blocked using the pan-caspase inhibitor ZVAD-FMK. Further studies in Caco2 cells demonstrate that these changes are associated with increases in myosin light chain kinase expression and activity and that the synergistic effects of TNF-α and IFNγ are mediated by IFNγ induced up-regulation of TNF receptor subtype 2 [65]. These fundamental data are supported in part by data from sigmoid colon biopsies form patients with active CD [66]. Biopsies taken before and 14 days after infliximab infusion clearly show enhanced electrical resistance (indicative of an improvement in barrier function), however in this case apopotosis was implicated, as improvements coincided with reduced apoptotic indices in the epithelium after therapy. In a separate study that examined the effect of infliximab on gut permeability, patients with CD were shown to have a reduced trans-intestinal flux of 51CrEDTA indicative of an improvement in barrier function [67]. Increases in the permeability of ileal mucosa to protein antigen in CD, have also been demonstrated to be, at least in part dependent on an increase in endocytic uptake. An increase in horseradish peroxidase uptake into endosomes in ileal mucosa from patients with CD has been shown [68]. This correlated within samples to the mucosal TNF-α level. To confirm a causal role for TNF-α, T84 colonic epithelial cells were cultured on filter supports and exposed to low dose TNF-α. The flux of horseradish peroxidase to the basolateral side of the filter was increased in response to TNF-α in the absence of a change in electrical resistance.

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Conflicting Evidence Regarding the Effect of TNF-α on Extracellular Matrix Remodelling in CD The major phenotypes in complicated CD are characterized by essentially opposing histopathological features in terms of extracellular matrix dynamics. The penetrating phenotype is characterized by the formation of fistulae which burrow in deep fissures from the mucosal epithelium to the serosa whereupon they form aberrant linkages between loops of bowel, the abdominal wall and/or the urinary bladder. In the stricturing phenotype, progressive accumulation of fibrous scar occurs in the muscular and serosal layers of the intestinal wall leads to luminal obstruction. Altered accumulation and activity of intestinal fibroblasts is implicated in mediating damage in both phenotypes [69, 70] . Although some reports had suggested that treatment with anti TNF-α neutralizing antibody led to an increased incidence of strictures, a more recent larger multivariate analysis of the data has discounted this [71] At the cellular level, conflicting evidence also exists regarding the role of TNF-α in fibroblast behaviour in CD. Collagen I and tissue inhibitor of matrix metalloproteinase I (TIMP-1) expression have been shown to be induced in vitro in intestinal myofibroblast cultures by TNF-α and insulin like growth factor 1 treatment, via a TNF-α receptor 2 dependent pathway [72] . Induction of myofibroblast proliferation in CD derived bowel explants has been shown to be increased by TNF-α via transcriptional induction of the DNAmismatch repair gene MutS homologue 2 [73]. Conversely it has been shown that TNF-α increases apoptosis in dermal fibroblasts via activation of caspase 8 downstream of death domain pathway activation [74]. TNF-α has also been shown to antagonize the transcriptional activation of connective tissue growth factor (CTGF) in colonic seromuscular fibroblast explant culture [75] . A recent study of a cohort of 205 patients with CD demonstrated that the TNFRSF1A +36 mutation was negatively associated with stricturing disease. However the functional consequences of this polymorphism are not as yet described, thus this observation cannot at present be considered as being supportive of either a positive or negative role for TNF-α signalling in intestinal extracellular matrix remodelling in CD [76] . In consideration of the role of TNF-α in fibroblast activity, it is important to bear in mind its potential indirect effects in vivo which are unable to be adequately modelled in the cell culture setting. For example, TNF-α is known to be a component of the TH1 response in which macrophage accumulation is a key feature. Macrophages in turn are well recognized to be a major source of TGF-β1, which in addition to contributing to clonal expansion of T H17 cells is well recognized to be a major pro-fibrotic cytokine in stricture formation [77].

Biological Therapy for CD Monoclonal antibodies are to date the most exploited of biological therapies, while soluble cell surface receptor fragments constitute another emerging class of protein based biological agent.

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The use of monoclonal antibodies in modern medicine stems from the original discovery that secreted, monospecific antibody clones could be produced by fusion of myeloma derived Blymphocytes with healthy murine spleen derived B-lymphocytes, a discovery credited to Köhler and Milstein in 1975, for which they were awarded a Nobel Prize in Physiology and Medicine in 1984 [78]. Such cells are termed hybridomas. Prior immunization of mice with the antigen to be targeted allows generation of a polyclonal response and following subsequent hybridoma production, clones with optimal antigen affinity and avidity are selected for purification. The retention of murine specific characteristics in the antibody structure are involved in generation of neutralizing antibody in treatment experienced patients and has driven efforts to humanize monoclonal antibodies, particularly in their FC domains, a process pioneered in 1988 by Greg Winter and colleagues at the University of Cambridge [79]. The first biological therapy to be licensed for use in humans was a murine CD3 specific transplant rejection drug, Muromonab, which was FDA approved in 1986 for use in steroid resistant organ transplant recipients [80]. This initial use of antibody mediated biological therapy in the control of the immune response in otherwise treatment resistant patients, draws an interesting parallel to the indications for use of TNF-α neutralizing antibodies in CD described below. Table 1 summarizes the range of TNF-α neutralizing therapies which have been the subject of clinical trials in CD, and highlights those that are now in clinical use following U.S. F.D.A. approval.

Infliximab (Remicade™) in Crohn’s Disease Management Development of infliximab Infliximab was the first biological agent to be approved for use in CD and remains the mainstay of anti-TNF-α based therapy in patients with CD. The antibody is a chimeric construct (75% human-constant regions /25% murine-variable regions) and was developed by researchers at New York University School of Medicine in the early 1990s and first used in the treatment of rheumatoid arthritis [81,82].

Phase II trials of infliximab in CD (1) Use in patients with actively draining fistulae In 1999 a study was published piloting the use of infliximab in CD patients with actively draining abdominal and perianal fistulae [83]. A total of 94 patients in these categories (90% perianal and 10% enterocutaneous) were randomized to receive intravenous (i.v) 5mg/kg or 10mg/kg infliximab or placebo administered in a 3 dose infusion regimen at 0, 2 and 6 weeks. Patients were then evaluated at 4 weekly intervals until week 18, with the primary endpoint established as a reduction of at least 50% in actively draining fistulae versus baseline for at least two consecutive visits. This endpoint was met in 68% of patients treated with the

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5mg/kg dose and 56% of patients with the 10mg/kg dose versus 26% in placebo treated patients. Rates for complete closure of fistulae for at least 4 weeks were 55% for the 5mg/kg group, 38% for the 10mg/kg group and 13% for the placebo group. Analysis of these results by the F.D.A. at the time of licensing application demonstrated that despite these very positive results, some of the data were less convincing, for example six patients who were originally reported to have achieved the primary end-point actually developed abscesses in the area of the draining fistula [84]. Table 1. TNF-α neutralizing therapies tested in CD. Biological Agent (trade name) Infliximab (Remicade)

Proprieter

Molecular Structure

Neutralizing Activity

Route of Delivery

Key trials for licensing

Johnson & Johnson

Neutralization of soluble and mTNF-α

i.v 5mg/kg bimonthly

ACCENT 1 and II

Adalimumab (Humira)

Abbott Laboratories

Chimeric 75% human IgG1 Fab/Human Fc Human IgG1

Certolizumab Pegol (Cimzia) CDP571 (Humicade)

UCB

s.c 40mg bimontly s.c 400mg monthly i.v 10mg/kg bimonthly

CLASSIC I and II CHARM PRECISE I and II

Etanercept (Enbrel)

Amgen/Wyeth

Neutralization of soluble and mTNF-α Neutralization of soluble and mTNF-α Neutralization of soluble and mTNF-α Reported not to affect T-lymphyocyte activation Neutralizes soluble TNF-α

Onercept (undisclosed)

Serono

CellTech

Pegylated humanised Fab 95% Human IgG4 (murine antigen determining region) Soluble TNF receptor p75/ human IgG1 Fc conjugate Soluble TNF receptor p55

Neutralizes soluble TNF-α

s.c 25mg (b.i.w) 8 weeks s.c 10,25,35 or 50mg (t.i.w) 8 weeks

Trials discontinued (lack of efficacy)

Trials discontinued (lack of efficacy) Trials Discontinued (lack of efficacy)

Table 1. describes the molecular nature, associated dosing regimens and trial outcomes of various TNF-α neutralizing therapies tested to date in CD.

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(2) Use in moderate to severe refractory disease Following successful pre-clinical trials of antibody mediated TNF-α neutralization in rodents [85], and a promising open label trial of infliximab involving 10 patients, 8 of whom achieved clinical remission (CDAI score indicating remission) after a single i.v dose of infliximab, [86], a larger study was undertaken. This was a multi-centre double blinded randomised controlled trial in which 108 patients with active CD were assigned to receive 5, 10 or 20mg/kg of infliximab or placebo in a single i.v infusion [87]. The primary end-point was to determine the percentages of patients achieving a reduction of 70 or more on the CDAI at one month after treatment. The results showed that in the 5mg/kg group 81% of patients achieved the primary end-point, of whom 40% achieved complete clinical remission. The primary end-point was achieved in 50% of the 10mg/kg group and 64% of the 20mg/kg group. Complete remission rates in both of these groups were 25%. These results demonstrated that infliximab induced a real and significant clinical response, as only 17% of the placebo group achieved the primary endpoint, of whom only 4% were found to be in complete remission. To examine how long remission might last in these patients, 73 responders were rerandomized at 12 weeks to receive 10mg/kg of infliximab or placebo every 8 weeks through to week 36 with follow-up out to week 48 [88]. In this study 53% of treated patients maintained remission through week 44 compared with only 20% in placebo treated patients. By week 48 the majority of treated patients had relapsed indicating that the effective duration of therapy lay around the 8-12 week mark. During this re-treatment study, the infusions were well tolerated and plasma levels of infliximab were stable. However, one case of lymphoma and one case of a lupus like syndrome were detected suggesting that there might be some concerns about the safety of anti-TNF-α therapy (discussed later).

Phase III Trials of Infliximab in CD A large randomised controlled trial led to FDA approval of infliximab as an induction and maintenance therapy for CD in June 2002. This study was entitled “A Crohn's Disease Clinical Study Evaluating Infliximab in a New Long Term Treatment Regimen” (ACCENT I) [89] The trial involved 55 sites across North America, Europe and Israel and recruited 573 patients with active CD as defined by a CDAI score between 220 and 400. All participants received 5mg/kg infliximab (i.v) and those who had a reduction of 70 points on the CDAI or an improvement equal or exceeding 25% over baseline score at 2 weeks were carried into randomized double blinded experiments consisting of 3 treatment arms. Of the 573 patients who received infliximab, 335 (59%) achieved this primary criterion. Following rerandomization, patients in the first group received placebo at weeks 2 and 6 and were then provided with 5mg/kg infliximab at 2 month intervals starting at 14 weeks and ending at week 30. Group 2 received 5mg/kg infliximab at weeks 2 and 6 and thereafter to week 30, while group 3 received infliximab 5mg/kg at weeks 2 and 6 followed by 10mg/kg dosing at 2 monthly intervals to week 30. Primary end-points were the percentage of patients in complete remission at week 30 and the median time to relapse in patients through week 54. In group 1,

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22% of patients were in remission at 30 weeks compared with 30% and 46% in groups 2 and 3 respectively. The median time to relapse was 19 weeks in the placebo group, 38 weeks in group 2 and greater than 54 weeks in group 3. A particular question posed was to examine the sustainability of remission in patients previously using steroids as measured at 54 weeks. Results were 9% for group 1, 24% for group 2 and 32% for group 3, indicating a steroid sparing effect for infliximab. In parallel to the ACCENT I trial, the ACCENT II trial [90] examined the effect of retreatment/maintenance therapy on patients with fistulae. A total of 306 patients were recruited from 45 sites in the United States of America, Europe and Israel and the study was carried out between January 200 and October 2001. Criteria for inclusion were that patients should be adults, infliximab naive at inclusion and presenting with single or multiple draining fistulae for a period of at least three months prior to the start of the study. The initial phase of the study was identical in randomization and treatment regimens to those used in the previously cited study that had led to infliximab licensing for fistulating disease [90]. Patients who responded to initial therapy (n=195 from 282 assessed) were re-randomized at week 14 to receive maintenance doses of placebo (n=99) or 5mg/kg infliximab every 8 weeks (n=96). Of the 87 patients who had no response in the primary study, 43 were assigned to receive 5mg/kg maintenance therapy and 44 assigned to placebo maintenance. Of the 99 responders originally assigned to receive placebo in phase 2, 50 crossed over to receive 5mg/kg infliximab after week 22 following relapse. In total, 195 patients were assessed for the primary end-point of the time to loss response through week 54 following withdrawal of therapy at week 46. In summary, the median time to a loss of response in placebo treated patients was 14 weeks after termination of the original 3 dose regimen (week 28 of overall study). In the maintenance therapy arm, median time to relapse was more than 40 weeks. At week 54, 36% of maintenance treated patients had retained a complete absence of fistulae versus 19% in the group who received placebo from week 14 onwards. As a result of this study, infliximab was FDA approved for use as a maintenance therapy for maintaining fistulae closure.

New Horizons on Infliximab Use in CD-Prevention of Recurrence Post-resection At the time of writing, a new application of infliximab has been developed by clinicians at the University of Pittsburgh [91]. As mentioned above, recurrence of disease following intestinal resection in patients with CD is a major cause of morbidity, for which medical therapy to date has proved to be of limited benefit. In this small single centre trial, 24 patients were randomly assigned to receive either placebo or 5mg/kg infliximab at 8 weekly intervals starting within 4 weeks of ileocolonic resection and continuing for 1 year. The percentage of patients with endoscopic recurrence at this time was set as the primary end-point. After 1 year, only 1 of 11 infliximab treated patients showed endoscopic recurrence compared to 11 of 13 patients treated with placebo. As a secondary end-point, histological evidence of recurrence occurred in 3 of the 11 patients treated with infliximab versus 11 of 13 in the

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placebo group. A larger trial is needed to establish whether infliximab may offer new theorapeutic options in post-surgical recurrence of CD.

Other TNF-α Neutralizing Drugs: Experience in CD Adalimumab (Humira™) Adalimumab is a fully humanized IgG1 which effectively neutralizes both soluble and membrane bound TNF-α. It was FDA approved for use in CD in February 2007 on the basis of the results obtained from rounds I and II of the “Clinical Assessment of Adalimumab Safety and Efficacy Studied as Induction Therapy in Crohn’s Disease” (CLASSIC) multicentre, double blinded, randomized controlled and dose ranging trials [92, 93]. In CLASSIC I, 299 anti-TNF-α naïve patients aged between 18 and 75, and with a CDAI score of between 220 and 450 were assigned to three treatment and one placebo arm. A total of 55 centres were involved in the trial with equally weighted participation. Treatments were administered at study entry and after 2 weeks, with the primary end-point of a reduction of at least 150 on the CDAI examined at week 4. Patients received dosages at week 0 and 2 of either: 40mg then 20mg s.c (group 1), 80mg then 40mg s.c (group 2) or 160mg then 80mg s.c. (group 3) or placebo (group 4). At week 4, all treatments regimens were significantly more effective than placebo in terms of the primary end-point (18% group 1, 24% group 2 and 36% group 3). In CLASSIC II, 276 patients who had a positive response to induction therapy in CLASSIC I were recruited to an open label study of the effectiveness of adalimumab as a maintenance therapy in CD. Patients in remission (CDAI<150) at week 4 of CLASSIC I, were assigned to receive weekly or bimonthly adalimumab (40mg s.c) or placebo. The primary end-point of sustained remission at week 56 was observed in 83% of the weekly and 79% of the bimonthly treated patients versus 44% of patients reassigned to placebo following successful induction therapy. A further study of the effectiveness of adalimumab as a maintenance therapy was examined in a 92 site open label trial (“Crohn’s Trial of the Fully Humanized Antibody Adalimumab for Remission Maintenance” (CHARM), [94] which aimed to quantify the numbers of responders to induction therapy who then achieved remission as measured at week 26 and 56. Following induction therapy with 80mg at time point 0, followed by 40mg at week 2, patients achieving at least a 70 point reduction in the CDAI at week 4 were randomized to receive 40mg weekly or bimonthly or placebo. At week 26 and 46, remission rates were measured (CDAI score of 150 or less) as a primary end-point. At week 26, 47% of the weekly administration group, 40% of the biweekly administration group and 17% of the placebo group were in remission. By week 56, these figures were 41%, 36% and 12% respectively.

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Certolizumab pegol (CDP870, Cimzia™) Certolizumab is a high affinity polyethylene glycolated humanized Fab’ fragment, which does not cause T-lymphocyte apoptosis or compliment fixation. Data from the “Pegylated Antibody Fragment Evaluation in Crohn’s Disease Safety and Efficacy” (PRECISE)) I and II trials [95, 96] led to FDA approval of its use in CD in April 2008. In PRECISE I, a 26 week, 171 centre randomized, double blinded, placebo controlled trial was carried out on a total of 664 patients. Eligible patients had a CDAI of between 220 and 450, disease duration of greater than 3 months and no exposure to other anti-TNF-α agents within the previous 3 months. Exclusion criteria included the presence of short bowel syndrome, ostomy, intestinal obstruction, cancer and a documented history of hypersensitivity reactions to TNF-α neutralizing therapy. Patients were stratified to receive 400mg (n=331) or placebo (n=329) at weeks 0, 2 and 4 and then monthly to week 22. Of the placebo group, 156 had a serum C-reactive protein (CRP) equal to or greater than 10mg/L at baseline while 146 of the treated group had a serum CRP of 10mg/L or higher at baseline. The primary end-point of a reduction of at least 100 points on the CDAI at week 6 and 26 was examined in patients with a baseline CRP of 10mg/L or higher was examined at week 26. As a secondary end-point, complete remission (CDAI<150) at week 6 and 26 was examined. At week 6, 37% of patients in the treatment group versus 26% of patients in the placebo group had achieved the primary end-point. Combing end-point achievement at week 6 and 26, these figures were 22% versus 12% respectively. No significant differences between groups were observed for the secondary end-point. In PRECISE II, a 147 centre trial with open label induction therapy followed by double blinded placebo controlled study of the effectiveness of certolizumab as a maintenance therapy was carried out. From a total of 668 eligible patients, 428 (64%) had an initial response to induction therapy of fortnightly 400mg certolizumab at week 6 (≥100 point reduction in CDAI). Of these patients, 215 were assigned to receive monthly certolizumab to week 24, while 210 patients received placebo. Assessment of sustained response at week 26 was set as the primary end-point, comparing particularly patients who had a CRP of 10mg/L or higher at baseline. At week 26, 62% of certolizumab treated patients versus 34% of placebo treated patients showed a sustained response coherent with achievement of the primary end-point. Of these patients, 48% were in complete remission in the certolizumab group versus only 29% of patients in the placebo group.

Anti-TNF-α Therapies That Failed to Meet F.D.A Criteria for Approval CDP571 (Humicade™) is a 95% humanized IgG4 monoclonal antibody targeting TNF-α in its soluble and membrane bound formats. A number of trials concluded that while short term treatment was useful, a significant benefit was not maintained [97, 98] . It was also ineffective as a steroid sparing agent in steroid dependent patients [99]. Interestingly, the limited efficacy of CDP571 in CD may be related to its apparent inability to prevent ongoing CD4 positive T-lymphocyte activation, as described in an open label study of 36 patients with rheumatoid arthritis [100] .

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Etanercept (Enbrel™) is a recombinant soluble p75 TNF-α receptor 2/IgG1 Fc fragment that was conclusively shown to not be effective in CD [101, 102] . This is likely to be due to the fact that the crucial effects of mTNF-α on the apoptotic resistance of activated Tlymphocytes are not affected by soluble receptor fragments which are only able to bind and neutralize soluble ligand. Similarly, onercept, the soluble p55 fragment of the type 1 TNF-α receptor has shown little promise for CD [103]

Adverse Effects of TNF-α Neutralizing Therapy: Data from the Phase III Trial of Drugs Licensed to Date Infliximab The relative propensity of patients to develop infusion reactions and anti-drug antibodies, dependent on the agent employed remains a major issue in maintaining the long-term clinical efficacy of biological therapeutics. Questions have been raised over the safety of anti-TNF-α therapies with regard to their potential to cause tuberculosis reactivation, intestinal fibrosis, and lymphoma, as well as their effects on the generation of anti-DNA antibodies with the attendant appearance of lupus like syndromes. With regard to infliximab, safety data from the ACCENT I trial [89], show that anti-drug antibodies developed in 14% of participants, particularly in those in whom treatment was interrupted and then reinitiated (28%) versus those in whom therapy was sustained to the end point of the study (9% with 5mg/kg and 6% with 10mg/kg regimens). Concomitant treatment with a combination of steroids and immunosuppressants significantly reduced anti-drug antibody development. Infusion reactions correlated positively with the dosage of infliximab administered. There were no significant differences in the incidence of serious adverse reactions between those who received single or multiple infusions. A serious infection rate of 4% was observed, including one case of tuberculosis. Neoplasms occurred in 1% of patients treated with infliximab and one death from intestinal obstruction also occurred. Sustained administration of infliximab caused a 34%-36% increase in the incidence of de novo auto-antibody (anti-nuclear-antibody (ANA) and anti-double stranded DNA antibody (d.s.DNAAb) generation. Two of the patients with positive ANA titres presented with arthralgia, suggesting that a lupus-like syndrome had developed. In ACCENT II [90], anti-drug antibodies developed in 24% of patients who were not receiving concomitant immunosuppression versus in 4% of those who were. Interestingly, response rates appeared similar between anti-infliximab positive and negative patients. Infusion reactions were 3 times more common in infliximab treated patients than in placebo treated patients (4% versus 1%). Serious adverse effects were limited, with 5% of randomized patients developing a serious infection requiring treatment. Neoplasms were similarily uncommon; on follow up 2 rectal adenocarcinomas were detected in patients treated with infiximab at 19 months and 2 years post infusion respectively.

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Auto-antibody generation was increased by infliximab (2-fold for ANA and 4-fold for d.sDNAAb). A single case of a lupus like syndrome was detected in a patient who was actually negative for the above auto-antibodies.

Adalimumab The CLASSIC I trial [92], did not report any significant difference in serious adverse effects between treatment and placebo arms. Infection rates were not different and no lymphomas, tuberculoses or opportunistic infections were discovered in any patient upon follow up. An increase in infusion reactions with higher drug concentrations was noted, however, in difference to infliximab, only 2 patients (<1%) developed anti-drug antibodies in the course of the study. In CLASSIC I, no mention was made of rates of auto-antibody generation. In CLASSIC II however [93], 19% of patients (33/172) with baseline negative auto-antibody titres became positive for ANA and d.sDNAAb during the course of the study.

Certolizumab In PRECISE I [95], anti-drug antibodies were found overall in 8% (26/331) of patients. Rates in patients with concomitant immunosuppression were lower at 4% versus 10% in nonimmunosuppressed patients. Certolizumab was associated with a doubling of serious infections versus placebo (2% (7/331) versus <1% (3/329). New generation of auto-antibodies (ANA) was also increased two-fold by certolizumab (2% (5/279) versus <1% (3/277). In PRECISE II [96], anti-drug antibodies were found in 9% [58] of the treated population. Of these 58, 54 became positive during the maintenance phase. There were no significant differences in serious adverse events although there was one case of new onset tuberculosis. ANA auto-antibodies were found in 8% of patients treated with certolizumab (16/192) versus 1% of patients treated with placebo ((2/178).

Conclusion From the first description of TNF-α in CD in 1990, the pharmaceutical industry, clinicians and most importantly patients with CD, have benefited greatly from the translational development of this original finding into the rational design of contemporary biological therapies. The variety of applications of TNF-α neutralizing therapy in CD has allowed for novel therapeutic entry points in the control of both active disease and in the maintenance of remission. Should encouraging recent data indicating that TNF-α neutralization is efficacious in preventing post-surgical relapse of CD be borne out in larger studies, it may lead to the

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possibility of it being used as a therapeutic prophylaxis, in an aspect of CD which to date, has proven difficult to control pharmacologically. The question of which of the three licensed drugs is most effective is important. The large evidence base for infliximab supports its continued use but this must be weighed against the distinct advantages offered by fully humanized antibodies, in terms of anti-drug antibody generation in the recipient. In this regard, adalimumab appears to offer a distinct advantage, as concomitant immunosuppressant therapy is not essential to prevent antibody development. The controversies surrounding the safety of TNF-α neutralization are an important consideration. It is clear that certain caveats should govern the use of this type of therapy, most importantly, the rigorous exclusion of tuberculosis. Reports that infliximab may be associated with intestinal fibrosis are not supported by the safety data in the large phase III trials while the potential neoplastic effects of TNF-α neutralization must be examined in terms of how favourably they compare in terms of relative risk analyses versus standard immunosuppressive therapy. A common finding across all of the drugs licensed to date is the propensity to cause autoantibody development, especially when used as maintenance therapy. The potential for this to lead to development of serious co-morbidities such as lupus-like syndrome requires more study. Further investigation of the underlying causes for TNF-α neutralization related autoantibody production may provide novel insights into the pathogenesis of systemic lupus erythrymatosis and other nuclear antibody mediated autoimmune pathologies. In closing, it is re-emphasized that TNF-α neutralization has represented a revolution in the treatment of CD, and on the basis of available to date, it can confidently be described as an excellent example of a “bench to bedside” success story in biological therapeutics.

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Neil Gerard Docherty and P. Ronan O’Connell randomised phase III trial. ATTRACT Study Group. Lancet, 1999 Dec 4, 354(9194), 1932-9. Present, DH; Rutgeerts, P; Targan, S; Hanauer, SB; Mayer, L; van Hogezand, RA; et al. Infliximab for the treatment of fistulas in patients with Crohn's disease. N Engl J Med., 1999 May 6, 340(18), 1398-405. Kornbluth, A. Infliximab approved for use in Crohn's disease: a report on the FDA GI Advisory Committee conference. Inflamm Bowel Dis., 1998 Nov, 4(4), 328-9. Powrie, F; Leach, MW; Mauze, S; Menon, S; Caddle, LB; Coffman, RL. Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4+ T cells. Immunity., 1994 Oct, 1(7), 553-62. van Dullemen, HM; van Deventer, SJ; Hommes, DW; Bijl, HA; Jansen, J; Tytgat, GN; et al. Treatment of Crohn's disease with anti-tumor necrosis factor chimeric monoclonal antibody (cA2). Gastroenterology, 1995 Jul, 109(1), 129-35. Targan, SR; Hanauer, SB; van Deventer, SJ; Mayer, L; Present, DH; Braakman, T; et al. A short-term study of chimeric monoclonal antibody cA2 to tumor necrosis factor alpha for Crohn's disease. Crohn's Disease cA2 Study Group. N Engl J Med., 1997 Oct 9, 337(15), 1029-35. Rutgeerts, P; D'Haens, G; Targan, S; Vasiliauskas, E; Hanauer, SB; Present, DH; et al. Efficacy and safety of retreatment with anti-tumor necrosis factor antibody (infliximab) to maintain remission in Crohn's disease. Gastroenterology, 1999 Oct, 117(4), 761-9. Hanauer, SB; Feagan, BG; Lichtenstein, GR; Mayer, LF; Schreiber, S; Colombel, JF; et al. Maintenance infliximab for Crohn's disease: the ACCENT I randomised trial. Lancet, 2002 May 4, 359(9317), 1541-9. Sands, BE; Anderson, FH; Bernstein, CN; Chey, WY; Feagan, BG; Fedorak, RN; et al. Infliximab maintenance therapy for fistulizing Crohn's disease. N Engl J Med., 2004 Feb 26, 350(9), 876-85. Regueiro, M; Schraut, W; Baidoo, L; Kip, KE; Sepulveda, AR; Pesci, M; et al. Infliximab prevents Crohn's disease recurrence after ileal resection. Gastroenterology. 2009 Feb, 136(2), 441-50 e1; quiz 716. Hanauer, SB; Sandborn, WJ; Rutgeerts, P; Fedorak, RN; Lukas, M; MacIntosh, D; et al. Human anti-tumor necrosis factor monoclonal antibody (adalimumab) in Crohn's disease: the CLASSIC-I trial. Gastroenterology, 2006 Feb, 130(2), 323-33; quiz 591. Sandborn, WJ; Hanauer, SB; Rutgeerts, P; Fedorak, RN; Lukas, M; MacIntosh, DG; et al. Adalimumab for maintenance treatment of Crohn's disease: results of the CLASSIC II trial. Gut., 2007 Sep, 56(9), 1232-9. Colombel, JF; Sandborn, WJ; Rutgeerts, P; Enns, R; Hanauer, SB; Panaccione, R; et al. Adalimumab for maintenance of clinical response and remission in patients with Crohn's disease: the CHARM trial. Gastroenterology, 2007 Jan, 132(1), 52-65. Sandborn, WJ; Feagan, BG; Stoinov, S; Honiball, PJ; Rutgeerts, P; Mason, D; et al. Certolizumab pegol for the treatment of Crohn's disease. N Engl J Med., 2007 Jul 19, 357(3), 228-38. Schreiber, S; Khaliq-Kareemi, M; Lawrance, IC; Thomsen, OO; Hanauer, SB; McColm, J; et al. Maintenance therapy with certolizumab pegol for Crohn's disease. N Engl J Med., 2007 Jul 19, 357(3), 239-50.

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[97] Stack, WA; Mann, SD; Roy, AJ; Heath, P; Sopwith, M; Freeman, J; et al. Randomised controlled trial of CDP571 antibody to tumour necrosis factor-alpha in Crohn's disease. Lancet, 1997 Feb 22, 349(9051), 521-4. [98] Sandborn, WJ; Feagan, BG; Radford-Smith, G; Kovacs, A; Enns, R; Innes, A; et al. CDP571, a humanised monoclonal antibody to tumour necrosis factor alpha, for moderate to severe Crohn's disease: a randomised, double blind, placebo controlled trial. Gut., 2004 Oct, 53(10), 1485-93. [99] Feagan, BG; Sandborn, WJ; Lichtenstein, G; Radford-Smith, G; Patel, J; Innes, A. CDP571, a humanized monoclonal antibody to tumour necrosis factor-alpha, for steroid-dependent Crohn's disease: a randomized, double-blind, placebo-controlled trial. Aliment Pharmacol Ther., 2006 Mar 1, 23(5), 617-28. [100] Choy, EH; Rankin, EC; Kassimos, D; Vetterlein, O; Garyfallos, A; Ravirajan, CT; et al. The engineered human anti-tumor necrosis factor-alpha antibody CDP571 inhibits inflammatory pathways but not T cell activation in patients with rheumatoid arthritis. J Rheumatol., 1999 Nov, 26(11), 2310-7. [101] Sandborn, WJ; Hanauer, SB; Katz, S; Safdi, M; Wolf, DG; Baerg, RD; et al. Etanercept for active Crohn's disease: a randomized, double-blind, placebo-controlled trial. Gastroenterology, 2001 Nov, 121(5), 1088-94. [102] D'Haens, G; Swijsen, C; Noman, M; Lemmens, L; Ceuppens, J; Agbahiwe, H; et al. Etanercept in the treatment of active refractory Crohn's disease: a single-center pilot trial. Am J Gastroenterol, 2001 Sep, 96(9), 2564-8. [103] Rutgeerts, P; Sandborn, WJ; Fedorak, RN; Rachmilewitz, D; Tarabar, D; Gibson, P; et al. Onercept for moderate-to-severe Crohn's disease: a randomized, double-blind, placebo-controlled trial. Clin Gastroenterol Hepatol., 2006 Jul, 4(7), 888-93.

In: Tumor Necrosis Factor Editor: Toma P. Rossard

ISBN: 978-1-60741-708-8 © 2009 Nova Science Publishers, Inc.

Chapter III

Tumor Necrosis Factor-α and Biliary Tract Diseases Hiroko Ikeda*1, Kenichi Harada1, Motoko Sasaki1, Yasunori Sato1 and Yasuni Nakanuma1 1

Department of Human Pathology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan

Abstract Tumor necrosis factor (TNF)-α is a pleiotropic cytokine involved in the pathophysiology of a variety of human diseases. This study reviews the roles of TNF-α in the pathogenesis of inflammatory biliary diseases such as primary biliary cirrhosis (PBC) and hepatolithiasis and also of cholangiocaricnoma. In PBC characterized by chronic destructive cholangitis of small intrahepatic bile ducts with their progressive loss, TNF-α which is produced by biliary epithelial cells and periductal inflammatory cells, induces the secretion of cytokines from biliary epithelial cells. TNF-α is also involved in the progressive bile duct loss via apoptosis and cellular senescence. Pathogenassociated molecular patterns which might originate from the gastrointestinal tract are involved in the secretion of TNF-α via interaction with TLR4. In hepatolithiasis, which is characterized by long-standing inflammation of intrahepatic large bile ducts with calculi formation, TNF-α is involved in the pathogenesis of intestinal metaplasia which is frequently seen in this disease. The expression of MUC2, an intestinal type mucin, and CDX-2, an intestine-specific transcription factor, characterize the intestinal metaplasia of the biliary mucosa. Human and cultural studies of biliary epithelial cells from polycystic kidney (PCK) rats which show chronic cholangitis with intestinal metaplasia, suggest that TNF-α is involved in the induction of CDX2 followed by an aberrant expression of MUC2, thereby playing a role in the pathogenesis of bile duct lesions of hepatolithiasis. TNF-α may also play an important role in the development and progression of cholangiocarcinoma. Matrix metalloproteinase-9 (MMP-9), an important enzyme in *

Department of Human Pathology, Kanazawa University Graduate School of Medicine, Kanazawa 920-8640, Japan, Fax: +81-76-234-4229, Tel: -81-76-265-2195, E-mail: [email protected]

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Hiroko Ikeda, Kenichi Harada, Motoko Sasaki et al. tumor invasion and metastasis, and cyclooxygenase-2 (COX-2) are frequently expressed in parallel in cholangiocarcinoma. In in vitro studies using cholangiocarcinoma cell line, a TNF-α/TNF-receptor 1 (TNF-R1) interaction induces COX-2 overexpression, MMP-9 production and activation, and increases the migration of cholangiocarcinoma cells. In conclusion, TNF-α plays a central role in the pathophysiology of inflammatory and neoplastic biliary diseases, and could be a target molecule in novel therapeutic strategic approaches for these biliary diseases.

Introduction Tumor necrosis factor (TNF)-α is one of the most important and pleiotropic cytokines in mediating inflammatory and immune responses and also cell survival [1-3]. TNF-α is synthesized as a transmembrane protein and it is secreted after cleavage by the metalloprotease TNF-α converting enzyme (TACE). TNF-α interacts with two distinct receptors, TNF-R1 and TNF-R2 that exist as transmembrane and soluble forms. TNF-R1 is found on most cells in the body, and TNF-R2 is primarily expressed on hematopoietic cells. TNF-R1 is activated by the soluble ligand, while TNF-R2 mainly by the membraneintegrated form. The activation of these receptors leads to the recruitment of intracellular adaptor proteins that activate multiple signal transduction pathways, which are associated with inflammation and cell survival [2,3]. Schematically, TNF-R1 mediates both cell-deathand cell-survival signals, whereas TNF-R2 primarily mediates cell-survival signals. Cell survival mainly involves the activation of the nuclear transcription factor NF-κB. Soluble complexes of TNF-α– TNFRs can be formed, thus altering the availability of TNF-α for its cell-surface receptors. There are many reports that TNF-α and its related molecules are significantly involved in the pathophysiology of hepatobiliary diseases [4-7]. This review will focus on the roles of TNF-α in both inflammatory and neoplastic biliary diseases.

TNF-αand Chronic Inflammatory Biliary Disease TNF-α is involved in the pathophysiology of many types of inflammatory biliary diseases. This review focuses on the roles of TNF-α in the pathogenesis of primary biliary cirrhosis (PBC) and hepatolithiasis. In PBC, the intrahepatic small bile ducts are selectively affected and in hepatolithiasis, the intrahepatic large bile ducts are mainly involved [8]. For a better understanding, the intrahepatic bile ducts are anatomically subdivided into large and small intrahepatic bile ducts in this review [8]. The former corresponds to the first to third branches of the right or left hepatic bile ducts which are physiologically associated with the peribiliary glands, while the latter consists of the septal and interlobular bile ducts.

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Figure 1 (a). An interlobular bile duct (*) is surrounded by dense lymphoplasmacytic infiltration (Chronic nonsuppurative destructive cholangitis, CNSDC). Primary biliary cirrhosis. HE., original magnification, x200. (b). TNF-α is expressed in biliary epithelial cells of interlobular bile ducts (*) showing CNSDC. Immunostaining of TNF-α and hematoxylin, original magnification, x200 (cited from reference #11).

Primary Biliary Cirrhosis PBC is an organ specific autoimmune disease which usually affects middle-aged women and eventually leads to liver failure and liver transplantation. PBC is histologically characterized by cholangitis of the small bile ducts (chronic non-suppurative destructive cholangitis; CNSDC) (Figure 1a) eventually followed by an extensive loss of such small bile ducts [9], and serologically by the presence of anti-mitochondrial antibodies (AMA) against pyruvate dehydrogenase E2 component (PDC-E2) [10]. CNSDC shows periductal infiltration of many lymphocytes and granuloma formation, and lymphocytes also exist in the biliary epithelial layer. Recent studies have shown that TNF-α may be involved in both the formation of chronic cholangitis and also progressive bile duct loss. Chronic cholangitis and TNF-α: One of the most important roles of TNF-α is a mediator in the inflammatory reactions of the innate immune system. While TNF-α is usually negative in normal biliary epithelial cells (BECs), the expression of TNF-α is increased in the cytoplasm of the BECs of damaged bile ducts in PBC (Figure 1b), and TNFα is also expressed in infiltrating mononuclear inflammatory cells around the damaged bile ducts, implicating that TNF-α produced and secreted locally in the microenviroment is involved in the bile duct damage [11]. In fact, TNF-α is frequently detected in gallbladder bile from PBC, and their titers are higher compared with other hepatobiliary diseases [11]. TNF receptors are also detectable in these damaged bile ducts, suggesting a receptormediated autocrine or paracrine effect of TNF-α in the pathogenesis of CNSDC [11]. For example, TNF-α secreted by periductal inflammatory cells and BECs themselves play a role in the up-regulation of the expression of fractalkine in the bile ducts. Fractalkine is a chemokine with both chemoattractant and cell-adhesive functions, and we recently showed that up-regulated fractalkine is involved with its receptor CX3CR1 in the chemoattraction

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and recruitment of intraepithelial lymphocytes of CNSDC [12]. Bacterial pathogens and many other noxious stimuli (pathogen associated molecular patterns, PAMPs) are known to induce TNF-α production via Toll-like receptors (TLR), particularly TLR4, which leads to activation of nuclear factor (NF)-κB signaling [13-16]. In fact, PAMPs are observed in the bile and periductal granulomas of PBC [17,18]. The enhanced translation of TNF-α prompts a subsequent biological cascade involving chemokines, cytokines, and endothelial adhesions followed by the development of CNSDC [1,14,16]. Progressive bile duct loss and TNF-α: Cytotoxic T lymphocytes (CTL) around CNSDC in PBC induce apoptosis in BECs by activation of the apoptosis receptor Fas and/or via the granzyme/perforin pathway [19]. BECs undergo apoptosis when cell surface Fas is activated with either cross-linking antibody or Fas ligand (FasL) -bearing effector cells [19]. Cooperation between Fas and CD40, another member of the TNF receptor superfamily, is also important in the loss of BECs in immune-mediated biliary diseases such as PBC [20]. TNF-related apoptosis-inducing ligand (TRAIL) has been recently proposed to be a new apoptotic inducer [21]. The death signal mediated by TRAIL receptor 2/death receptor 5 (DR5) may be a key regulator of PBC and primary sclerosing cholangitis (PSC) [22]. Agonistic anti-DR5 monoclonal antibody treatment has been shown to trigger the apoptosis of BECs, and thus subsequently inducing cholangitis and cholestatic liver injury. Human BECs constitutively expressed DR5, and TRAIL expression and apoptosis are significantly elevated in BECs of PBC and PSC. Therefore, TRAIL/DR5-mediated apoptosis may substantially contribute to PBC and PSC. In fact, BECs of intrahepatic bile ducts express TRAIL receptors which are up-regulated by increased levels of bile acids [21]. In fact, soluble TRAIL (sTRAIL) levels have been shown in the serum of PBC patients [21]. Therefore, TRAIL systems may mediate the apoptotic disappearance of the bile duct in PBC. Cellular senescence and TNF-α：We recently showed that BECs of the small bile ducts in PBC, especially those involved in CNSDC, frequently expressed the cellular senescence features, the increased activity of SA-β-gal and also the increased expression of senescence-associated p16 INK4a and p21WAF1/Cip1 [23]. The impaired replacement and nonproliferative properties of senescent biliary epithelial cells are prone to be attacked by another cell injuries and are likely to be followed by the loss of bile ducts in PBC. Oxidative stress causes cellular senescence and there have been several studies reporting the involvement of oxidative stress in the pathogenesis of PBC [24,25]. The cellular senescence of small bile ducts in PBC, especially at an early stage, is closely associated with the infiltration of myeloperoxidase (MPO)-positive cells inside biliary epithelial cell layers. MPO-positive cells provide reactive oxygen species (ROS) and nitric oxide (NO)-derivatives and then cause ROS- and NO-mediated tissue injuries. Therefore, the oxidative and nitrosative stress caused by the infiltration of MPO-positive cells in biliary epithelial layers may play a critical role in the induction of cellular senescence of BECs in PBC. A number of proinflammatory cytokines, such as TNF-α, have been shown to increase in the inflammatory lesions around the damaged bile ducts in PBC [11,26]. Accumulating data suggest that ROS is a critical chemical mediator of cell signaling induced by cytokines such as

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TNF-α. Particularly, the ATM/p53/p21WAF1/Cip1 pathway is suggested to be involved in cellular senescence related to oxidative stress. Inflammatory cytokines such as TNF- α, generate ROS, activate the ATM-signaling pathway, and the subsequent induction of cellular senescence in BECs. TNF-α and IFN-γ are increased in inflammatory lesions around the damaged bile ducts in PBC. It is conceivable that inflammatory cytokines such as TNF-α play a role in the generation of endogenous ROS and therefore the induction of cellular senescence in BECs. In conclusion, the activation of the ATM/p53 pathway by proinflammatory cytokines such as TNF-α and oxidative stress is involved in the increased expression of p21WAF1/Cip1 in PBC and then in the induction of cellular senescence in BECs. This may be, at least partly, responsible for the progressive bile duct loss in PBC. Hepatolithiasis Hepatolithiasis is infrequently encountered in the Far East, while rare in Western countries. This disease is characterized by the formation of stones, particularly brown pigment stones, in the intrahepatic large bile ducts. The affected bile ducts show luminal dilatation and stenosis, dense fibrosis with chronic inflammatory changes, and proliferation of the peribiliary glands (proliferative cholangitis) (Figure 2a). Suppurative changes are variably admixed in this lesion. A bacterial infection has been implicated as an important and crucial factor for the development of chronic proliferative cholangitis and lithogenesis of hepatolithiasis [27]. Interestingly, intestinal metaplasia with goblet cell metaplasia, is also known to develop in the affected bile ducts of hepatolithiasis, as seen in other types of chronic cholangitis such as PSC [27-29].

Figure 2 (a). Intrahepatic large bile duct shows dense fibrosis, proliferation of peribiliary glands, and lymphyoid cell infiltration (chronic proliferative cholangitis). Hepatolithiasis, HE., original magnification, x50. (b). MUC2, an intestinal mucin, is aberrantly expressed in the cytoplasm of lining epithelium of the intraheaptic large bile duct of hepatolithiasis. Immunostaining of MUC2, original magnification, x300 (cited from reference #28).

Intestinal metaplasia showing goblet cells, Paneth cells, and endocrine cells, occurs in non-neoplastic and neoplastic conditions in extra-intestinal organs such as the pancreas,

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stomach, and esophagus [30-32]. Mucus core protein (MUC) are the backbone structure of the mucin, and among them, MUC2 is physiologically expressed in epithelial cells of the intestine [28]. Intestinal metaplasia is characteristically associated with aberrant expression of MUC2 and CDX2, a caudal-related homeobox gene encoding an intestine-specific transcription factor, in the biliary system [28,33-35], and such phenotypes are also aberrantly expressed in hepatolithiasis (Figure 2b). While bacterial components such as lipopolysaccharide (LPS) are known to induce MUC2 and CDX2 expression in cultured BECs [33,35], LPS-induced TNF-α up-regulation through activation of nuclear NF-κB is observed in human BECs [36]. Interestingly, TNF-α is strongly expressed in affected bile ducts (lining epithelia and peribiliary gland) and also in the infiltrating mononuclear inflammatory cells in hepatolithiasis [37]. Human hepatolithiatic livers and polycystic kidney (PCK) rats, an animal model of Calori’s disease, both show chronic cholangitis of the intrahepatic bile ducts, with goblet cell metaplasia and aberrant expression of MUC2 [33,34,38]. An in vitro study, using cultured BECs was used to elucidate the relationship between TNF-α and intestinal metaplasia in chronic cholangitis. TNF-α and intestinal metaplasia in animal model: In the liver of PCK rats, progressive multiple and segmental dilatation of the intrahepatic bile duct and fibrous expansion of the portal tract occur [33,38]. At around 6 weeks, spontaneous chronic cholangitis with bacterial infections can be observed in PCK rats. Intestinal metaplasia containing goblet cells in the lining epithelia of the bile duct gradually increases with progression of chronic cholangitis [33,37,38]. The cytoplasmic expression of MUC2 and the nuclear expression of CDX2 are detectable in the metaplastic mucosa, respectively [33,37]. MUC2 is more frequently found in the goblet cells and CDX2 is more diffusely found. The expression of TNF-α is negligible in the bile ducts without cholangitis in this rat liver, whereas, in the livers with chronic cholangitis, TNF-α is expressed frequently and extensively in the luminal and lateral membranes and in the cytoplasm of the lining epithelia. The expression of TNF-α is also seen in a majority of the infiltrating mononuclear cells around the bile ducts. TNF-R1 and TNF-R2 are constantly expressed in the cytoplasm and luminal cell membranes in the BECs with cholangitis, respectively. Interestingly, the level of TNF-α in PCK rat liver tissue, as analyzed by ELISA, is increased with the severity of cholangitis and the frequency of goblet cell metaplasia. The co-expression of MUC2, CDX2, and TNF-α in the intestinal metaplastic mucosa of the bile duct is observed by double immunostaining in chronic cholangitis of PCK rats (Figure 3), suggesting that expression of these molecules are closely and causally related to each other in the bile ducts showing cholangitis [37]. TNF-α-induced MUC2 and CDX2 expression in cultural studies: The expression of MUC2, CDX2 and TNF-α is not seen in cultured BECs isolated from the liver of PCK rats, whereas the receptors of TNF-α is constantly expressed at mRNA level (Figure 4a). Interestingly, CDX2 mRNA is induced after treatment with interleukin (IL)-1β, IL-6, interferon (IFN)-γ, and TNF-α, whereas the up-regulation of MUC2 mRNA is evident only

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by TNF-α treatment (Figure 4b). A semiquantitative analysis shows expression of CDX2 mRNA by TNF-α to increase in a dose-dependent manner, while MUC2 mRNA is induced at high dose TNF-α treatment (Figure 4c). CDX2 mRNA is upregulated 3hr after treatment of TNF-α, whereas the increase of MUC2 mRNA is evident after 6hr (Figure 4d) [33,37]. These findings strongly suggest that the overexpression of CDX2 followed MUC2 upregulation is induced by TNF-α. The mechanism of TNF-α-associated intestinal metaplasia in chronic cholangitis: Based on the observations in human tissue and culture studies described above, it seems likely that TNF-α binds to its receptor on cytoplasmic membrane and the signal is introduced to NF-κB pathway. Thereafter, the signal activates CDX2 thus leading to MUC2 expression. The expression of TLR4, which is known as a receptor that recognizes LPS, is shown in human and rat BECs of the liver tissue. LPS-induced upregulation of TNF-α has been demonstrated in human and rat cultured BECs [26,33,37]. Therefore, the source of TNF-α seems to be the BECs themselves, and also inflammatory cells infiltrating the biliary mucosa, because the expression of TNF-α is found in mononuclear inflammatory cells [37]. Taken together, the aberrant expressions of CDX2 and MUC2 in the affected bile ducts seem to be closely and causally related to TNF-α expressed on BECs of the affected ducts and periductal inflammatory cells in human hepatolithiasis and PCK rat liver, and TNF-α is able to induce MUC2 and CDX2 via NF-κB signaling pathway in cultured BECs. These data suggest that TNF-α is responsible for intestinal metaplasia and it is therefore likely to be a key cytokine in pathogenesis of chronic cholangitis.

Figure 3. Expression of MUC2 or CDX2 at intestinal metaplasia of the intrahepatic bile ducts of polycystic kidney (PCK) rats at 10 months. a: MUC2 is expressed in the cytoplasmic mucin of goblet cells (arrow) and also secreted in the lumen. Counterstained with hematoxylin, original magnification, x400. b: CDX2 is expressed in the nuclei of goblet cells (arrow) and of surrounding columnar biliary epithelial cells (BECs) (arrowhead). Counterstained with hematoxylin, original magnification, x400. c: Double fluorescence immunostaining for MUC2 (green) and CDX2 (red). In goblet cells, MUC2 in the cytoplasm and CDX2 in the nucleus are co-expressed. In nuclei of some columnar BECs around goblet cells, CDX2 is expressed while MUC2 expression is not evident. *Lumen of the bile duct. Original magnification, x400 (cited from reference #15).

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Figure 4. TNF-α induce the expression of MUC2 and CDX2 in biliary epithelial cells. a: The expression of TNF-R1 and TNF-R2 are constantly seen in these cells. b: CDX2 expression is induced by either of interleukin IL-1β, IL-6, IFN-γ, or TNF-α. Induction of MUC2 expression is only seen in the treatment with TNF-α. c: TNF-α-induced MUC2 and CDX2 expression is dose-dependent. d: TNF-α-induced CDX2 expression is followed MUC2 expression (cited from reference #15).

TNF-α and Cholangiocarcinoma There is now substantial evidence that TNF-α is significantly involved in the development and progression of malignant tumors [39]. Particularly, TNF-α plays reportedly an important role in the migration/invasion of malignant cells via mediating and regulating other biological active molecules [40-42]. Among these molecules, matrix metalloproteinases (MMPs), a family of zinc-dependent proteinases, are well known to degrade various components of the extracellular matrix (ECM). Particularly, MMP-9 plays an important and necessary role in the catalytic activity of tumor cell invasion and metastasis [43,44]. Stromal cell-derived factor-1 (SDF-1: CXCL12), and its specific receptor CXCR4 are another molecules also involved in the cancer cell migration observed. [45,46]. Cholangiocarcinomas arise from the intrahepatic, hilar and extrahepatic bile ducts. Once cholangiocarcinoma begins invasive growth, it aggressively invades into the surrounding tissue commonly associated with metastasis, finally resulting in a dismal prognosis [47,48]. TNF-α also seems to be an important cytokine in the promoting the invasion of

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cholangiocarcinoma cells (Figure 5). Herein, the roles of TNF-α in the migration and invasion of cholangiocarcinoma are mainly described with respect to MMP-9 and SDF-1. TNF-α and MMP-9 in cholangiocarcinoma: TNF-α-dependent MMP-9 production in cholangiocarcinoma cell lines has been reported in several studies [49,50]. Cyclooxygenase (COX)-2 which is induced by a variety of stimuli, is a rate-limiting enzyme that catalyzes the conversion of arachidonic acid to prostaglandins, including prostaglandin E2 (PGE2). In cholangiocarcinoma, COX-2 expression is associated with tumor growth and invasion in carcinoma cells and PGE2 has many biological activities such as cell proliferation, cell invasion and angiogenesis [51-53].

Figure 5. There are many TNF-α positive mononuclear cells infiltrating at the interface of ICC (C) and surrounding liver (H). A majority of them correspond to infiltrating macrophages. Kupffer cells in the surrounding liver are also strongly positive. Immunostaining for TNF-α counterstained by hematoxylin, original magnification, x200 (cited from reference #5).

Figure 6. Expression of MMP-9 and COX-2 in cholangiocarcinoma. a: MMP-9 is strongly and diffusely expressed in the cytoplasm of almost all CC cells. Immunohistochemistry of MMP-9, original magnification, x200. b: COX-2 is expressed strongly and diffusely in the cytoplasm and membrane of almost all CC cells. Immunohistochemistry of COX-2, original magnification, x200 (cited from reference #54).

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We recently examined the roles of COX and PGE2 with respect to the production and activation of MMP-9 in cholangiocarcinoma induced by TNF-α, using human tissue specimens and also a human cholangiocarcinoma cell line, HuCCT-1 [54]. While the expression of MMP-9 and COX-2 is either faint or absent in the nonneoplastic bile ducts, MMP-9 and COX-2 were evidently and frequently expressed in carcinoma cells in cholangiocarcinoma, and the expression of COX-2 is significantly correlated with that of MMP-9 (Figure 6). In fact, co-expression of both molecules was observed in cholangiocarcinoma cells by double immunofluorescent staining. In cholangiocarcinoma tissue, zymography reveals latent MMP-9 constantly, and also active MMP-9 infrequently. An in vitro study showed that TNF-α induced latent MMP-9 production and also active MMP-9 in a dose-dependent manner. TNF-R1, TNF-R2 and MMP-9 mRNA were detected in cholangiocarcinoma by RT-PCR analysis. Anti-TNF-R1 neutralizing antibodies inhibited the TNFα-induced MMP-9 production and activation, while anti-TNF-R2 neutralizing antibodies alone failed to inhibit TNF-α-induced MMP-9 upregulation, suggesting that TNF-R1 is crucial in TNF-α-induced MMP-9 upregulation. TNF-α treatment also induced COX2 expression, as well as PGE2 oversecretion. Pretreatment with COX2 inhibitors such as indomethacin or NS398 inhibits TNF-α-induced MMP-9 production and activation (Figure 7). Interestingly, pretreatment with PGE2 receptor blocking peptides clearly reduce the TNF-αinduced MMP-9 production and activation in culture cells. These findings suggests that the induction of COX-2, followed by PGE2 oversecretion, seems to be essential for TNF-αinduced MMP-9 activation in cholangiocarcinoma cells. A cell migration assay showed that TNF-α treatment increased significantly the migration index of the cultured cholangiocarcinoma cells, and such increased cell migration is significantly inhibited by pretreatment with MMP-9 inhibitor and also COX-inhibitors. The results suggest that TNFα is associated with migration activity in cholangiocarcinoma cells and the upregulation of MMP-9 via COX activation is involved in TNF-α-inducible cell mobility of cholangiocarcinoma cells. A transfection study using siRNA of COX-2 and COX-1 confirmed the necessity of COX-2 and participation of COX-1 in TNF-α-induced MMP-9 upregulation and cell migration in HuCCT-1 cells, a cholangiocarcinoma cell line.

Figure 7. The effects of COX inhibitors and PGE2 blocking peptides in TNF-α-induced MMP-9 upregulation. Indomethacin (Indo, non-selective COX inhibitor), SC560 (SC, selective COX1 inhibitor), NS398 (NC, selective COX-2 inhibitor), AH6809 (BP1, blocking peptide of EP1), SC19220 (BP1/2, blocking peptide of EP1/2), and AH23848 (BP4 blocking peptide of EP4). (a) Gelatin zymograpghy. (b) Real-time PCR. (cited from reference #54).

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Figure 8. Schema of TNFα-induced up-regulation of COX-2 and MMP-9, and their interaction in cholangiocarcinoma. (cited from reference #54).

Upregulation of COX-2 and MMP-9 by TNF-α, and their interaction are summarized in Figure 8. The binding of TNF-α to TNF-R1 leads to direct or indirect MMP-9 overexpression followed by MMP-9 activation, which results in migration of cholangiocarcinoma cells. In addition to the active participation of COX-2, COX-1 may also be partly involved via PGE2 production in this process. The involvement of another signaling pathways, including extracellular signal-signal regulated kinase 1/2 (Erk1/2), p38 mitogenactivated protein kinase (p38MAPK), or translocation of NF-κB in TNF-α-induced MMP-9 up-regulation in cholangiocarcinoma cells, has also been demonstrated using specific inhibitors [4]. TNF-α and SDF-1 in cholangiocarcinoma: We also examined the roles of CXCR4 and its ligand, SDF-1, and their interaction with TNF-α in cholangiocarcinoma [5]. The expression of TNF-α was found in infiltrating macrophages at the interface of intrahepatic cholangiocarcinoma tissue (Figure 5), whereas CXCR4 was expressed in cholangiocarcinoma cells and SDF-1 in the fibroblast-like stromal cells in cholangiocarcinoma. Cholangiocarcinoma cell lines expressed CXCR4 mRNA and protein, and WI-38 fibroblasts express SDF-1 mRNA and protein. The migration of cultured cholangiocarcinoma cells in Matrigel is induced by a co-culture with WI-38 fibroblasts and by incubation with SDF-1. Anti-SDF-1 antibody suppresses this migration, thus demonstrating that SDF-1 released from WI-38 fibroblasts is responsible for this migration. TNF-α pretreatment of cholangiocarcinoma cells up-regulated CXCR4 mRNA and protein expression in a concentration-dependent manner. The administration of SDF-1 and TNF-α synergistically increase cholangiocarcinoma cell migration, which is suppressed by the CXCR4 antagonist. Taken together, it seems likely that the interaction of SDF-1 released from fibroblasts and CXCR4 expressed on cholangiocarcinoma cells are actively involved in cholangiocarcinoma cell migration, and that TNF-αenhances cell migration by increasing the CXCR4 expression.

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Conclusion TNF-α plays important roles in both the pathogenesis of chronic cholangitis of the intrahepatic small and large bile ducts and also in the migration and invasion of cholangiocarcinoma. In PBC, TNF-α induces the secretion of cytokines from BECs. TNF-α is also involved in the progressive bile duct loss via apoptosis and cellular senescence. The pathogen-associated molecular patterns such as LSP which might originate from the gastrointestinal tract are involved in the secretion of TNF-α via interaction with TLR4 expressed in the bile ducts in PBC. TNF-α seems also to up-regulate the intestine-related molecules such as MUC2 and CDX2 in the response to bacterial components in hepatolithiasis. TNF-α appears to promote invasion of cholangiocarcinoma cells, as stimulators of cell migration, producing MMP-9, and interacting with SDF-1/CXCR4. It is important to understand the molecular mechanisms of TNF-α in the pathophysiology of inflammatory and neoplastic biliary diseases and to develop novel strategies using TNF-α and its related molecules for these intractable diseases.

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In: Tumor Necrosis Factor Editor: Toma P. Rossard

ISBN: 978-1-60741-708-8 © 2009 Nova Science Publishers, Inc.

Chapter IV

Tumoricidal Effect of Tumor Necrosis Factor-Alpha in Isolated Limb Perfusion Treatment of Human Cancers Chandrakala Menon and Douglas L. Fraker Harrison Department of Surgical Research, University of Pennsylvania School of Medicine, Philadelphia, PA 19104

Abstract Tumor necrosis factor-alpha (TNF-alpha), first named for its anti-tumor property, is a pleiotropic cytokine implicated in many physiological and pathological reactions including cell death, cell survival, immune response, and inflammation. The human TNFalpha gene is located on the short arm of chromosome 6. The gene was cloned in 1984 and recombinant human TNF-alpha (rhTNF-alpha) was successfully produced in Escherichia coli but could not be used as an effective systemic anticancer agent because of its dose-limiting toxicity. It is currently effectively used in regional therapy for melanomas and soft-tissue sarcomas using isolated limb perfusion (ILP), a surgical procedure which circulates high-dose therapeutics within an extremity (arms and legs), thus limiting systemic toxicity while avoiding the organs of metabolism. Objective response rates of 90-100% with 65-90% complete response rates have been reported in patients with in-transit metastases from melanoma. Overall response rates with soft-tissue sarcoma range from 58%-82% with 15-30% complete responses. These results, achieved with a single 90-minute ILP using rhTNF-alpha and melphalan, are significantly better than the best results achieved with combination systemic chemotherapy of 30% overall response rates and 0-5% complete responses for melanoma and 15-20% overall response rates with essentially no complete responses for sarcoma. The precise mechanism underlying the remarkable anti-tumor effect of rhTNF-alpha in conjunction with melphalan against solid tumors using ILP has not been fully elucidated. During ILP treatment, all of the normal tissues immediately surrounding the tumor i.e. skin, subcutaneous fat, muscle, bone, and peripheral nerve are subject to the same drug concentration as the tumors present in the extremity. Remarkably however, the combination of rhTNF-alpha and melphalan affects only tumor and not normal tissue and

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Chandrakala Menon and Douglas L. Fraker is known to work synergistically. ILP with melphalan-alone has activity for melanoma when the tumor burden (size and number of nodules) is limited. For large tumors or extensive disease, the response to melphalan-alone ILP is limited, but is significantly improved by adding rhTNF-alpha to the treatment regimen. It is widely accepted that melphalan, a nitrogen-mustard, forms damaging DNA adducts within dividing tumor cells. This chapter critically reviews recent data that provide insights into plausible mechanisms by which rhTNF-alpha asserts its anti-tumor activity.

Discovery of TNF-Alpha - A Historical Perspective The history of the role of TNF-alpha as an anti-cancer agent dates back to the 1700's when it was observed that cancer patients who developed a concurrent bacterial infection also experienced simultaneous remissions of their malignant disease [1, 2]. In the late 19th century, a surgeon named William B. Coley and his colleagues at the Memorial Hospital in New York had modest success in treating certain cancer patients by infecting them with live bacteria [2, 3]. Ironically, Dr. Coley's interest in this method of cancer treatment grew from the loss of his first cancer patient, a young girl with a sarcoma in her right arm. Despite the radical surgery, the patient developed metastatic disease and died. Dr. Coley researched the medical records of his hospital and discovered a seven-year-old record of a patient with a four-time recurrent inoperable sarcoma of the neck. This patient was reported to have experienced a regression of the disease under the influence of an erysipelas infection (a superficial, streptococcal infection of the skin). Dr. Coley located this patient, and found him free from any clinical evidence of malignancy. He then found a substantial number of publications containing similar observations [1]. Since these were pre-antibiotic times, the difficulty of controlling the induced bacterial infection was serious cause for concern. In 1892, Dr. Coley developed vaccines using killed bacteria, a mixture of Streptococcus pyogenes and Serratia marcescens, which came to be known as "Coley's toxins" [1, 3-7]. This mixture of dead bacteria produced many of the same symptoms of bacterial infections such as fevers of up to 102 - 105° F that could last several weeks or even months due to multiple serial injections in the treatment regimen. However, the mixture could be administered without fear of producing an actual infection. Tumors in some of the patients treated with Coley's toxins regressed or completely disappeared. However, despite the effort of a committed group of physicians, success of this method which used killed microbes as treatment for cancer was modest at best. It did not work in all cancer patients and neither was it successful against all types of cancers (Table 1) [3]. Only about 25-30% of the data shown in Table 1 was gathered from Dr. Coley, the rest coming from other participating contemporary physicians who were willing to attempt to treat cancer with infectious agents [3]. This collaborative effort between several physicians was viewed at the time as a measure of the confidence of Coley's peers in his clinical success. The second striking feature about the data gathered in Table 1 was that soft tissue sarcomas represented a greater than 50% of the study population and also registered a greater than 50% complete response rate (Table 1, column E) although soft tissue sarcomas represent a less than 1% of cancers in the general population. This was not a random occurrence. Dr. Coley deliberately selected such patients to be part of the study because, unfortunately, his earlier

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observations had showed a greater success rate of his treatment method among sarcoma patients than other more prevalent cancers such as carcinomas. A more careful examination of Dr. Coley's original papers and those of his peers, however, expressed optimism about the potential of this treatment method thus, "….if by the expression of certain bacterial toxins, we can cause the degeneration, death, and absorption of one variety of cancer -- sarcoma, it is not unreasonable to suppose that by the use of some other forms of bacterial toxins, we may succeed in destroying or inhibiting the growth of the other and more common variety -carcinoma" [3]. Table 1. Summary of Patients Treated with Coley's Toxins before 1940 Type of Cancer Soft Tissue Sarcoma Lymphosarcomas (Lymphomas) Osteosarcoma Ovarian Carcinoma Cervical Carcinoma Testicular Renal Multiple Myeloma Colorectal Carcinoma Breast Carcinoma Melanoma

Total 104 50 3 4 2 18 6 1 2 14 6

A 38 24 2 1 0 10 3 0 1 8 2

B 12 7 1 2 1 2 0 0 1 4 3

C 17 4 0 0 0 3 1 1 0 2 0

D 15 7 0 0 0 2 1 0 0 0 1

E 22 8 0 1 1 1 1 0 0 0 0

The patients in the table above were considered inoperable at the time of treatment with Coley's toxins and had received no therapy other than the vaccine. Column A: Patients that had no beneficial response to treatment. Column B: Patients that showed an initial response and either relapsed or were lost to follow-up within 5 years. Column C: Patients were free of disease but were lost to follow-up between 5 and 10 years. Column D: Patients were free of disease but were lost to followup between 10 and 20 years. Column E: Patients were free of clinical evidence of disease for at least 20 years. (Source of data is a series of papers by H. C. Nauts and G. A Flower in the Cancer Research Institute Monograph between 1969 and 1975).

Dr. Coley died in 1936 and his treatment method would have died with him but for the efforts of his daughter, Helen Coley Nauts [2]. As a consequence of her perseverance, it was not only determined that many different types of infectious agents could produce anti-cancer effects in animals but also that injection of live or dead strains of gram-negative bacteria could cause mouse tumors to undergo hemorrhagic necrosis by which tumors bled into themselves causing tumor cells to be killed [6-9]. In 1943, M. J. Shear and his colleagues at the National Cancer Institute established that the active component of the gram-negative bacteria that produced the above effect was a complex lipid and polysaccharide containing compound [10]. Subsequent work showed that this compound was a component of the outer wall of the bacterium, a lipopolysaccharide (LPS) that was named endotoxin. In the latter part of the 1950's, B. Benacerraf and L. J. Old found that an attenuated form of a microbe that caused tuberculosis called bacillus Calmette-Guerin (BCG) could also induce an infection in mice that not only prevented a subsequent bacterial infection but also inhibited tumor growth [11]. Although animal studies showed that injection of bacterial components could cause

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tumor regression while leaving normal cells untouched, in the test-tube however, neither LPS nor BCG killed tumor cells. This finding was seminal in concluding that the injected bacterial component was acting indirectly via another agent in the host. Moreover, in searching for molecules in the body that would inhibit the growth of cancers while leaving normal cells unharmed, L. J. Old, E. A. Carswell, R. L. Kassel and B. D. Williamson found that blood drawn from mice that were first injected with BCG and some days later with LPS induced hemorrhagic necrosis in tumors of different mice. Such blood was highly toxic to cancer cells in the test-tube as well. Since this host factor appeared to kill mouse tumors by hemorrhagic necrosis, it was called "tumor necrosis factor (TNF)" [12]. Table 2. The Discovery of TNF-alpha Time-period/Year

Credits

1700's

Observation/Event Cancer patients with concurrent bacterial infection experience tumor remission

Late 1800's to 1936

Coley

Treated cancer patients with "Coley's Toxins" with moderate success

1936 - 1975

Nauts

Infection with gram negative bacteria causes hemorrhagic necrosis in mice

1943

Shear and colleagues

Active ingredient in gram negative bacteria is in bacterial wall later found to be LPS

1950's

Benacerraf, Old

Attenuated form of tuberculosis microbe (BCG) could inhibit tumor growth in mice. Neither LPS nor BCG killed tumor cells in test tube. Host factor responsible was called "Tumor Necrosis Factor".

1960's - 1975

Green and colleagues

Isolation of TNF from mouse blood

Haranaka

Isolated and purified TNF from mice and rabbits.

Mannel, Mergenhagen

Macrophages produce TNF in mice

Carswell, Old, Rubin

Macrophages as well as many other cell types could secrete TNF

Goeddel, Fiers

Cloning of the gene, identification of amino acid sequence of protein and production of large quantities of recombinant TNF

1984

The next step was to determine the source and biochemical identity of TNF. It was known at the time that both LPS and BCG could activate macrophages resulting in an increase in the number of macrophages in the body. It was also known that a mixing of activated macrophages and tumor cells was lethal to tumor cells. The logical conclusion from the above observations was that TNF was probably produced by macrophages [13]. Similar processes were then shown to exist in rabbits, rats, and guinea pigs.

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The isolation of TNF from mouse blood was initiated by S. Green and his colleagues at the Memorial Sloan Kettering Cancer Center [14]. It was shown that the factor was a single protein with anti-tumor activities in the animal as well as in the test-tube. Later, K. Haranaka and N. Satomi from the University of Tokyo isolated and purified TNF, a single polypeptide, from mice and rabbits [15, 16]. Earlier, D. N. Mannel and S. E. Mergenhagen from the National Institute of Dental Research and N. Matthews from the University of Wales had proved that macrophages produce TNF in mice [17]. The work with human cells that produce TNF was mainly carried out by W. Carswell, L. J. Old, B. D. Williamson and B. Y. Rubin who showed that macrophages as well as many other cell types could secrete TNF [18-22]. The decade-long effort to purify TNF finally culminated in 1984 with the cloning of the gene, the identification of the amino acid sequence of the protein, and the production of large quantities of recombinant TNF by several groups associated with biotechnology companies including D. V. Goeddel and his colleagues at Genentech, Inc., [23, 24] and W. Fiers and his co-workers at the State University of Ghent and at Biogen SA [25, 26]. This process allowed for the elucidation of TNF's primary role in inflammation and immunity and also in its use as a limited scope anti-cancer agent. Around this time, it was recognized that another molecule whose discovery predates TNF and is secreted mostly by T-lymphocytes called lymphotoxin had similar but not identical structural, biological, and anti-neoplastic activities as TNF. It was also determined that it was coded for by a separate gene than TNF. It was necessary, therefore, to distinguish these two molecules by nomenclature. TNF, also characterized as "cachectin" for its role in the wasting syndrome and mainly secreted by myeloid cells was named TNF-alpha and Lymphotoxin, mainly secreted by lymphoid cells was named TNFbeta [24, 27-30]. This review chapter focuses on the role of TNF-alpha as an anti-cancer agent. The important milestones in the discovery of TNF-alpha are summarized in Table 2.

Biology of TNF-Alpha Endogenously produced TNF-alpha is a pleiotropic cytokine that has a short circulating serum half-life of 6-20 minutes. It is released in response to bacterial toxins such as LPS, viruses such as HIV and influenza, mycobacteria, fungi, parasites, products of complement activation, antigen-antibody complexes and other cytokines [31, 32]. The cellular sources of TNF-alpha in the body mainly include macrophages, lymphocytes, eosinophils, and polymorphonuclear leucocytes, and to a lesser extent include astrocytes, Langerhans cells, Kupffer cells, fibroblasts, smooth muscle cells and tumor cells [31, 32]. Endogenously produced TNF-alpha is a central mediator of inflammation in the body resulting in the activation of leukocytes, in the adherence of monocytes and neutrophils to the endothelium and migration of these cells into the intercellular matrix. It also causes the local production of proinflammatory cytokines that mediate the downstream effects of inflammation resulting in all the beneficial effects of TNF-alpha such as curing infections and causing wound healing. As mentioned earlier, TNF-alpha is selectively cytotoxic to cancer cells. However, TNF-alpha is also a known mediator of the pathobiological effects of diseases. For example, in cancer it is also known to induce cachexia, stimulate tumor growth and increase metastatic potential. In AIDS, it is known to induce cachexia, stimulate viral

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proliferation and mediate central nervous system injury. In transplantation, it acts as a key mediator of allograft rejection and in host versus graft disease. It also triggers veno-occlusive disease. In multiple sclerosis, it is cytotoxic to oligodendrocytes and induces inflammatory plaques. In diabetes, it promotes the destruction of islets and mediates insulin resistance. In rheumatoid arthritis, it causes tissue inflammation and is implicated in joint destruction. It also plays a role in the pathobiology of trauma, meningitis, malaria, hepatitis, ischemiareperfusion injury, and adult respiratory distress syndrome. [31, 32] Endogenous TNF-alpha is produced by the activation of a single-copy gene that is located on the short arm of chromosome 6 in humans (6p21.3) at the boundary of class III and class I major histocompatibility genes. The gene is approximately 3 kilobase pairs long and consists of four exons and three introns. The fourth exon codes for greater than 80% of the mature protein. Human TNF-alpha is a 17 kDa protein that has 157 amino acids in its mature state (Figure 1). In the native form, it exists as a tightly but non-covalently bound homotrimer. Each of the three subunits in the homotrimer contains a disulphide bridge between the cysteine residues at position 69 and 101 of the mature protein which does not appear to be important for biological activity. The pro-form of the molecule has a 76 residue long precursor sequence (Figure 1) containing both hydrophobic as well as hydrophilic regions that allow the protein to anchor to the plasma membrane of the cell.

Figure 1. Schematic representation of the sequence structure of human TNF-alpha gene, mRNA and protein. A. Sequence structure of human TNF-alpha gene with 4 exons and 3 introns; B. mRNA with 5' and 3' untranslated regions (clear bars), a 76aa code for a leader sequence (diagonal bars) and a 157aa code for the mature protein (vertical bars); C. pro-protein with a 76aa leader sequence (diagonal bars) and a 157aa protein sequence (vertical bars); D. mature protein with 157aa (vertical bars).

The hydrophobic region between -44 and -26 in the pro-form of the polypeptide enables the transmembrane nature of the pre-sequence. In human TNF-alpha, the cleavage of the proform to yield the mature protein usually occurs between alanine (-1) and valine (+1). The 26 kDa pro-form is proteolytically cleaved by the metalloprotease, TNF-alpha converting enzyme (TACE), to the mature 17 kDa form. The cDNA corresponding to the mature TNF-

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alpha is conserved across mammalian species showing that this protein has a very specific and vital function in the body [23-25, 27, 33, 34] . The direct biological activity of endogenous TNF-alpha is facilitated mostly by two TNF receptors, Type I (TNFR-55) and Type II (TNFR-75) that are 55 kDa and 75 kDa, respectively. TNF-alpha has a much greater affinity for TNFR II than TNFR I. However, it appears that most of the biological activities of TNF-alpha are initiated by TNFR I that is essentially found on all cell types except erythrocytes. TNFR II is mostly confined to the cell surfaces of immune cells and ECs. The extracellular portions of these two receptors are very similar in sequence and contain 4 characteristic domains with evenly spaced cysteine residues. TNFR I which initiates most of TNF's biological activities, has 182 amino acids in the extracellular domain whereas TNFR II has 235 amino acids. In contrast, the intra-cellular portions of these receptors do not show any homology or domain similarities suggesting that they activate separate signal transduction pathways. TNFR I has 220 amino acids in the intracellular portion of the protein whereas TNFR II has 174 residues. While both receptors are N-glycosylated, only TNFR II is O-glycosylated. Receptor-ligand binding is believed to cause the clustering of three receptors together and recruitment of other proteins. Although the presence of TNF receptors is a prerequisite for biological activity, there appears to be no correlation between the number of receptors on the cell surface and the extent of response. However, it appears that there is a positive correlation between the number of ligand-receptor complexes that are internalized and the extent of the response. The number of receptors appears to vary between 200 and 10,000 per cell [25, 31, 35-40]. Full-length human TNF-alpha cDNA was easily expressed in Escherichia coli with high efficiency. Unlike other heterologous proteins, rhTNF-alpha was sufficiently soluble in the bacterial cell and, therefore, could be readily purified from bacterial extracts without the denaturation-refolding steps that could have decreased yields. This rhTNF-alpha had all the expected biological properties including in vivo and in vitro anti-tumor cell activity. This resulted in rhTNF-alpha being made available for crystal structure elucidation, biological, biochemical, and preclinical as well as clinical studies. [23-25, 41-45]

Use of RhTNF-Alpha as a Selective Anti-Cancer Agent Acute systemic exposure to exogenously injected TNF-alpha in amounts that are physiologically possible during infection results in shock, tissue injury, capillary leak syndrome, hypoxia, pulmonary edema and multiple organ failure resulting in death that closely resembles the after-effects of septic shock syndrome [46, 47]. In animal studies, it has been shown that the septic shock-like syndrome that is caused by the systemic administration of TNF-alpha is mediated by the toll like receptor 4 (TLR4) [48-50]. Chronic systemic exposure to exogenous TNF-alpha causes cachexia characterized by anorexia, weight loss, dehydration and depletion of proteins and lipids in the body [31, 32]. Due to the extensive toxic effects of systemically administered TNF-alpha, the enthusiasm about its effectiveness as an anti-cancer agent was diluted. Clinical Phase I and Phase II studies in which rhTNF-alpha was administered systemically confirmed that the dose

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limiting toxicity was due to the septic shock-like syndrome called systemic inflammatory response (SIRS) that lead to multi-organ failure [51, 52]. The focus, therefore, shifted to using rhTNF-alpha as a limited scope cancer therapeutic either by itself in relatively high doses or in conjunction with other chemotherapeutic agents by direct infusion into the tumor in the liver, extremity or brain, under conditions of hyperthermia. Based on a completely different rationale, antibodies/antagonists to endogenous TNF-alpha have also been used to inhibit its role in tumor growth and progression [53]. By the mid-1980's, the role for rhTNF-alpha as a cancer therapeutic was very diminished because it was determined in preclinical studies that an effective antitumor dose of rhTNFalpha in mice was around 50 mg/kg which would translate into a dose that was at least a tenfold higher in humans than the maximum tolerated dose (MTD). Most Phase I studies showed that the MTD for rhTNF-alpha was 30 - 200 μg/m2 when injected daily for 5 days or 200 545 μg/m2 during a 24-hour protracted infusion [52, 54-58]. In Phase II studies, the use of a dose of up to 300 - 400 μg/m2 produced infrequent and minimal tumor regression [59, 60]. Moreover, many preclinical tumor models showed that when rhTNF-alpha was used in conjunction with other chemotherapeutic agents or with gamma interferon and hyperthermia, the extent of tumor regression was much better than when rhTNF-alpha was used alone [52, 61, 62]. By the late 1980's, the use of rhTNF-alpha was restricted to the treatment of locally advanced malignancies by a surgical technique called isolated limb perfusion (ILP) which could deliver very high doses of drug to the extremities. This surgical procedure was first developed with a combination of two new emerging technologies in the 1950's [63]. Antineoplastic agents such as nitrogen mustards were being developed as medical treatment for malignancies. While these early drugs had some activity against hematological malignancies, they had very little activity when administered systemically for the treatment of solid tumors. Cardiopulmonary bypass had been developed by this time and was being standardized by cardiac surgeons as a way to sustain circulation to the body during open heart procedures [64]. Early surgical oncologists saw the opportunity to combine cardiopulmonary bypass with available antineoplastic drugs to develop regional perfusion procedures for localized cancers. The benefit of this approach was primarily to escalate the dose of drugs to increase the tissue concentration while minimizing systemic toxicity. Isolated perfusion procedures were developed and performed on the liver, kidney and extremities. The isolation perfusion procedure of solid organs had considerable technical complications and did not develop into a standard technique. Fortunately, the initial patient treated in New Orleans in the 1950's with ILP had a complete and sustained response of multiple melanoma metastases [63]. From this beginning, ILP has been used worldwide mostly for patients with soft tissue sarcomas or with in-transit metastases from primary melanomas. The rationale of ILP procedures is to gain total control over the circulation of an upper or lower extremity in which advanced tumor is located such that very high doses of the chemotherapeutic can be administered and re-circulated leading to very high tissue levels. The ability to dose-escalate chemotherapy with ILP occurs for two reasons: First, the target organs of toxicity which for most standard chemotherapeutics are the bone marrow and gastrointestinal tract are eliminated from the perfusion circuit. The small amount of bone

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marrow that is exposed in the long bones of the lower extremity accounts for only a very small fraction of the hematopoietic tissue. Second, the organs of metabolism and excretion of chemotherapeutics (i.e. liver and kidney) are also eliminated from the exposure to the intravascular drug in the ILP circuit. These two features combine to increase the area under the curve for tissue exposure many times more than what can be achieved with maximal systemic administration. The final additional benefit of ILP is that it allows regional hyperthermia to be administered. There is extensive literature documenting that hyperthermia alone has antineoplastic effects and generally augments the antineoplastic effects of chemotherapeutics [65]. Hyperthermia can be created in the extremity by external warming and by heating the perfusion circuit leading to high tissue temperatures simultaneous with high drug exposures. The surgical technique for isolation perfusion involves dissection of blood vessels to treat the entire extremity with intravascular chemotherapy without systemic exposure. Even for lesions that are in the distal extremity, such as the calf and forearm, because the primary nodal basin is the inguinal region and the axilla, respectively, the entire extremity needs to be treated for perfusions targeting extremity melanoma. The standard cannulation site for lower extremity limb perfusion is the external iliac vessels, which can be dissected by a retroperitoneal approach in the lower abdomen. An alternative area for cannulation would be the common femoral vessels. The isolation that can be achieved with common femoral vessels and the extent of proximal perfusion is somewhat limited. Furthermore, often these patients have had previous superficial inguinal lymph node dissections and operating to dissect the common femoral vessels is a reoperative field, which is much more technically demanding. For upper extremity perfusions, the axillary vessels are cannulated and this approach is used even when patients have had prior axillary dissections [66]. The principles of the surgical isolation are circumferential dissection of the entire external iliac or axillary vessels, including ligation and division of multiple side branches of the artery and vein. These vessels are cannulated such that the tip of the perfusion cannula is in the proximal extremity at a level above distal to where a tourniquet will be applied to complete the isolation. The isolation perfusion circuit typically consists of a pediatric cardiac surgery bypass circuit. After the cannula is attached to the perfusion circuit, a tourniquet is placed around the root of the extremity externally and this prevents leak between the systemic circulation and the perfusion circuit through subcutaneous and skin vessels [66]. Once the perfusion circuit has been developed, flow is begun and flow rates are typically 300 - 500 mL/minute for lower extremity perfusions and 150 - 300 mL/minute for upper extremity perfusions. One important concept in a successful and safe ILP is controlling and monitoring perfusate leak from the circuit to the systemic circulation. One benchmark for blood leak from the perfusion circuit is looking at the reservoir volume where venous blood leaves the extremity and goes to a static container. If this container increases in volume, it is indicative of blood leaving the systemic circulation and going into the perfusate circuit, which means blood loss for the patient. If the venous reservoir loses volume, it is indicative of leak from the perfusate circuit to the body. After high-dose chemotherapeutics have been administered in the perfusate circuit, this leak causes systemic exposure of the antineoplastic, and this is particularly toxic when high-dose rhTNF-alpha is used as a perfusion agent. Older techniques to monitor perfusate leak include injection of fluorescein and identification by

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Wood's light in the systemic tissues. However, this technique is imprecise and cannot be quantified. Techniques have been developed using radioactive tracers with a gamma counter to assay cardiac blood volume continuously as a representative of the systemic circulation. This radioactive tracer system is very precise and detection of leak of perfusate and the amount of leak can be quantified during a perfusion [67]. Theoretically speaking, rhTNF-alpha is an ideal drug for ILP because it acts quickly with a single high dose application and because its systemic use is limited by significant toxicity. The oncological conditions that specifically lend themselves to ILP are melanoma in-transit metastases which occur in about 6 - 10% of melanoma patients and soft tissue sarcomas that occur in about 10% of sarcoma patients. In both these instances, tumor spreads extensively and exclusively for a time in the limb [48]. A complete surgical resection of these tumors is either not possible or is not effective treatment. The first site of metastases from primary cutaneous melanoma is most commonly through the lymphatics to regional lymph nodes. Occasionally, lesions may develop in the skin and subcutaneous tissue within lymphatic vessels in conjunction with metastases to the primary nodal basin or in the absence of any nodal metastases. These lesions will appear as visible dermal lesions or palpable subcutaneous nodules typically between the location of the primary and the nodal basin. However, particularly in the lower extremities in which there may be an influence of gravity, in-transit metastases may occur distal to the site of the primary lesion. An initial staging system for extremity melanoma developed at MD Anderson Cancer Center (Houston, TX) identified in-transit metastases within 3 cm of the primary melanoma site as satellite lesions and identified this as stage II disease. In-transit metastases more than 3 cm from the primary melanoma were considered stage IIIa and nodal metastases was considered stage IIIb. Patients who had in-transit lesions and nodal metastases were considered to be stage IIIab [68]. The current American Joint Committee on cancer staging systems recognizes that these are all part of a regional lymphatic spread of disease and include them as stage III disease. In-transit metastasis without regional nodal disease is considered to be N2c and is stage IIIb. In-transit metastasis with any regional nodal metastases is N3 disease and is stage IIIc [69]. Appropriately, in the present classification schema, there is no distinction between satellite nodules and in-transit metastases because these represent a continuum of disease. The best data on the incidence of in-transit metastases come from prospective randomized trials in which patients with high risk or intermediate risk primary melanoma of the extremities are randomized to an adjuvant ILP versus observation [70, 71]. In the observation arm, patients are followed long-term and patterns of recurrence in survival are recorded. In a recently reported large randomized trial for adjuvant ILP for melanoma larger than 1.5 mm in thickness, there was a 9.6% incidence of in-transit metastases [71]. There are less data available regarding the incidence of in-transit metastases for patients with thinner melanomas. However, because it is clearly evident that thinner melanomas have a better overall prognosis; the incidence of in-transit metastases from melanomas smaller than 1.5 mm in thickness will be considerably less than this level of 9%.

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One of the arguments put forth by opponents of ILP is that in-transit metastases represent systemic disease and regional treatments are doomed to failure. However, because these lesions represent spread within the regional lymphatics as opposed to hematogenous subcutaneous metastases, in-transit disease is still a regional disease (stage IIIb or IIIc) similar to patients who have positive nodal disease in which no one would oppose regional node dissection. Furthermore, a proportion of patients that have this pattern of disease spread despite developing bulky disease within the extremity may not develop hematogenous metastases. The best evidence for this comes from older surgical literature regarding amputation for patients with locally advanced extremity melanoma [72]. Before the description of ILP and its widespread use, patients who had bulky regional melanoma often had considerable morbidity secondary to their disease and the only surgical option was an ablative amputation. Several series primarily reported from Memorial Sloan-Kettering (New York, NY) indicate a 5-year disease-free survival rate ranging from 25-33% for patients who undergo a major amputation for melanoma [72]. Because, by definition, patients would not undergo an amputation unless they had very bulky disease, this argues that the natural history in a certain subgroup of these patients is for lymphatic spread only with no hematogenous metastases. Soft tissue sarcomas (STS) constitute approximately 1% of all malignancies [73, 74] and account for 2% of all cancer related deaths. STS can occur anywhere in the body but predominantly affect the limbs (59%), the trunk (19%), the retroperitoneum (15%), and the head and neck (9%). They are a heterogenous group of tumors with nearly 50 different histological types that arise mainly from mesenchymal tissue. They can originate in fibrous tissue, fatty tissue, smooth muscle, skeletal muscle, blood and lymph vessels, perivascular tissue, synovial tissue, or peripheral nerves. The most common of these are malignant fibrous histiocytoma (28%), leiomyosarcoma (12%), liposarcoma (15%), synovial sarcoma (10%), and malignant peripheral nerve sheath tumors (6%). Rhabdomyosarcoma is the most common childhood STS. The current American Joint Committee on Cancer staging of STS is based on location (depth), size, grade of tumor, and presence of distant metastases. In the 2002 American Joint Committee on Cancer staging system, four tumor grades were designated: grade 1 - well differentiated, grade 2 - moderately differentiated, grade 3 - poorly differentiated and grade 4 - undifferentiated. Grades 1 and 2 are considered low grade whereas grades 3 and 4 are considered high grade. The metastatic potential of STS varies from 5 - 10 % for low grade tumors to 50 - 60% for high grade tumors. The size of an STS is another important prognostic variable. T1 lesions are defined as those STS that are smaller than 5 cm and T2 lesions are greater than 5 cm. The size of the tumor is generally considered alongside the anatomic location of the tumor for prognostic predictions. Tumors located above the superficial investing fascia or trunk are designated as "a" and those invading or deep to the fascia are designated by a "b" with the "a" types having a better prognosis. The dominant route of metastases is hematogenous and lymph node metastasis is rare (less than 5%). Nodal spread is designated as stage IV disease. Distant metastases occur most often to the lung and are considered stage IV disease. Other potential sites of metastases include bone, brain, and liver. Surgical resection followed by adjuvant external beam radiotherapy after marginal resection is the recommended course of treatment for STS of the extremities [73]. However, limb salvaging surgery may be difficult when the tumor burden is large or the

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tumor's proximity to vital structures limits the benefits of such surgery. Under such circumstances, ILP is a more desirable option when amputation of the limb is the only alternative [68, 75]. Regional intravascular therapy against human melanomas and soft-tissue sarcomas using high-dose rhTNF-alpha and melphalan, administered by ILP, has proven to be extremely effective. Objective response rates of 90-100% with 65-90% complete response rates have been reported in patients with in-transit metastases from melanoma. A representative melanoma patient's and soft tissue sarcoma patient's tumor burden before and after ILP with rhTNF-alpha plus melphalan are shown in Figures 2 and 3 respectively. Overall response rates with soft-tissue sarcoma range from 58%-82% with 15-30% complete responses. These results, achieved with a single 90-minute ILP using rhTNF-alpha and melphalan, are significantly better than the best results achieved with combination systemic chemotherapy of 30% overall response rates and 0-5% complete responses for melanoma and 15-20% overall response rates with essentially no complete responses for sarcoma. These results are especially significant given that the treatment of established solid human malignancies with systemic administration of currently available chemotherapeutic agents leads to relatively poor outcomes with few exceptions [66, 68].

Figure 2. Pre-op (left panel) and 13-month post-op photographs after a 90-min ILP with rhTNF-alpha and melphalan (right panel) showing effectiveness of treatment in a melanoma patient. In the intervening months, this patient received no other therapy.

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Figure 3. A: Photographs of a patient with multifocal angiosarcoma of the ankle and calf region. An appropriate above-knee amputation was recommended for this patient. B: The patient underwent an ILP with rhTNF-alpha plus melphalan and had complete resolution of disease 4 months after the operation. The patient remained free of disease more than five years after ILP.

The precise mechanism underlying this remarkable anti-tumor effect against solid tumors with ILP has not been fully elucidated. During ILP treatment, all of the normal tissues immediately surrounding the tumor i.e. skin, subcutaneous fat, muscle, bone, and peripheral nerve are subject to the same drug concentration as the tumors present in the extremity. Remarkably however, the combination of rhTNF-alpha and melphalan affects only tumor and not normal tissue and is known to work synergistically. ILP with melphalan-alone has activity for melanoma when the tumor burden (size and number of nodules) is limited. For large tumors or extensive disease, the response to melphalan-alone is limited, but is significantly improved by adding rhTNF-alpha to the treatment regimen. It is widely accepted that melphalan, a nitrogen mustard, forms DNA adducts within dividing tumor cells. The next section critically reviews recent data that provide insights into plausible mechanisms by which rhTNF-alpha asserts its anti-tumor activity.

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Mechanism of Selective Tumor Destruction by RhTNF-Alpha In trying to understand the gross and molecular mechanisms underlying the tumoricidal effects of high-dose rhTNF-alpha in combination with melphalan during an ILP, it would be beneficial to recall the important observations made during Coley's time and in the years immediately following his seminal discovery. First, a relatively strong and sustained release of endogenous TNF-alpha, probably acting synergistically with other cytokines, was necessary to obtain a lasting anti-tumor effect. Second, different types of cancer cells were not equally sensitive to the tumoricidal effects of TNF-alpha even at high doses. In fact, TNF-alpha was found to be cytotoxic to some types of cancer cells, cytostatic toward others, and showed no effect toward yet other types of cancer cells. When normal non-cancerous cells were exposed to TNF-alpha, it appeared to promote cell growth and survival. Third, the differences in sensitivity to the cytotoxic effects of TNF-alpha were not dependant on the differences in the number of TNF-alpha cell surface receptors on cancer cells. Fourth, experiments in preclinical animal models showed that tumors were destroyed by a process of bleeding into themselves called "hemorrhagic necrosis". In fact, it was widely believed at the time that TNF-alpha damaged tumors mostly by destroying the tumor's blood supply. In normal tissue, however, TNF-alpha was known to support the process of angiogenesis. Lastly, the tumoricidal effect of TNF-alpha was independent of nuclear activity showing that de novo protein synthesis was not essential for TNF-alpha's cytotoxicity toward malignant cells. In fact, the use of either actinomycin D (a transcription inhibitor) or cycloheximide (a protein synthesis inhibitor) actually increased the sensitivity to TNF-alpha approximately 50 to 100-fold showing that exposure to TNF-alpha concurrently activated a "resistance-type phenotype". In non-cancer cells as well as in less sensitive cancer cells, TNF-alpha was known to induce mitogenic cell-cycling activity as well as the synthesis of a wide array of proteins that could be protective in nature and thus reduce the toxic effects of the molecule. In the absence of these proteins, TNF-alpha could be lethal. In the years that followed, studies have attempted to further elucidate the mechanism underlying the anti-tumor effect of high-dose TNF-alpha grossly and at the molecular level. Some of these studies have attempted to do so by primarily analyzing the direct cytotoxic effects on tumor cell lines in an in vitro setting. While one can rationalize the use of such in vitro direct tumor cell cytotoxicity studies to test secondary hypotheses, there are several reasons that call for the use of in vivo data from solid tumors or in vitro endothelial cell (EC) models that simulate conditions in solid tumors to test primary hypotheses to arrive at the most plausible molecular mechanisms that result in the tumoricidal effects of rhTNF-alpha in an ILP setting. First, in the ILP treatment of extremity tumors, high-dose rhTNF-alpha is circulated in the affected area via local blood vessels. Therefore, the first tissue type that encounters rhTNF-alpha is the blood vascular endothelium and the cell type that is first exposed to high doses of rhTNF-alpha is the EC and not the tumor cell. So, it is imperative to study the effect of high-dose rhTNF-alpha on tumor associated ECs and the tumor vasculature. Second, some tumor cell types that are resistant to the direct cytotoxicity of rhTNF-alpha plus melphalan in vitro have been found to be sensitive to ILP treatment with rhTNF-alpha plus melphalan in vivo indicating that the tumor milieu is an important

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participant in determining the therapeutic outcome of rhTNF-alpha ILP [62, 76]. Third, ILP treatment with high-dose rhTNF-alpha plus melphalan is therapeutically effective against larger well-vascularized tumors indicating that direct tumor cell cytotoxicity is not the primary cause of tumor destruction [77-79]. Fourth, direct tumor cell cytotoxicity occurs in vitro within 48 - 72 hours post-treatment and this is much longer than the time it takes to see tumor necrosis in ILP in tumors that are greater than 5 cm in diameter [76]. Fifth, the treatment of tumors with melphalan-alone ILP, in general, has yielded less significant results when compared with the combination therapy of high-dose rhTNF-alpha plus melphalan [68]. Analyses of tumor tissue samples treated with melphalan-alone or rhTNF-alpha plus melphalan showed an increase in tumor tissue accumulation of melphalan in the latter instance [80, 81]. Also, studies in mice and rats have shown that a low dose of rhTNF-alpha augments anti-tumor activity of pegylated liposomal doxorubicin [82, 83]. The plausible conclusion from this increased uptake of melphalan and liposomal doxorubicin is that there is an increased leakiness of the blood vessels following exposure to rhTNF-alpha. Sixth, in the syngeneic model of methylcholanthrene induced fibrosarcoma, tumor cells are resistant to rhTNF-alpha in vitro whereas in vivo systemic administration of rhTNF-alpha consistently causes hemorrhagic necrosis of subcutaneous (vascular) but not intraperitoneal (avascular) tumors [84-87]. Taken together, the above observations indicate that in order to decipher the molecular mechanism underlying the tumoricidal effects of rhTNF-alpha in an ILP setting, it is important to study its effect primarily in solid tumors, complete with tumor vasculature and surrounding tumor microenvironment or in in vitro models of tumor plus tumor microenvironment, simulating the in vivo setting rather than in tumor cell lines alone. It is imperative to be vigilant about not making sweeping mechanistic extrapolations and conclusions about the human ILP effect from in vitro and/or preclinical animal models unless such observations have been specifically tested in the human system. It is also important to distinguish between effects produced as a result of exposure to physiologically relevant levels of endogenously produced TNF-alpha versus the effect of very high doses of rhTNF-alpha as seen in the case of ILP. A good example of the importance of the effect of dose in the context of rhTNF-alpha is seen in the work of Fajardo et al (1992) in which a murine disc angiogenesis system designed to quantify microvessel proliferation at low and high doses of rhTNF-alpha was used. At lower doses (0.01 to 1 ng), rhTNF-alpha supported angiogenesis which was maximum at 0.1 ng and at higher doses (1 and 5 μg) the treatment inhibited it [88]. At a gross level, a general consensus has emerged that ILP with rhTNF-alpha plus melphalan predominantly destroys the tumor as a result of three types of pathological changes to the tumor endothelium. First, there is an inflammation type reaction including oedema and hemorrhage followed by leukocyte extravasation and erythrostasis. Second, there is a definite destruction of the existing tumor vascular endothelium, and third, there appears to be an inhibition of the process of neovascularization within the tumor thus preventing the rapid regeneration of the tumor post-treatment with rhTNF-alpha. Although the evidence is not robust, preclinical work from our laboratory at the Department of Surgery, University of Pennsylvania indicates that there may be a secondary component to the process of tumor destruction post-rhTNF-alpha ILP which involves a direct tumor cell kill by tumor infiltrating macrophages [89]. The focus of the more recent ILP-related mechanistic research has shifted

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toward determining the molecular events that lead to the above described tumor destroying gross characteristics post-ILP. Some of the candidate molecules explored in this context are enumerated below and are summarized in Figure 4.

Figure 4. Schematic representation of the effect of high-dose rhTNF-alpha on the tumor vasculature showing the thus far identified molecular mediators of destruction of the tumor vasculature.

Cell Adhesion Molecules (CAMS) It is generally accepted that an acute inflammation reaction in which ECs swell, protrude into the lumen, and become leaky to macromolecules is elicited in the tumor vasculature immediately after exposure to rhTNF-alpha. Such a reaction also involves EC activation which is a term used for the expression of a specific set of molecules by the exposed EC followed by leukocyte adhesion and extravasation between tumor EC. In mid-1994, a group of scientists lead by Ferdy Lejeune at the University of Brussels in Belgium showed that early endothelium activation and polymorphonuclear cell invasion preceded necrosis of human melanoma and sarcoma treated intravascularly with rhTNF-alpha plus alkylating agents [90]. Activation of the EC under pathophysiological conditions in general, as in the case of the treatment with rhTNF-alpha, includes the expression of EC activation antigens also referred to as CAMS which act as markers of the EC activated state. It also includes morphologic changes in the post-capillary venular ECs culminating in a vascular leak syndrome. In the University of Brussels study, biopsies from 27 human cancer patients, 20 of whom had melanoma in-transit metastases and seven had high-grade soft

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tissue sarcoma, were analyzed before and after ILP with rhTNF-alpha by immunohistochemistry for platelet EC adhesion molecule (PECAM), vascular EC adhesion molecule (VCAM-1), E-selectin and intercellular cell adhesion molecule (ICAM-1). This study reported observing a swelling in the ECs of the tumor capillaries in the first hours after perfusion. In the melanoma cases, they observed tumor cell necrosis within 3 hours after perfusion. The overall predominant feature was coagulative necrosis with or without hemorrhagic necrosis. Treatment with rhTNF-alpha induced an increased expression of Eselectin and VCAM-1 on tumor vascular ECs and the activated tumor vasculature was progressively destroyed. There was increased tumor infiltration by polymorphonuclaer cells (PMNs) and macrophages. Melanoma in-transit metastases that were treated solely with the alkylating agent did not show necrosis or an upregulation of VCAM-1 and E-selectin and did not show tumor infiltration with PMNs or with macrophages [90]. In 1996, Nooijen et al. arrived at a different conclusion about E-selectin upregulation on vascular endothelial cells after rhTNF-alpha plus melphalan ILP in responsive melanoma and sarcoma patients. They analyzed immunohistochemically stained sequential biopsies from tumor and normal distant skin before ILP and at 30 minutes and 24 hours after ILP by light and electron microscopy and compared the results with those obtained using an in vitro system consisting of human umbilical vein EC exposed to rhTNF-alpha plus melphalan. They found that ICAM-1 and PECAM-1 were constitutively expressed on vascular EC both in normal tissues and in tumors. E-selectin was found to be moderately increased in tumor EC and markedly increased in perfused normal skin. They found no drastic changes in expression of VCAM-1, ICAM-1 or PECAM-1. Since the upregulation of E-selectin was not restricted to the tumor vasculature and they did not observe hemorrhagic necrosis following this upregulation, they concluded that the above microvascular events were not a decisive tumorspecific mechanism for tumor regression in ILP [91]. However, later studies reported by Eggermont et al. (1997) , by Lejeune et al. (1998) and by the Italian group, Ferroni et al. (2001) [92] all confirm the upregulation of E-selectin and VCAM-1 in human ILP tumors post- rhTNF-alpha treatment which is believed to set off a cascade of events leading to tumor destruction.

Von Willebrand Factor (VWF) VWF is a multimeric glycoprotein protein that is exclusively synthesized and stored in ECs and megakaryocytes. Its displacement from the EC is considered a marker for EC damage. In 1995, another study from the Belgium group lead by D.J. Ruiter [93], examined sequential biopsies from 29 stage IIIA/B melanoma patients before and after treatment with ILP with high-dose rhTNF-alpha, interferon gamma, and melphalan. Addition of interferon gamma to the perfusate is known to produce marginal increments in the overall response rate in human patients. The tissue samples from the above study showed no necrosis, hemorrhage or fibrin thrombi before perfusion. Within 3 hours post-treatment, a change in the distribution of VWF occurred from a discrete endothelial pattern in the untreated lesions to a fuzzy perivascular and subendothelial pattern in the treated lesions. The above study showed that within 24 hours post-ILP, VWF displacement was accompanied by intravascular thrombocyte

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aggregation and erythrostasis, in the absence of tissue factor and fibrin deposits. It is now known that when there is damage in the vascular endothelium, VWF binds to the platelet membrane receptor (glycoprotein Ib-IX complex) and to the components of the subendothelial connective tissue and mediates initial platelet binding to the subendothelium. These findings indicate that the thrombocyte aggregation observed is not caused by local procoagulant activity, but is rather the result of the therapy-associated vascular damage or haemostasis. In 1996 [94], the same group used a preclinical isolated rat sarcoma limb perfusion model and showed that ILP with rhTNF-alpha caused vascular leakiness which preceded vascular occlusion due to platelet aggregation. This was an important observation because it established that platelet aggregation was the result of increased vascular permeability rather than an initiator of vascular damage in the rat ILP system.

Endothelial Monocyte-Activating Polypeptide II (EMAP II) In the mid- to late 1990's and in 2000 and 2001 investigators looked for alternative molecules that could potentially mediate an enhanced anti-tumor response to ILP with rhTNF-alpha. One such candidate molecule was the endothelial EMAP II which was at the time considered a novel tumor-derived cytokine. It was first identified by Clauss et al. in 1990 [95] in the supernatant of methlycholanthrene A-induced fibrosarcoma cells. The protein in the supernatant was cloned and characterized by Stern D. and his colleagues in 1994 [96] at Columbia University's College of Physicians and Surgeons to be a unique leaderless single polypeptide chain with predicted molecular mass of 34 kDa with a mature form of 20 kDA. The work from this group showed that local injection of EMAP II into a tumor that was resistant to the effects of rhTNF-alpha such as murine mammary carcinoma rendered it sensitive to subsequently administered rhTNF-alpha. These data suggest that tumor-derived EMAP II has properties of a proinflammatory mediator with the capacity to prime the tumor vasculature for a locally destructive process in the murine system. Libutti S. K. and his team at the Surgery Branch of the National Cancer Institute showed using cell lines and preclinical animal models that EMAP II expression post-rhTNF-alpha treatment appeared to increase TNF-alpha receptor expression on tumor endothelium and enhance the induction of tissue factor on endothelial cells resulting in an anti-angiogenic effect [96, 97]. It was shown that in vivo sensitivity of the tumor vasculature to rhTNF-alpha is determined by the tumor production of EMAP II [98]. These and other similar studies also showed that EMAP II is produced in various levels and that one can increase the sensitivity of tumor to TNF-alpha therapy in vivo by up-regulating or increasing EMAP II production [99, 100]. Apart from being an anti-angiogenic molecule, EMAP II is also considered a modulator of inflammatory reactions [101]. Under pathophysiological conditions, EMAP II appears to have several functions. It is known to trigger the recruitment of macrophages by elevating cytosolic-free Ca2+ concentrations [96]. It also causes the release of VWF from EC, it stimulates leukocyte chemotaxis, induces the expression of endogenous TNF-alpha and tissue factor by monocytes and myeloperoxidase by polymorphonuclear cells [96]. In human patients, Eggermont A. M. and colleagues in The Netherlands found that for melanomas there was an up-regulation of EMAP II directly after ILP and such an increase in expression

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significantly correlated with a complete tumor response [102]. No such correlation was found for sarcoma patients [102].

Alphavbeta3 Integrin In 1998, Ferdy Lejeune [103] and his colleagues showed that an exposure of human endothelial cells to a combination of TNF-alpha and IFN-gamma resulted in a reduced activation of alphavbeta3 integrin which is an adhesion receptor that plays a key role in angiogenesis leading to a decreased alphavbeta3-dependant adhesion and survival. The study also showed detachment and apoptosis of angiogenic endothelial cells in vivo in melanoma metastases in patients treated with rhTNF-alpha and IFN-gamma. However, the study also showed that pre-treatment of human endothelial cells with either cytokine alone was ineffective in inhibiting adhesion. These results, therefore, only implicate alphavbeta3 in the anti-vascular activity of the specific combination of rhTNF-alpha and IFN-gamma. It must also be pointed out that several studies have found either no significant role or only a marginal role for IFN-gamma in the preclinical models of ILP with TNF-alpha and/or melphalan or in ILP patients in the clinic [104-107].

Fas and Fas Ligand In their work published in Nature Medicine in 1998, Sata M. and Walsh K. from the Tufts University School of Medicine analyzed the role of endothelial Fas Ligand expression in controlling leukocyte extravasation after rhTNF-alpha treatment [108]. While Fas receptor is expressed on many cell types, Fas ligand expression is very restricted. Fas Ligand induces apoptosis in cell types that carry its receptor, Fas also known as CD95 or APO-1. In their work reported in 1998, Sata M. and Walsh K. used in vitro EC models and demonstrated that there was constitutive expression of functional Fas Ligand on EC. Also, ECs exhibited a cytotoxic activity toward co-cultured Fas bearing cells which was diminished when ECs were pre-treated with rhTNF-alpha or were treated with neutralizing anti-Fas Ligand antibody. They also used the central artery of the rabbit ear as their preclinical animal model for leukocyte extravasation in which a tourniquet was temporarily applied to the base of the ear to interrupt blood flow and to allow the infusion of rhTNF-alpha. Data collected from this set-up showed that Fas Ligand expression was vastly downregulated by rhTNF-alpha in a dose-dependant manner. The functional significance of this finding was a blockage of rhTNF-alpha induced leukocyte extravasation when Fas Ligand was constitutively expressed on the endothelium. This model also allowed the endothelium of the isolated central ear artery to be infected with a replication defective adenoviral construct that would constitutively express Fas Ligand from a viral promoter called adeno-FasL or a control adenoviral construct that expressed the bacterial marker protein, beta-galactosidase called adeno-betagal. When central ear arteries were isolated and rhTNF-alpha was administered locally, Fas Ligand was found to be downregulated and adhesion molecules were found to be upregulated. These changes were accompanied by a robust infiltration of the vessel wall by

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leukocytes. When the rabbit ear arteries were infected with adeno-FasL alone, Fas Ligand expression was maintained following rhTNF-alpha stimulation, whereas cell adhesion molecule expression was induced. Under conditions of constitutive Fas Ligand expression, leukocyte invasion of the vessel wall was reduced, whereas vessels infected with adenobetagal showed extensive leukocyte extravasation. Analyses of the adeno-FasL infected vessels at the earlier time-points showed that leukocytes adhered but underwent apoptosis rather than migration to the subendothelial tissue. Taken together, these findings indicate that Fas Ligand expression by the vascular endothelium prevents leukocyte infiltration and a downregulation of the Fas Ligand is a necessary prerequisite to leukocyte extravasation after rhTNF-alpha treatment [109]. Although the data obtained in the above study is very compelling, the signals that connect rhTNF-alpha to the downregulation of Fas Ligand in tumors have not been worked out. Also, the results have not been confirmed in the human ILP system.

VEGF Receptors, Flk-1 and Flt-1 Our team at the University of Pennsylvania's Department of Surgery developed a human melanoma rat xenograft model for ILP and demonstrated in 2003 using VEGF-transduced human melanoma tumors in the rat that rhTNF-alpha downregulated the expression of the VEGF receptor, Flk-1 [110]. Earlier work had shown that rhTNF-alpha downregulates VEGF-specific receptors, Flk-1 and Flt-1, on endothelial cells in vitro and that the rhTNFalpha effect was transcriptionally mediated and was accompanied by a decrease in immunoprecipitable Flk-1 protein [111]. RhTNF-alpha has been shown to selectively shutdown tumor vasculature by causing increased tumor vascular leakiness and erythrostasis while sparing normal vasculature in the limb [112, 113]. Vascular shutdown has been shown to increase hypoxia and, therefore, to increase VEGF production within the tumor [114]. VEGF is a known endothelial mitogen and a potent inducer of angiogenesis that works via its two receptors, Flk-1 and Flt-1. Therefore, in order to prevent tumor recovery after ILP with rhTNF-alpha and melphalan, it would be important to not only shutdown existing vasculature in the tumor but to also prevent angiogenesis induced by the resulting hypoxic milieu. There appears to be a direct correlation between Flk-1 expression levels and angiogenic activity because highest levels of Flk-1 expression are observed during vasculogenesis and angiogenesis and pathological processes associated with neovascularization such as tumor angiogenesis [115-117]. Also, suppression of VEGF activity by inhibiting its receptors has been shown to suppress retinal neovascularization in a murine model of ischemic retinopathy [117], and deletion of the Flk-1 gene by homologous recombination has been shown to abolish vasculogenesis in mice [118]. Several studies have also shown a direct correlation between Flk-1 inhibition and tumor regression/cell kill [119-123]. These findings, taken together, indicate that any factor that can decrease the levels of expression of Flk-1 in tumors is likely to directly affect the process of tumor angiogenesis and, therefore, tumor growth. One obstacle in evaluating the effect of TNF on VEGF receptor expression in vivo was the relatively low baseline level of these receptors in many tissues, including tumors. The

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VEGF over-expressing melanoma tumors, NIH1286/15 and NIH1286/3 which developed larger, more prominent and greater numbers of blood vessels provided an amplified system to carry out this study. Using these human tumors grown in nude mice, we found in our study that rhTNF-alpha downregulates Flk-1 expression at the mRNA and protein levels in vivo in a time dependent manner. Specifically, a reduction in Flk-1 mRNA expression was seen as early as 0.5 h post-TNF treatment, expression was 20% of baseline levels at 6 h and returned to baseline levels by 12-24 h. Protein levels were reduced as early as 2 h post-TNF and were barely detectable at 6-12 h post-treatment. Onset of tumor necrosis in the TNF treated tumors was seen between 24 and 72 h post-treatment. Downregulation of Flk-1 occurred without gross damage to tumor blood vessels or decrease in expression of PECAM, another endothelial cell-specific antigen. Post-treatment Flk-1 mRNA and protein expression pattern correlated with the half-life of rhTNF-alpha which has been determined to be between 11 and 30 min. It appears that the transient decrease in Flk-1 expression after rhTNF-alpha treatment works in conjunction with other established tumor selective anti-vascular effects of TNF such as increased vascular leakiness and blood vessel occlusion, to produce tumor necrosis. The time-course of Flk-1 expression changes between 30 min and 12 h after rhTNF-alpha administration appears to support this hypothesis because it precedes the onset of tumor necrosis. In order to demonstrate that a downregulation of Flk-1 by rhTNF-alpha independently contributes to the therapeutic effect of TNF, its effect on Flk-1 will have to be separated from its effect on vascular permeability and blood vessel occlusion. Such a separation of TNF effects is almost impossible to achieve with currently available techniques and resources. However, there is some indirect evidence in the literature to show that a transient decrease in Flk-1 expression impacts neovascularization. Cyclic angiogenic processes in the ovarian corpus luteum of mono-ovulatory species are characterized by distinct phases of blood vessel growth, vessel maturation, and vessel regression. The VEGF/VEGF receptor system is expressed through most of the ovarian cycle and is only transiently downregulated during luteolysis, which has been shown to directly lead to a regression of the neovasculature before the 21-day cycle repeats itself [124]. Conversely, in a rat infarct model, an initial rapid rise in VEGF and its receptors (275-400%) was observed about 1 h after an acute myocardial infarction and was sustained above baseline levels until about 6 h post-infarct. New vessels were found infiltrating the infarct at 3-7 days after the infarction. The increase in Flk-1 was due to tissue hypoxia [125]. If it is assumed, for the sake of argument, that TNF causes erythrostasis without decreasing Flk-1 in the human melanoma xenograft used in this study, then increased tumor hypoxia that will follow erythrostasis could upregulate Flk-1 and allow for tumor recovery. It is therefore significant that TNF downregulates Flk-1 rather than upregulates it. There is also ample evidence in the literature to suggest that Flk-1 upregulation and activation is an absolute requirement for tumor angiogenesis. There is also abundant evidence in the literature to show that inhibition or downregulation of Flk-1 can, by itself, significantly inhibit tumor growth by inhibiting tumor angiogenesis [119-123]. One can infer from this information that a transient decrease in Flk-1 expression, coincident with vascular shutdown caused by TNF, will delay the process of angiogenesis and tumor recovery. Since tumor necrosis begins in the time after this transient Flk-1 decrease, it could be rationalized that a delay in tumor recovery due to Flk-1 downregulation could be therapeutically beneficial. Taken together, evidence in the

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literature and our present work shows that rhTNF-alpha can be anti-vascular and antiangiogenic in tumors. Significant upregulation of VEGF receptor expression is seen in the neovasculature of tumors compared to established normal tissue [117, 121]. This could be one explanation for the specificity of intravascular TNF targeting tumor vessels while having minimal effects on adjacent normal vessels during isolated perfusion procedures. Also, VEGF as a growth factor is present more in the microenvironment of the tumor than in quiescent normal tissue. It has been shown that VEGF protein diffusion does not extend beyond 50 microns from the tumor border [126]. If the downregulation of Flk-1 by rhTNF-alpha is important in its anti-tumor response, this localization of VEGF in the tumor microenvironment would provide a mechanism by which the response would be seen only in the tumor vasculature. An area that has remained largely unexplored, due to the complexity of the process involved, is the regulation of VEGF receptor expression by rhTNF-alpha. There is some evidence to show that AP-1 and NFkB, the common regulators of TNF action, are not involved in this regulation [111] and that some novel trans-acting factors are involved. It is also not known whether the TNF receptors, TNFR-I and/or TNFR-II and the TNF receptor associated factors (TRAFs) are involved or whether these effects are mediated in a TNFRindependent pathway. It is also not clear as to what other factors within the tumor, if any, interact with TNF in producing the anti-vascular effect seen in tumors. Also, an artificial amplified preclinical system was used in our study and the results we obtained have not been confirmed in the human ILP system. Clearly, these are areas of research that need to be pursued and can further our understanding of the effect of high-dose rhTNF-alpha in tumors.

Vascular Endothelial Cadherin (VE-Cadherin) While tumors are known to be more hypoxic and acidic than normal tissue [127], there are very many important differences between the two vasculatures. Normal tissues have a regular predictable pattern of blood vessels while tumors are known to have highly abnormal, distended capillaries with leaky endothelial linings and sluggish blood flow [128]. There is evidence that TNF-alpha causes increased permeability specifically within tumors compared to normal tissues. Studies using radiolabeled antibodies suggested that the delivery of these large molecules to tumors was augmented significantly by pretreatment with rhTNF-alpha suggesting a further increase in vascular permeability [129]. The precise cellular and molecular changes that occur in the tumor vasculature to cause this increase in vessel permeability following TNF-alpha is not known. At a structural level, vascular endothelial adherens junctions promote intercellular adhesion and contribute to the control of vascular integrity and leakiness in any given tissue, including tumors [130]. At a molecular level, the vascular adherens junctions are formed by a transmembrane and cell specific adhesive protein, vascular endothelial (VE)-cadherin, which is linked by its cytoplasmic tail to intracellular proteins [131]. VE-cadherin is a 130 kDa cell surface glycoprotein that is constitutively expressed in the vascular endothelium and mediates Ca2+ - dependent cell-cell adhesion. It is composed of an N-terminal extracellular domain and a relatively small cytoplasmic domain at the C-terminal side. A single membrane-

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spanning region connects the two domains. The extracellular domains of VE-cadherin molecules from neighboring cells establish a homophilic type of binding resulting in cell-cell adhesion. On the cytoplasmic end, VE-cadherin interacts with beta-catenin, plakoglobin and p120. Beta-catenin and plakoglobin bind alpha-catenin, which is homologous to vinculin. The cadherin-catenin complex is in contact with the actin cytoskeleton intracellularly via vinculin. This complex organization involving the cadherin-catenin complex and the actin cytoskeleton is known to be important for angiogenesis and neovascularization and thus, for tumor growth and metastases [132]. There is also evidence in the literature to show that VEcadherin expression is important for transferring signals between neighboring cells, [93] and for cell-cell adhesion and to regulate vascular permeability [133]. It is conceivable, therefore, that any disruption of tumor VE-cadherin would alter the integrity of the vasculature and have an impact on tumor viability. Evidence in the literature indicates that an increase in vascular permeability is almost always associated with a disruption of the adherens junction complexes that are glued together via cadherin homodimers [134-136]. Our study carried out at the Department of Surgery, University of Pennsylvania [137] showed that rhTNF-alpha selectively damages the integrity of tumor vasculature by disrupting VE-cadherin complexes at vascular endothelial cell junctions leading to gapping between endothelial cells, causing increased vascular leak and erythrostasis in tumors. This study demonstrated using electron microscopic analyses of human melanoma biopsies, pre- and post-melphalan perfusion, that the addition of rhTNFalpha caused gapping between endothelial cells by 3-6 h post-treatment followed by vascular erythrostasis in treated tumors. In human melanoma xenografts raised in mice, tumor necrosis factor-alpha selectively increased tumor vascular permeability by 3 h and decreased tumor blood flow by 6 h post-treatment relative to treated normal tissue. In an in vitro tumor endothelial cell model, tumor necrosis factor-alpha caused vascular endothelial adherens junction protein, VE-cadherin, to re-localize within the cell membrane away from cell-cell junctions leading to gapping between endothelial cells by 3-6 h post-treatment. Phosphotyrosinylation was a prerequisite for movement of VE-cadherin away from endothelial cell junctions and for gapping between endothelial cells. Clinical ILP tumor specimens, at 3 h post-perfusion, showed a discontinuous and irregular pattern of VEcadherin expression at endothelial cell junctions when compared with normal (skin) or pretreatment tumor tissue. This study utilized clinical specimens from before and after rhTNF-alpha plus melphalan ILP treatments besides utilizing preclinical in vivo models, and in vitro tumor endothelial cell models to demonstrate that TNF causes tumor selective damage to the vascular integrity mediated by VE-cadherin, which appears to be a molecular target in ILP-tumor therapy. Our in vitro and in vivo data indicate that the tumor microenvironment is less conducive to strong vascular adherens junctions than normal tissue environments. Using cultured microvascular endothelial cells, tumor supernatant from human melanoma plus hypoxia led to a moderate disruption of cell-cell junctions. The combination of conditioned medium from melanoma cell cultures and hypoxia was used to reproduce the tumor microenvironment. The addition of rhTNF-alpha at the levels utilized in ILP to this simulated tumor microenvironment caused a complete breakdown of cell-cell contact. This rhTNF-alpha effect was greatly decreased in normoxia without tumor supernatant, thus offering some explanation to the selectivity of the effect on tumor endothelium versus normal

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tissues. Further evidence for the specificity of rhTNF-alpha to tumor microvasculature comes from the confocal studies of ILP specimens. RhTNF-alpha appears to disrupt the integrity of the tumor vasculature represented by a complete loss of or abnormal/discontinuous VEcadherin expression at tumor endothelial junctions in relation to skin perfused under the same conditions. Our in vitro data suggests that VE-cadherin molecules from endothelial cell junctions do not undergo degradation but move within the cell membrane away from junctional areas in response to TNF treatment. It is hypothesized that such a change in the endothelial junctions could result in the gapping seen between tumor endothelial cells in vitro and in vivo. The damaged endothelial lining could in turn lead to increased vascular leakiness, decreased blood flow and resulting hemostasis or erythrostasis. Evidence from angiograms, primarily of human soft tissue sarcomas done before and after perfusion with rhTNF-alpha plus melphalan show specific obliteration of blood flow in tumor vessels compared to surrounding normal vessels [77]. The specific factor in the tumor supernatant in the in vitro studies or in the tumor microenvironment in vivo that is responsible for such an effect is not known. Clearly, the rhTNF-alpha effect is augmented by hypoxia and the tumor supernatant and the impact these conditions have on endothelial cells is under investigation. Evidence in the literature and our in vitro data also indicate that movement of VE-cadherin away from endothelial cell junctions and gapping between endothelial cells is preceded by increased phosphorylation of junctional proteins [135]. Similar results have been obtained in studies using inflammatory mediators such as histamine and TNF on human placental vasculature [138] and also by other vascular hyperpermeability inducing factors [139]. Our results vary considerably from those published in 2003 by Freidl J. and Alexander H. R. in the International Journal of Oncology in which they used an in vitro system consisting of human vein umbilical endothelial cell monolayer exposed to hyperthermia (40 deg C) with or without rhTNF-alpha in which permeability was assessed by quantifying flux of albumin bound Evan's Blue dye across the EC monolayer. They also measured VE-cadherin expression by immunohistochemical methods. Their results showed increased permeability across the EC barrier with hyperthermia but not with rhTNF-alpha alone. This result is not in keeping with the generally accepted notion that TNF-alpha affects a change in EC layer permeability. This study also showed a decrease in VE-cadherin expression in vitro with hyperthermia but not with rhTNF-alpha alone [140].

Nitric Oxide (NO) and Nitric Oxide Synthase (NOS) There has been a range of results reported about the role of NOS and NO in the mechanism underlying TNF-alpha ILP. NO is a noxious, stable free radical gas that is highly reactive and if produced in sufficient pathological quantities (μmolar) can create extensive damage in tissues. It has been known for a while that the effects of TNF-alpha mediated septic shock in tissues are mediated by NO which is produced from L-arginine and the reaction is catalyzed by NOS. There are 3 types of NOS in tissues. EC derived eNOS and neuronal/brain cell derived nNOS are constitutively expressed and produce small amounts of NO in response to normal physiological stimuli. In contrast, inducible NOS (iNOS) produces

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NO in relatively large quantities over sustained periods of time and is implicated in many pathophysiological reactions of NO. In their work published in the American Journal of Physiology in 1997, Joseph R. Williamson and his team at the Washington University School of Medicine used a rat lung quantitative vascular permeation assay and selective iNOS inhibitor, aminoguanidine, as well as a nonselective NOS inhibitor, N-monomethyl- L-arginine (L-NMMA), to demonstrate that TNF-alpha impairs vascular barrier integrity and that such a dysfunction was associated with an increase in expression of iNOS mediated NO production. Selective inhibition of iNOS reduced vascular permeation whereas nonselective inhibition of NOS increased vascular barrier dysfunction. Until this time, the role of NO produced by iNOS and constitutively expressed NOS in TNF-alpha mediated vascular dysfunction was not clearly defined. In the above study, NO was measured by the conventional indirect method by quantifying nitrate and nitrite in the tissue and a low dose of TNF-alpha was used than seen in ILP [141]. In 2000, Eggermont A. M. M. and his colleagues in The Netherlands used a BN175 rat soft tissue sarcoma model of ILP in which the tumor was concurrently treated with the nonspecific NOS inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME) (80 mg/kg) in combination with rhTNF-alpha and melphalan. They reported that L-NAME when used alone caused growth delay of tumors when compared with sham perfusions (p = 0.02). When used with rhTNF-alpha, tumor responses increased from 0 - 64% when compared with rhTNFalpha-alone (p < 0.001) and when used with melphalan tumor responses increased from 0 63% when compared with melphalan-alone (p < 0.001). An additional anti-tumor effect was reported when L-NAME was added to the combination treatment of rhTNF-alpha and melphalan when tumor responses increased from 70 - 100% [142]. In work done at the University of Pennsylvania and published in 2008 [89], we investigated the role of tumor NO and vascular regulation in tumor ulceration following highdose rhTNF-alpha treatment in a preclinical mouse model. Using TNF-responsive (MethA) and non-responsive (LL2) mouse tumors, tumor NO concentration was measured in real-time with an electrochemical sensor and tumor blood flow was measured by Doppler ultrasound. The direct local NO measurements carried out in this study are far more reliable than the indirect measurements by plasma nitrate and nitrite. Mice were also pre-treated with a selective iNOS inhibitor, 1400W at a dose of 5mgs/kg. Tumors harvested from rhTNF-treated mice were cryosectioned and immunostained for murine macrophages, or/and iNOS. Another set of MethA tumor-bearing mice were depleted of macrophages, pre- and post-TNF-alpha tumor NO levels were measured continuously and mice were followed for gross tumor response. In MethA tumors, rhTNF-alpha caused a 96% response rate and tumor NO concentration doubled to pathophysiological μmolar concentrations. Tumor blood flow decreased to 3% of baseline by 4 hours and was sustained at 24 hours and 10 days post-TNF. Selective NO inhibition with 1400W blocked NO rise and decreased response rate to 38%. MethA tumors showed tumor infiltration by macrophages post-TNF-alpha and the pattern of macrophage immunostaining overlapped with iNOS immunostaining showing that the macrophages were the source of iNOS mediated NO burst. Depletion of macrophages inhibited tumor NO increase and response to TNF. LL2 tumors had a 0% response rate to TNF-alpha and exhibited no change in NO concentration. Blood flow decreased to 2% of baseline by 4 hours, recovered to 56% by 24 hours and increased to 232% by 10 days. LL2

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tumors showed no infiltration by macrophages post-TNF-alpha. We concluded that high-dose rhTNF-alpha caused tumor infiltrating macrophage-mediated iNOS-derived tumor NO rise and sustained tumor blood flow shutdown resulting in tumor ulceration in the responsive tumor. This work introduced the possibility that although rhTNF-alpha may primarily express its anti-tumor activity via an anti-vascular mechanism, there could likely be a secondary component that causes tumor cell kill via a selective tumor infiltrating macrophage-mediated pathway. Tumor infiltrating macrophages and pathologically toxic levels of the free radical NO produced by these macrophages in response to rhTNF-alpha treatment appear to be common intermediates in both the anti-vascular as well as direct tumor cell kill pathways. In a related study carried out in our laboratory, we found that inhibiting NOS using a nonselective inhibitor such as L-NAME (10 mg/kg) prior to rhTNF-alpha treatment was highly lethal in both groups and led to ulceration in 83% of MethA tumors and 93% of LL2 tumors (Bauer, Fraker, unpublished data, 2000). The results from our above murine study have been summarized in Figure 5. Our results using L-NAME appears to mirror data obtained by Eggermont and his team from the systemic administration of L-NAME with rhTNF-alpha plus melphalan in a rat renal subcapsular CC531 adenocarcinoma in which the combination therapy was significantly effective in tumor regression but was also found to be responsible for considerable impairment of renal function.

Figure 5. Schematic representation summarizing the data obtained in the Fraker laboratory using a mouse model consisting of a TNF-alpha sensitive MethA fibrosarcoma and a resistant Lewis Lung carcinoma.

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Although we have a considerable amount of information in in vitro systems as well as from in vivo preclinical animal models, the role of NO and NOS in the human ILP system with rhTNF-alpha plus melphalan therapy is yet to be fully elucidated. In one study from the University Hospital Groningen lead by Armand R. J. Girbes in the Netherlands [143], it was reported that there was no increase in plasma nitrate or nitrite levels in the human ILP system after rhTNF-alpha plus melphalan. They also rightly point out that plasma concentrations of nitrite and nitrate may not reliably reflect activation of the inducible form of NOS. Local concentrations of NO may have been sufficiently increased to generate profound vascular effects without producing a demonstrable increase in nitrite or nitrate in the plasma. Further work is warranted in this area to conclude one way or the other regarding the role of NO and NOS in the rhTNF-alpha plus melphalan anti-tumor therapy in human ILP patients.

Future Directions In 1997, Xu and colleagues at the MD Anderson Cancer Center developed an adenovirus vector with tetracycline-regulatable TNF-alpha gene expression. A variety of human tumor cells and T lymphocytes transduced by this vector secreted high-titer (5,000-100,000 pg/106 cells/24 hours) and biologically active TNF-alpha in the absence of tetracycline. Expression of TNF-alpha in the transduced cells was not detectable when the culture medium contained as little as 0.1 microgm/ml of tetracycline. At least a fraction of the clonogenic cells from human peripheral blood stem cell concentrates were also transducible by this vector [144]. More recently, the group lead by Libutti S. K. at the Surgery Branch of the National Cancer Institute have focused their research efforts to target rhTNF-alpha to the tumor vasculature using specially designed hybrid adeno-associated virus phage vector. They found that a systemic administration of targeted vector containing rhTNF-alpha to human melanoma xenografts in mice produced the specific delivery of rhTNF-alpha to tumor vasculature. In contrast, the non-targeted vector did not target to the tumor vasculature. Moreover, targeted vector delivery resulted in expression of rhTNF-alpha, induction of apoptosis in tumor vessels and significant inhibition of tumor growth. No systemic toxicity to normal organs was observed [145]. This is an important first step toward a clinical translation to expand the scope of this limited use therapy with rhTNF-alpha. Other possibilities include use of tumor EC specific antibodies or tumor EC specific ligands that can target rhTNF-alpha specifically to the tumor vasculature. Recent years have witnessed important strides in the deciphering of some of the molecular players in the processes leading to tumor destruction after treatment with high dose rhTNF-alpha in an ILP setting as described in the previous section. However, not all of these candidate molecules have been tested and confirmed as participants in the human ILP rhTNFalpha system. Also, the contiguous timeline of events and detailed signaling mechanisms involving each of the above described molecules leading to tumor destruction is yet to be delineated. A complete elucidation of the molecular pathway leading to tumor destruction would be critical to expanding the scope of this limited use and yet very efficacious therapy to potentially include more types of cancers and to reduce systemic toxicity. It is also imperative, in this context, for us to try to understand the molecular basis underlying the

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differential response of tumor types to rhTNF-alpha therapy. It appears that some tumor vasculatures are more susceptible to rhTNF-alpha than others. What factor(s) contribute to such a susceptibility/resistance is an area that has remained largely unexplored and warrants more focus. It would also be useful to explore the rhTNF-alpha molecule further to arrive at relevant mutations that would improve the therapeutic efficacy of the drug and/or reduce its systemic toxicity. Alternatively, it may also help to focus future efforts on developing synthetic molecular mimics of rhTNF-alpha that retain its therapeutic effects in tumors and do not have its systemic toxicity.

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In: Tumor Necrosis Factor Editor: Toma P. Rossard

ISBN: 978-1-60741-708-8 © 2009 Nova Science Publishers, Inc.

Chapter V

Tumor Necrosis Factor Antagonist Induced Psoriatic Skin Lesions Angelique N. Collamer and Daniel F. Battafarano Department of Rheumatology, Brooke Army Medical Center, Fort Sam Houston, Texas

Disclosure: The opinions or assertions contained herein are those of the authors and are not to be construed as official policy of the Department of the Army, Department of the Air Force, or the Department of Defense.

Tumor Necrosis Factor (TNF) antagonists have proven to be highly effective in treating a variety of inflammatory autoimmune diseases including psoriatic skin disease and psoriatic arthritis [1]. Several side effects of these medications were evident during clinical trials and were not unexpected, including infusion and injection site reactions and an increase in infections [2-4]. Surprising reports of patients developing de novo psoriatic-like skin lesions or worsening of baseline psoriatic skin disease began to surface as early as 2003, and to date there are greater than 180 such patients reported in the literature [5-56]. The mechanism of action of this perplexing response remains elusive and there have been several theories postulated to explain this autoimmune phenomenon. The worsening or new-onset of psoriasiform skin lesions occurs in only a minority of patients exposed to TNF antagonists; however the number of reported cases indicates that it is not an uncommon occurrence. The following case depicts the typical clinical scenario facing the physician: A 40-year-old woman with a 5-year history of rheumatoid arthritis has been treated with infliximab infusions for the past 3 years. The patient responded well to the TNF antagonist therapy, achieving clinical remission of her disease within 12 weeks of drug initiation. Three months ago she developed pustular lesions on both palms that were thought to be contact dermatitis. The lesions persisted and worsened over the next 6 weeks and the patient was referred to a dermatologist. Biopsy of the lesions was consistent with pustular psoriasis. The patient had no personal or family history of psoriasis. She chose to continue the TNF antagonist and the lesions were treated with topical therapies with partial resolution. Ultraviolet phototherapy was added and the lesions resolved.

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At present, no predisposing factors for development of this secondary autoimmune reaction have been identified. Reports of anti-TNF induced psoriasiform lesions include patients treated with the three currently available TNF antagonists (infliximab, adalimumab, etanercept) for a multiplicity of rheumatic and non-rheumatic disease states including rheumatoid arthritis, psoriasis, inflammatory bowel disease, ankylosing spondylitis, psoriatic arthritis, Behçet’s disease and juvenile idiopathic arthritis [5-56]. Psoriasiform skin lesions can develop at any time in therapy, from within days of the first anti-TNF injection or infusion or after years of treatment. The majority of patients described in the literature have responded quite well to the TNF antagonist therapy, apart from the development of the skin lesions. Males and females appear to be equally affected and no predilection for any age group has been identified. A few patients have reported remote histories of a psoriasis-like skin rash; however the vast majority have no personal or family history of psoriasis. Certain medications, infections, and psychological stressors are known to trigger psoriasis in the predisposed individual [57-58]; to date no linkage between any infection, trauma or other influencing factor has been identified in these cases. A subset of the reported patients were concurrently receiving other immunosuppressive medications used to treat psoriasis, such as methotrexate and azathioprine, at the time of the psoriasiform eruption. The coexistence of psoriasis with rheumatoid arthritis is rarely reported, however the incidence is known to be increased in patients with spondyloarthopathies such as enteropathic arthritis [59]. In a study comparing 9826 rheumatoid arthritis patients treated with anti-TNF therapies with 2880 patients receiving traditional therapies from The British Society for Rheumatology Biologics Register (BSRBR), 25 cases of new-onset psoriasis were reported in a 6-year period in the anti-TNF cohort. In contrast, there were no reported cases in over 5000 person years of follow-up in the patients not receiving biologic therapy [60]. Thus it appears that TNF antagonism in a predisposed individual is the unifying cause for the development these psoriasiform skin lesions. Three types of psoriatic type skin lesions have been described in affected patients: plaque, pustular and guttate. Nail changes typical of psoriasis with onycholysis, discoloration and nail pitting have also been reported. The morphology of the psoriatic lesions is often atypical or of multiple types, and may occur on unusual areas of the body such as the flexural and inguinal regions. The majority of patients develop pustular or plaque lesions, with guttate lesions occurring in approximately 15% of reported cases [44]. The pustular lesions are often painful, erythematous and pruritic. There has been no correlation made between the type of lesion found and the disease being treated. However, those with psoriasis or psoriatic arthritis and psoriatic skin lesions who present with this reaction represent a unique group of patients. These patients tend to develop lesions of a new and different morphology than their original lesions (often guttate or pustular) and experience worsening of their baseline skin disease as well. On biopsy, the psoriasiform skin lesions demonstrate typical findings of psoriasis such as epidermal hyperplasia, parakeratosis, epidermal lymphocytic infiltrates, dilated capillaries and intraepidermal pustulosis. This is in contrast to pustular drug eruptions, in which eosinophils are visualized in the inflammatory infiltrate of the lesions [61]. Immunohistochemical analysis of a TNF-antagonist induced psoriatic lesion with staining for T and B cells, macrophages, interferon mRNA, TNF mRNA and vascular endothelial growth factor (VEGF) was indistinguishable from idiopathic psoriasis [52]. Skin biopsies of psoriatic

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patients with paradoxical worsening of their psoriasis during TNF-antagonist treatment are similar or identical to biopsies prior to the initiation of the offending drug [52]. Keratinocytes and inflammatory skin cells such as activated TH1 cells are known to produce large amounts of TNFα [58]. This powerful inflammatory cytokine suppresses the development of plasmacytoid dendritic cells (PDCs) that produce high levels of interferon-α (INFα). PDCs are found in the skin of psoriasis patients and have also been identified in patients with other autoimmune diseases such as systemic lupus erythematous but not in healthy controls [62-63]. Psoriatic skin lesions have been shown to develop when skin PDCs produce INFα, stimulating the activation and amplification of pathogenic T cells [32, 62-68]. It has been hypothesized that the pathogenesis of TNF antagonist induced psoriasiform lesions involves a disruption in cytokine balance by TNF blockade, allowing unopposed INFα production by PDCs in genetically predisposed individuals [32]. Increased INFα has been demonstrated in the lesional dermal vasculature and perivascular lymphocytic infiltrate of biopsied TNF antagonist induced psoriatic plaques when compared to spontaneous psoriasis [32]. INFα has been shown to induce expression of the chemokine receptor CXCR3 on T cells [65]. CXCR3 induces T cell migration to the psoriatic dermis as well as subsequent homing to the epidermis [69]. Patients receiving systemic TNFα inhibition have increased TH1 lymphocytes in the peripheral circulation, likely secondary to decreased trafficking to the previous site of inflammation, such as the joint or gut [65]. In the predisposed patient, this T cell population may activate and home to the cutaneous sites when their CXCR3 receptors are activated by INFα [32]. It is likely increased INFα expression is only one piece of an inflammatory cascade that leads to the unexpected development of psoriasis. It has been noted that palmoplantar pustular psoriasis differs from psoriasis vulgaris not only in clinical appearance, but also immunohistochemically and genetically [32, 69-70]. While TNFα is a well-recognized contributor to the development of plaque psoriasis, decreased expression of this inflammatory cytokine has been demonstrated in the eccrine palmar sweat gland and skin in patients with palmoplantar pustulosis [16]. However, not all patients with TNF antagonist induced psoriatic skin lesions present with pustular lesions, and TNF antagonists have been successfully utilized to treat some patients with pustular psoriasis [71]. Thus despite our new molecular understanding, many questions remain regarding the pathogenesis of this unusual reaction. It has been suggested that the pustular lesions associated with TNF antagonism are more closely related to the rare psoriasis variant acute palmoplantar pustular psoriasis of Andrews, rather than true palmoplantar pustular psoriasis [32, 72]. There are also recent case reports of the unexpected appearance of psoriasiform lesions or worsening of baseline psoriasis in patients treated with other biologic therapies such as rituximab, efalizumab and anakinra [73-76]. Further study of the immunosuppressive and immunostimulatory mechanisms responsible for this psoriasis response will likely assist with the elucidation of the pathogenesis of TNF antagonist induced psoriasis. In general, the induction of psoriatic-like skin lesions is considered a drug class effect, as it has been reported with all three currently available anti-TNF agents. It is unclear if there is some variance in the pathophysiology of the reaction between the three agents, as patients who develop the skin lesions with one agent are often successfully switched to another TNF antagonist without recurrence of the skin lesions. Many patients demonstrate improvement or

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resolution of the skin lesions over time, especially with dermatological treatment, while maintaining the original TNF antagonist therapy. Therefore TNF antagonist discontinuation or switch is not required if the patient tolerates the skin lesions. Patients with any personal or family history of psoriasis, especially pustular psoriasis, should be warned of the possibility of development or worsening of psoriatic skin disease prior to initiation of TNF antagonist therapy. Patients with suspected TNF antagonist induced psoriasis should be referred for dermatology evaluation and histological confirmation to exclude other skin disorders. TNF antagonists have been associated with a variety of skin eruptions and reactions including injection site reactions, eczema-like eruptions, cutaneous vasculitis, acute generalized exanthematous pustulosis, dermatitis herpetiformis, urticarial skin reactions, drug-induced cutaneous lupus erythematosus, erythema multiforme, lichenoid eruptions, viral, bacterial and fungal skin reactions, and cutaneous malignancies [31, 77-78]. A treatment algorithm for TNF antagonist psoriasiform skin eruptions has been proposed [44]. Patients with confirmed TNF antagonist induced psoriasis often respond to conventional treatments and may have resolution of the lesions without a change of TNF antagonist. A minority of patients require anti-TNF discontinuation and the psoriasiform lesions typically resolve. Many of the reported patients with this cutaneous reaction had the TNF antagonist immediately discontinued, thus it is difficult to estimate the percentage of patients who are able to successfully continue the TNF antagonist therapy. Patients with severe lesions, lesions significantly affecting quality of life, or which the patient finds intolerable, should have the TNF antagonist discontinued and the psoriasis aggressively treated, by a dermatologist. These patients may be able to tolerate an alternative TNF antagonist agent without reappearance of the psoriasiform lesions. If psoriasis is affecting <5% of body surface area and the patient wishes to continue treatment, we recommend topical therapies to include corticosteroids, keratolytics and vitamin D analogs. Methotrexate is often beneficial as is ultraviolet phototherapy (PUVA). Any patient presenting with pustular psoriasis or skin lesions covering more than 5% of body surface area should be given topical therapies as well offered PUVA and systemic treatments including methotrexate, retinoids, and cyclosporine. TNF antagonist switch or discontinuation of therapy should be considered if the patient does not respond well to these therapies. As experience with TNF antagonist therapy increases so does the recognition and reporting of unexpected and unusual adverse reactions. Other reported paradoxical autoimmune reactions observed with TNF antagonist therapies include the development of antinuclear and anti-double-stranded DNA antibodies, as well as drug induced lupus, cutaneous and systemic vasculitis, and the induction of demyelinating diseases and inflammatory bowel disease. The perplexing evidence that a medication class used to treat a disease such as psoriasis may exacerbate the condition, or induce psoriasis in patients with other autoimmune conditions, underscores that the pathophysiology of autoimmune diseases and their treatments is incompletely understood. Further observations and investigations are necessary to better elucidate this phenomenon.

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[49] Chan, CY; Browning, JC; Larsen, F; Hsu, S. Development of new-onset psoriasis in a patient receiving infliximab for treatment of rheumatoid arthritis. Dermatol Online J., 2008, 14, 12. [50] Richetta, AG; Mattozzi, C; Carlomagno, V; Maiani, E; Carboni, V; Giancristoforo, S; et al. A case of infliximab-induced psoriasis. Dermatol Online J., 2008, 14, 9. [51] Costa-Romero, M. Guttate psoriasis induced by infliximab in a child with Crohn’s disease. Inflamm Bowel Dis., 2008, 14, 1462-3. [52] Cuchacovich, R; Espinoza, CG; Virk, Z; Espinoza, LR. Biologic Therapy (TNF-α antagonists)-induced psoriasis. A cytokine imbalance between TNF-α and IFN-α? J Clin Rheumatol., 2008, 14, 353-56. [53] Wollina, U. Tumor necrosis factor-alpha inhibitor-induced psoriasis or psoriasiform exanthemata: first 120 cases from the literature including a series of six new patients. Am J Clin Dermatol., 2008, 9, 1-14. [54] Wendling, D; Balblanc, JC; Briancon, D; Brousse, A; Lohse, A; Deprez, P; Humbert, P; et al. Onset or exacerbation of cutaneous psoriasis during TNFα antagonist therapy. Joint Bone Spine, 2008, 75, 315-18. [55] Bosch, RI; Amo, Ndel, V; Manteca, CF; Cortina, EL; Polo, RG; Courel, LG. Psoriasis Induced by anti-TNF probably not so uncommon. J Clinical Rheumatol., 2008, 14, 128. [56] Bal, A; Gurcay, E; Aydog, E; Umay, E; Tatlican, S; Cakci, A. Onset of psoriasis induced by infliximab. J Clinical Rheumatol., 2008, 14, 129. [57] Menter, A; Gottlieb, A; Feldman, SR; Van Voorhees, AS; Leonardi, CL; Gordon, KB; et al. Guidelines of care for the management of psoriasis and psoriatic arthritis: Section 1. Overview of psoriasis and guidelines of care for the treatment of psoriasis with biologics. J Am Acad Dermatol., 2008, 58, 826-50. [58] Turkiewicz, A; Moreland, L. Psoriatic arthritis: current concepts on pathogenesisoriented therapeutic options. Arthritis Rheum., 2007, 56, 1051-66. [59] Bernstein, CN; Wajda, A; Blanchard, JF. The clustering of other chronic inflammatory diseases in inflammatory bowel disease: a population-based study. Gastroenterology, 2005, 129, 827-36. [60] Harrison, MJ; Dixon, WG; Watson, KD; King, Y; Groves, R; Hyrich, KL; et al. Rates of new-onset psoriasis in patients with rheumatoid arthritis receiving anti-tumour necrosis factor-α therapy: results from the British Society for Rheumatology Biologics Register. Ann Rheum Dis., 2009, 68, 209-15. [61] Spencer, JM; Silvers, DN; Grossman, ME. Pustular eruption after drug exposure: is it pustular psoriasis or a pustular drug eruption? Br J Dermatol., 1994, 130, 514-9. [62] Farkas, L; Beiske, K; Lund-Johansen, F; Brandtzaeg, P; Jahnsen, FL. Plasmacytoid dendritic cells (natural interferon-α/β-producing cells) accumulate in cutaneous lupus erythematosus lesions. Am J Pathol., 2001, 159, 237-43. [63] Gilliet, M; Conrad, C; Geiges, M; Cozzio, A; Thurlimann, W; Burg, G; et al. Psoriasis triggered by toll-like receptor 7 agonist imiquimod in the presence of dermal plasmacytoid dendritic cell precursors. Arch Dermatol., 2004, 140, 1490-5. [64] Palucka, AK; Blanck, JP; Bennett, L; Pascual, V; Banchereau, J. Cross-regulation of TNF and IFN-α in autoimmune diseases. Proc Natl Acad Sci., U S A 2005, 102, 3372-7.

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[65] Aeberli, D; Seitz, M; Juni, P; Villiger, PM. Increase of peripheral CXCR3 positive T lymphocytes upon treatment of RA patients with TNF-α inhibitors. Rheumatology, 2005, 44, 172-5. [66] Nestle, FO; Conrad, C; Tun-Kyi, A; Homey, B; Gombert, M; Boyman, O; et al. Plasmacytoid predendritic cells initiate psoriasis through interferon-α production. J Exp Med., 2005, 202, 135-43. [67] Aeberli, D; Oertle, S; Mauron, H; Reichenbach, S; Jordi, B; Villiger, P; et al. Inhibition of the TNF-pathway: use of infliximab and etanercept as remission-inducing agents in cases of therapy-resistant chronic inflammatory disorders. Swiss Med Wkly., 2002, 132, 414-22. [68] Eriksen, KW; Lovato, P; Skov, L; Krejsgaard, T; Kaltoft, K; Geisler, C; et al. Increased sensitivity to interferon-α in psoriatic T cells. J Invest Dermatol., 2005, 125, 936-44. [69] Rottman, JB; Smith, TL; Ganley, KG; Kikuchi, T; Krueger, JG. Potential role of the chemokine receptors CXCR3, CCR4, and the integrin alphaEbeta7 in the pathogenesis of psoriasis vulgaris. Lab Invest., 2001, 81, 335-47. [70] Eriksson, MO; Hagforsen, E; Lundin, IP; Michaelsson, G. Palmoplantar pustulosis: a clinical and immunohistological study. Br J Dermatol., 1998, 138, 390-398. [71] Routhouska, SB; Sheth, PB; Korman, NJ. Long-term management of generalized pustular psoriasis with infliximab: case series. J Cutan Med Surg., 2008, 12, 184-8. [72] Dutz, JP. Tumor necrosis factor-α inhibition and palmoplantar pustulosis: Janus- faced therapy? J Rheumatol., 2007, 34, 247-49. [73] Dass, S; Vital, EM; Emery, P. Development of psoriasis after B cell depletion with rituximab. Arthritis Rheum., 2007, 56, 2715-18. [74] Mielke, F; Schneider-Obermeyer, J; Dörner, T. Onset of psoriasis with psoriatic arthropathy during rituximab treatment of non-Hodgkin lymphoma. Ann Rheum Dis., 2008, 67, 1056-57. [75] Balato, A; La Bella, S; Gaudiello, F; Balato, N. Efalizumab-induced guttate psoriasis. Successful management and re-treatment. J Dermatolog Treat., 2008, 19, 182-4. [76] González-López, MA; Martinez-Taboada, VM; González-Vela, MC; Fernández-Llaca, H; Val-Bernal, JF. New-onset psoriasis following treatment with the interleukin-1 receptor antagonist anakinra. Br J Dermatol., 2008, 158, 1146-48. [77] Devos, SA; Van Den Bossche, N; De Vos, M; Naeyaert, JM. Adverse skin reactions to anti-TNF-alpha monoclonal antibody therapy. Dermatology, 2003, 206, 388-90. [78] Nikas, SN; Voulgari, PV; Drosos, AA. Urticaria and angiedema-like skin reactions in a patient treated with adalimumab. Clin Rheumatol., 2007, 26, 787-8.

In: Tumor Necrosis Factor Editor: Toma P. Rossard

ISBN: 978-1-60741-708-8 © 2009 Nova Science Publishers, Inc.

Chapter VI

A New Promising Role of Melatonin in Promoting Tumor Necrosis Factor Toxicity in Cancer Cells Rosa M. Sainz*1,2, Juan C. Mayo1,2, Dun-Xian Tan2 and Russel J. Reiter2 1

Departamento de Morfología y Biología Celular, Instituto Universitario de Oncología del Principado de Asturias (IUOPA), Universidad de Oviedo, Spain. 2 Department of Cellular and Structural Biology University of Texas Health Science Center, San Antonio, 7703 Floyd Curl Drive, San Antonio TX, 78229-3900 USA.

Abstract Melatonin, a neuroindole mainly produced by the pineal gland, has antioxidant, antiproliferative and anti-inflammatory properties which can be responsible to its beneficial effects on human health. It is well known that melatonin serum levels are reduced in cancer patients since late 80’ies and its role on the growth of cancer cells has been clearly demonstrated. Melatonin directly inhibits the growth of several cell types from diverse embryological origins including breast, prostate, melanoma, lung, kidney or brain; additionally, melatonin is a modulator of the immune function. Physiologically, melatonin administration results in a functional enhancement of immune cells which might play a key role against cancer cells in vivo. In fact, there is a direct crosstalk between the pineal gland and the immune system in several ways. Previous data suggest that the increase in circulating tumor necrosis factor-alpha (TNFα), after a defense response transiently blocks nocturnal melatonin production. In fact, the transcription of arylalkylamine-N-acetyltransferase, the rate-limiting enzyme in melatonin biosynthesis, together with the synthesis of the melatonin precursor N-acetylserotonin, was inhibited by TNFα. It is also clear that cytokine production caused by infection or inflammation, including TNFα, is reduced by melatonin pretreatment. The remarkable ability of TNFα *

Departamento de Morfología y Biología Celular, Facultad de Medicina, C/Julián Clavería 6, 33006 Oviedo, SPAIN. Phone # 34 985103000 ext 5223. Fax # 34 985103618, E-mail: [email protected]

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Rosa M. Sainz, Juan C. Mayo, Dun-Xian Tan et al. to inhibit the growth of malignant tumor cells is unfortunately limited by its systemic toxicity. New strategies are being tested in order to reduce TNFα toxicity without losing its antitumor efficiency. On the other hand TNFα, induced by a wide range of pathogenic stimuli induces other inflammatory mediators and proteases that act as tumor promoters. The role of TNF in cancer has been linked to all steps of carcinogenesis including carcinogenesis, cellular transformation, promotion, survival, proliferation, angiogenesis and metastasis and how the cytokine works in this intricate link is actually a matter of debate. Since melatonin has been claimed to prevent the toxicity of several anticancer drugs and more recently, to enhance the toxicity of TNFα in cancer cells, we will discuss here the innovative idea of the employment of melatonin in combination with TNFα in cancer treatments. The possible use of melatonin in preventing the toxicity of TNFα without losing its antitumor properties as well as its capacity to promote ability to kill cancer cells especially resistant to TNFα treatment is an idea which needs to be deeply explored.

Commentary 1. Melatonin: A Pineal Indole with Multiple Biological Functions Melatonin is the main product of the pineal gland in vertebrates but its synthesis has been found in several extrapineal tissues and organs including retina, Harderian gland, brain, thymus, bone marrow, ovary, testicle, gut, airway epithelium, placenta, lymphocytes and skin [1]. In all the organisms studied, melatonin synthesis follows a circadian rhythm, being produced and released mainly by pineal gland during darkness. Classically melatonin has been considered a chemical signal to adjust circadian rhythms and to control seasonal reproduction in some vertebrates. In fact, melatonin is the physiological signal in adjusting reproduction of animals with a photoperiod-dependent seasonal reproduction [2]. Although this has been highlighted as its main role in nature, melatonin is now considered a more versatile and important molecule. Since, the antioxidant properties of melatonin were discovered in the early 90´ies, it is considered one of most beneficial molecules in humans’ health. Melatonin has been found in bacteria, unicellular algae, yeast and plants. Its role on the physiology of these organisms is far from adjusting seasonal reproduction although it could be related to circadian changes in their biology [3]. In 1993, Tan and co-workers found that melatonin is able to scavenge hydroxyl radical (OH•) and thus, to protect cell components, including DNA or proteins, from its harmful activity [4]. Since then, a complete research about melatonin scavenging and antioxidant properties has been followed by several research groups. Overall, melatonin has been demonstrated to be a powerful scavenger but also an indirect endogenous antioxidant increasing the expression or activity of the principal antioxidant enzymes [5]. In this regard, the role of melatonin in unicellular organisms or in plants has been more related with a possible protective function than with its role in the adjustment of cell physiology to circadian rhythms. The major urinary metabolite of melatonin is 6-hydroxymelatonin in vertebrates. However, in unicellular organisms and several metazoas, 6-hydroxymelatonin has not been found. Instead, N1-acetyl-N2-formyl5methoxykynuramine (AFMK) has been considered its original and primitive metabolite [6].

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Recently, a complete pathway of melatonin metabolism to this type of kinuramine derivates, including AMFK, has been described. In fact some of these compounds share with melatonin some of its biological properties, including scavenging properties [7] Several cell transduction pathways have been implicated in melatonin signaling in target cells. Some of melatonin actions are mediated by its binding to specific receptors. Melatonin membrane receptors belong to the superfamily of seven transmembrane domains. MTNR1A and MTNR1B melatonin receptors, formerly called MT1 and MT2 respectively, are linked to heterotrimeric inhibitory G-proteins and melatonin binding promotes its dissociation into α and βγ dimmers which interact with various downstream effectors including adenylyl cyclase, phospholipase C, phospholipase A2 or potassium channels [8]. Melatonin receptors have been found and fully characterized in retina, suprachiamastic nucleus, pars tuberalis, cerebral and peripheral arteries, kidney, pancreas, adrenal cortex, testes and immune cells and have been related to many of its physiological functions. MTNR1A melatonin receptors modulate neuronal firing, vasoconstriction, reproductive and metabolic functions and cell proliferation of cancer cells. On the other hand, MTNR1B activation modulates circadian rhythms of neuronal the suprachiasmatic nucleus, inhibits dopamine release in retina or enhance immune responses. Other functions of melatonin are receptor-independent; melatonin seems to cross easily all biological barriers given its amphyphylic nature although a recent publication have shown that there is a more complex system to cross cell membranes at least in some particular cell models [9]. Once inside the cell, melatonin can work through its binding to cytosolic proteins like calmodulin [10] or through its interaction with reactive oxygen species (ROS) [11]. Since ROS play a key role on multiple cellular processes, through redox regulation, melatonin can modify multiple cellular functions including cell growth, differentiation or death by scavenging ROS

2. Melatonin Has a Role on Tumor Growth and Progression Melatonin has been considered a hormonal regulator of neoplastic cell growth since early 80´ies. In fact, melatonin reduces cell growth in several cellular or animal models. First, serum melatonin levels were found to be depressed in cancer patients, particularly in endocrine-dependent tumors like mammary, endometrial or prostate cancer but also, in endocrine-independent as lung, gastric or colorectal cancer patients when compared with its sex and age-matched controls [12]. Later on, it was showed that melatonin exerts a direct antiproliferative activity in tumor cells when it was employed either at physiological or pharmacological concentrations, depending on the cellular type. In particular, estrogendependent mammary tumor cells, MCF-7, responded basically restricted to the physiological range and no or little response at subphysiological or pharmacological concentrations of melatonin was found while melanoma, colocarcinoma, glioma or prostate cancer cells respond to pharmacological concentrations of the indole [13]. On the other hand, several mechanisms have been proposed to explain how melatonin reduces tumor growth, and none of them seem to explain completely all the abilities of the indole reported. In endocrinedependent tumors, melatonin mechanism of action has been related to its interaction with both estrogen and androgen-receptor signaling pathways; thus, it avoids hormone functioning

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by inhibiting estrogen and androgen-receptors binding activity in breast and prostate cancer cells [14, 15]. Also, melatonin impedes the uptake of linoleic acid that enters tumor cells and is converted to the mitogenically active metabolite 13-hydroxyoctadecadienoic acid (13HODE) base on the evidence obtained on hepatoma 7288CTC and on human breast xenografts [16]. At pharmacological concentrations, melatonin was proven to be also an inhibitor of cell proliferation in several cell types [17]. Direct or indirect antioxidant properties of melatonin could participate in its antitumoral properties. First, several carcinogens work through the generation of free radicals, but also ROS can act as tumor growth promoters. In fact, growth factors increase the intracellular concentration of ROS to stimulate mitogenesis via the induction of the phosphorylation of receptor protein tyrosine kinases as well as extracellularregulated kinases (ERK). In fact, high concentrations of melatonin reduce human umbilical vein endothelial cells (HUVEC) by inhibiting MAPKs including ERK phosphorylation [18]. In general, pharmacological concentrations of melatonin reduce cancer cells proliferation by inhibiting MAPKs, reducing cyclin dependent protein kinases or increasing cyclin-dependent protein kinases inhibitors such as p21 or p27 but the exact mechanism is still under debate and differs depending on the cell type.

3. Melatonin Promotes Immune Function in Vitro and in Vivo In vivo, the role of melatonin in tumorigenesis could be more complex since melatonin has a potent stimulatory activity on immune function. The inhibition of melatonin synthesis results in the attenuation of cellular and humoral responses in mice. Thus, pinealectomy accelerates thymus involution while exogenous melatonin counteracts immunosuppression caused by stress. Mainly, melatonin affects T-lymphocyte proliferation, enhances antigen presentation by macrophages to T cells and restores Th-cell activity in immunosupressed mice. Melatonin enhances the production of cytokines, including interleukin(IL-)2, interferon (IFN)-γ and IL-6 in cultured mononuclear cells; and IL-2 and IL-12, in macrophages [19]. The ability of melatonin to enhance inflammatory cytokine production suggests that its most relevant role in the immune enhancement is mediated by controlling Th1 response. Also, the influence of melatonin on NK cells is of great interest since NK cells are killers of virusinfected cells in addition to a variety of tumor cells. Daily dietary administration of melatonin on mice bearing erythroleukaemia resulted in a 2.5 fold increase in NK cell number. Moreover, one third of those animals fed with melatonin in their food, survived 3 months after tumor initiation while controls died after 16-27 days after tumor injection [20]. The potential clinical employment and importance of melatonin in cancer treatment has been anticipated in several ways. First, the preventive role of melatonin has been proposed in those tumors whose incidence increase with age. Secondly, melatonin treatment as a pharmacological compound, able to inhibit directly tumor growth and finally, as a coadyuvant of conventional treatments. Melatonin protects cells and patients from the toxicity of chemotherapy but it is still unclear the role of the indole in the effect of those drugs on tumor cells. It is conceivable that melatonin enhancement of cytokines production play a critical role in those aspects. Thus, Lissoni and cols. have demonstrate in several

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occasions that melatonin treatment helps IL-2 immunotherapy and improves the efficiency of treatment or reduces collateral damage improving quality of life of patients [21]. It is possible that this role of melatonin in immunotherapy might be due to its potential ability to improve Th1 priming or NK-cell activation.

4. Crosstalk between Melatonin and TNFα Tumor necrosis factor (TNFα) has been found to be one of the principal molecules in the link between neuroendocrine and immune system. It is known that cytokine production in human blood exhibits a diurnal rhythmicy; therefore, there is a peak production of proinflammatory cytokines such as IFN-γ, TNFα, IL-1 and IL-12 during the night and early morning. In addition, TNFα synthesis is increased in virus infected mice after melatonin treatment [22] but also, the release of TNFα from macrophages suppresses the nocturnal melatonin and IL-2 production. In fact, the transcription of arylalkylamine-Nacetyltransferase, the rate limiting enzyme in melatonin biosynthesis, together with Nacetylserotonin was inhibited by TNFα [23]. Although, the antitumor activity of TNFα is known in several cellular models, TNFbased patients’ treatment is compromised by its high toxicity. In fact, sophisticated drugdelivered systems are necessary to avoid its systemic toxicity. Moreover cancer cells have developed survival pathways to respond to TNFα, which interfere with its antitumoral properties [24]. After treatment, the increment in NFκB binding activity promotes the expression of anti-apoptotic proteins. Thus, NFκB drives translation of bcl-2 and apoptosis inhibiting proteins (IAPs) like survivin [25]. Several preventive agents like some antioxidants, including curcumin or melatonin, have been used in combination with TNFα. The role of curcumin or melatonin in preventing NFκB activation seems to be the mechanism implicated in promoting TNF-induced cell death by these agents. Melatonin inhibits NFκB binding activity induced by several agents including γ-irradiation or TNFα [26] and more recently, we found that it stimulates apoptosis induced by TNFα in prostate cancer cells by inhibiting NFκB binding activity [27]. It is not known the mechanism by which melatonin reduces NFκB binding activity caused by TNFα treatment. In other occasions, the antioxidant properties of melatonin play a critical role in its ability to interrupt NFκB signaling. Also, it is believed that melatonin prevents collateral toxicity of some chemotherapeutic drugs like doxorubicin due to its antioxidant properties. However, our results indicate that melatonin is only able to promote TNFα toxicity because its toxicity is independent of ROS production. In other words, the combination of melatonin with those antitumoral agents like irradiation, which generate free radicals, either in cancer or normal cells, might not be so clear and perhaps a matter of debate elsewhere, there is not doubt that its combination with TNFα could be of great importance. Also, it is noteworthy that in vivo TNFα is mainly produced by Th1 cells but the ability of the indol to improved TNFα immunotherapy against cancer models is still under study. In addition, preliminary data obtained by our group demonstrate that melatonin only promotes apoptosis induced by TNF or TNF-related apoptosis-inducing ligand (TRAIL) without affecting the ability of other ROSrelated drugs to induce apoptosis in a resistant cellular model of prostate cancer cells.

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Conclusion Based on the previous mentioned results, the role of melatonin in preventing the toxicity of TNFα without affecting its ability to induce cell death in other cellular models need to be explored. It opens a new field of research to study deeply how the indole interacts with TNFα signaling pathways in normal or in cancer cells. The important role of melatonin to stimulate immune system must be always considered because previous in vitro results suggest a more important role of this effect in animal models where immunity can play a crucial role in the function of melatonin as antitumoral agent.

References [1]

Pandi-Perumal, SR; Srinivasan, V; Maestroni, GJM; Cardinali, DP; Poeggeler, B; Hardeland, R. Melatonin: Nature´s most versatile biological signal?. FEBS J, 2006, 273, 2813-2838. [2] Reiter, RJ. (1991). Melatonin: cell biology of its synthesis and of its physiological interaction. Endocr Rev., 49, 151-180. [3] Hardeland, R; Poeggeler, B. (2003). Non-vertebrate melatonin. J Pineal Res., 34, 233241. [4] Tan, DX; Chen, LD; Poeggeler, B; Manchester, LC; Reiter, RJ. Melatonin: a potent endogenous hydroxyl radical scavenger. Endocr J., 1993, 1, 57-60. [5] Mayo, JC; Sainz, RM; Antolin, I; Herrera, F; Martin, V; Rodriguez, C. Melatonin regulation of antioxidant enzyme gene expression. CMLS, 2002, 59, 1703-1713. [6] Tan, DX; Manchester, LC; Terron, MP; Flores, LJ; Reiter, RJ. One molecule, many derivatives: A never-ending interaction of melatonin with reactive oxygen and nitrogen species?. J Pineal Res., 2007, 42, 28-42. [7] Ressmeyer, AR; Mayo, JC; Zelosko, V; Sainz, RM; Tan, DX; Poeggeler, B; Antolin, I; Zsizsik, BK; Reiter, RJ; Hardeland, R. Antioxidant properties of melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK): scavenging free radicals and prevention of protein destruction. Redox Rep., 2003, 8, 205-213. [8] Dubocovich, ML; Markowska, M. Functional MT1 and MT2 melatonin receptors in mammals. Endocrine., 2005, 27, 101-110. [9] Hevia, D; Sainz, RM; Blanco, D; Quiros, I; Tan, DX; Rodriguez, C; Mayo, JC. Melatonin uptake in prostate cancer cells: intracellular transport vs simple passive diffusion. J Pineal Res., 2008, 45, 247-257. [10] Del Rio, B; Garcia-Pedrero, JM; Martinez-Campa, C; Zuazua, P; Lazo, PS; Ramos, S. Melatonin, an endogenous-specific inhibitor of estrogen receptor alpha via calmodulin. J Biol Chem., 2004, 279, 38294-38302. [11] Sainz, RM; Mayo, JC; Tan, DX; Lopez-Burillo, S; Natarajan, M; Reiter, RJ. Antioxidant activity of melatonin in Chinese hamster ovarian cells: changes in cellular proliferation and differentiation. BBRC, 2003, 302, 625-634. [12] Bartsch, C; Bartsch, H. Melatonin in cancer patients and in tumor-bearing animals. Adv Exp Med Biol., 1999, 467, 247-264.

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[13] Blask, DE; Sauer, LA; Dauchi, RT. Melatonin as a chronobiotic/anticancer agent: Cellular, biochemical and molecular mechanisms of action and their implications for circadian-based cancer therapy. Curr Top Med Chem., 2002, 2, 113-132 [14] Rato, AG; Garcia-Pedrero, JM; Martinez, MA; del Rio, B; Lazo, PS; Ramos, S. Melatonin blocks the activation of estrogen receptor for DNA binding. FASEB J, 1999, 13, 857-868. [15] Rimler, A; Culig, Z; Levi-Rimler, G; Lupowitz, Z; Klocker, H; Matzkin, H; Bartsch, G; Zisapel, N. Melatonin elicits nuclear exclusion of the human androgen receptor and attenuates its activity. Prostate, 2001, 49, 145-154. [16] Blask, DE; Brainard, GC; Dauchy, RT; Hanifin, JP; Davidson, LK; Krause, JA; Sauer, LA; Rivera-Bermudez, MA; Dubocovich, ML; Jasser, SA; Lynch, DT; Rollag, MD; Zalatan, F. Melatonin-depleted blood from premenopausal women exposed to light at night stimulates growth of human breast cancer xenografts in nude rats. Cancer Res., 2005, 23, 11174-11184. [17] Sainz, RM; Mayo, JC; Tan, DX; Leon, J; Manchester, LC; Reiter, RJ. Melatonin reduces prostate cancer cell growth leading to neuroendocrine differentiation via a receptor and PKA independent mechanisms. Prostate, 2005, 63, 29-43. [18] Cui, P; Luo, Z; Zhang, H; Su, Y; Li, A; Li, H; Zhang, J; Yang, Z; Xiu, R. Effect and mechanism action of melatonin`s action on the proliferation of human umbilical vein endothelial cells. J Pineal Res., 2006, 41, 358-362. [19] Miller, SC; Pandi-Perumal, SR; Esquifino, AI; Cardinali, DP; Maestroni, GJM. The role of melatonin in immuno-enhacement: potential application in cancer. Int J Exp Path, 2006, 87, 81-87. [20] Currier, NL; Miller, SC. Echinacea purpure and melatonin augment natural-killer cells in leukemic mice and prolong life span. J Altern complement Med., 2001, 7, 241-251. [21] Lissoni, P. Modulation of anticancer cytokines IL-2 and IL-12 by melatonin and the other pineal indoles 5-methoxytryptophol in the treatment of human neoplasms. Ann NY Acad Sci., 2000, 917, 560-567. [22] Bonilla, E; Valero, N; Chacin-Bonilla, L; Medina-Leendertz, S. Melatonin and viral infections. J Pineal Res., 2004, 36, 73-79. [23] Markus, RP; Ferreira, ZS; Fernandes, PA; Cecon, E. The immune-pineal axis: a shuttle between endocrine and paracrine melatonin source. Neuroimmunology, 2007, 14, 126-133. [24] Royuela, M; Rodriguez-Berriguete, G; Fraile, B; Paniagua, R. TNFα/IL-1/NFκB transduction pathway in human cancer prostate. Histology Histopathol, 2008, 23, 12791290. [25] Beg, AA; Baltimore, D. An essential role for NF-κappaB in preventing TNFalphainduced cell death. Science, 274, 782-784. [26] Mohan, N; Sadeghi, K; Reiter, RJ; Meltz, ML. The neurohormone melatonin inhibits cytokine, mitogen and ionizing radiation induce NFkappaB. Biochem Mol Biol Int., 1995, 37, 1063-1070. [27] Sainz, RM; Reiter, RJ; Tan, DX; Roldan, F; Natarajan, M; Quiros, I; Hevia, D; Rodriguez, C; Mayo, JC. Critical role of glutathione in melatonin enhancement of tumor necrosis factor and ionizing radiation-induced apoptosis in prostate cancer cells. J Pineal Res., 2008, 45, 258-270.

In: Tumor Necrosis Factor Editor: Toma P. Rossard

ISBN: 978-1-60741-708-8 © 2009 Nova Science Publishers, Inc.

Chapter VII

Circulating TNF- α and Oral Health Condition in Elderly Japanese Hideaki Hayashida1, Toshiyuki Saito1*, Reiko Furugen1, Noboru Yamaguchi2, Akihiro Yoshihara3, Hiroshi Ogawa3 and Hideo Miyazaki3 1

Department of Oral Health, Unit of Social Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan 2 Section of Pediatric Dentistry, Division of Oral Health, Growth and Development, Kyushu University Faculty of Dental Science, Fukuoka, Japan 3 Division of Preventive Dentistry, Department of Oral Health Science, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan

Abstract Objectives The oral cavity, especially area around teeth, is a hotbed for bacteria, which compose a biofilm that becomes a significant source of continuous subclinical infection. Tumor necrosis factor-α (TNF-α), which is secreted from adipose tissue and developing type 2 diabetes, is also known to be secreted in periodontal inflammation. Moreover, there is a two-way relationship between diabetes and periodontal disease. We hypothesized that the circulating level of TNF-α is associated with the oral health condition, including periodontal disease. The purpose of this study was to assess the relationship between serum levels of TNF-α and the oral health condition in elderly Japanese people.

*

Department of Oral Health, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan, Tel: +81 95 819 7662 Fax: +81 95 819 7665, e-mail: [email protected]

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Among 418 Japanese subjects, 76 years old, who attended complete oral and general health examinations held in Niigata, Japan, in 2004, the serum TNF-α levels of 198 subjects were measured by ELISA. Among them, 76 subjects with healthy gingiva (maximum probing depth ≤ 5 mm, at least 10 teeth), 85 subjects with periodontitis (maximum probing depth ≥ 6 mm, at least 10 teeth), and 37 edentulous subjects were selected. Serum levels of TNF-α were compared among the groups using the KruskalWallis test. The proportions of subjects with detectable levels of TNF-α (≥ 0.5 pg/mL) were compared by chi-square test.

Results The median serum TNF-α levels in subjects with healthy gingiva, those with periodontitis, and edentulous subjects were 0.64 pg/mL (range 0–16.36), 0.72 pg/mL (range 0–4.53), and 0.00 pg/mL (range 0–1.83), respectively. The frequencies of detectable TNF-α levels were 48/76 (63.2%), 53/85 (62.4%), and 6/37 (16.2%), respectively. There were no significant differences between subjects with and without periodontitis. However, TNF-α levels and the frequency of subjects with detectable levels of TNF-α were significantly lower in edentulous subjects than in other groups (P < 0.001).

Conclusion In this study of elderly Japanese, the circulating TNF-α levels were not affected by current periodontal status. However, they were significantly lower in edentulous subjects than in the other two dentate groups, suggesting that the oral cavity in elderly people with teeth could be a source of systemic subclinical inflammation, which may have negative effects on general health.

Introduction Periodontitis is an inflammatory disease and a major cause of tooth loss and tooth decay. Periodontitis is clinically characterized by the formation of deep periodontal pockets around teeth, leading to alveolar bone loss. Several Gram-negative bacterial species, such as Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Tannnerella forsyensis harbored in periodontal pockets have been identified as periodontal pathogens [1-4]. The cell wall component, lipopolysaccharide (LPS), of periodontal pathogens induces the secretion of pro-inflammatory cytokines, including necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) from several cell types, such as monocytes, macrophages, and neutrophils [5]. A recent in vitro study using RAW264.7, which is a cell line derived from mouse monocyte/macrophage, demonstrated that LPS and secreted proinflammatory cytokines, including TNF-α, induced cell fusion at a later stage of osteoclastogenesis [6].

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There is increasing evidence that low-grade inflammation such as periodontitis plays an important role in the pathogenesis of systemic diseases, such as coronary heart disease, diabetes mellitus, and obesity [7]. Circulating inflammatory mediators may be modulated by the inflammatory response of periodontal tissue to oral pathogens, spreading to the surrounding area. Stashenko et al. reported that the concentrations of TNF-α in diseased periodontal sites were higher than those of healthy periodontal sites [8]. Previous studies have reported that the severity of periodontitis is positively correlated with the TNF-α level in the serum, as well as with that at periodontal diseased sites [9, 10]. Additionally, periodontal treatment decreases the levels of circulating TNF-α, as well as in periodontal tissue [9]. Conversely, other studies have reported that periodontitis is positively correlated with local TNF-α levels, but not with circulating TNF-α [11, 12]. Bretz et al. assessed the relationship between periodontitis, periodontal infections and systemic inflammatory markers such as TNF-α, IL-6, C-reactive protein (CRP), and plasminogen activator inhibitor type-I in an elderly cohort. The extent of periodontal disease was strongly associated with higher levels of circulating TNF-α, after the adjustment of some risk factors associated with periodontitis and systemic inflammatory markers [13]. Recently, we reported that no significant difference was observed in the serum TNF-α levels between elderly Japanese subjects with and without periodontitis [14]. In this present report, we have appended edentulous subjects to the previous study population and reanalyzed the relationship between the serum levels of TNF-α and oral health condition in elderly Japanese people.

Subjects and Methods Subjects In 1998, a total of 4542 registered people who were 70 years old in Niigata, Japan, were sent a written request to participate in the survey and were informed of the purpose of this survey. After two requests, 81.4% (3695) responded positively to participate in the survey. After considering the availability of resources, 600 subjects were randomly selected. The participants signed informed consent forms for this study, approved by the Ethics Committee of Niigata University Graduate School of Medical Dental Sciences. Among a total of 418 subjects who attended the 2004 examination, 76 subjects with healthy gingiva (probing depth ≤ 5 mm, at least 10 teeth), 85 subjects with periodontitis (probing depth ≥ 6 mm, at least 10 teeth), and 37 edentulous subjects were selected.

Periodontal Examination The periodontal examinations were performed by trained dentists. The probing pocket depth (PD), attachment level (AL), and bleeding on probing (BOP) were measured using a plastic periodontal probe scaled at 1 mm intervals and calibrated at a pressure force of 20 g. All functioning teeth were assessed except those that were partially erupted. A calibration of

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examiner was carried out and the κ (kappa value) ranged from 0.81 to 1.00 for probing pocket depth, and from 0.74 to 1.00 for AL were obtained.

TNF-α Measurements Blood samples were obtained from the anticubital vein in the morning, then frozen and stored at −80°C until needed. The TNF-α levels in the serum samples were examined in duplicate using an ELISA kit (KHC3014, Biosource International Inc., CA, USA) according to the manufacturer's protocol. In addition, each plate was checked before use to ensure that the calibration curve measuring the standard was accurate. The absorbance of the assay samples was measured using a Microplate manager (Bio-Rad Laboratories, Hercules, CA, USA) at 450 nm. The detectable limit for TNF-α was 0.5 pg /mL under these conditions.

Demographic and Medical Variables Demographic and medical variables including gender, smoking habit, body mass index (BMI), fasting glucose, leukocyte count, platelet count, monocyte ratio and neutrophil ratio were used for the analyses.

Data Analysis The serum TNF-α concentrations were compared between subjects with healthy gingiva, periodontitis and no dentition using the Kruskal-Wallis test. The proportions of subjects with detectable levels of TNF-α (0.5 pg/mL) were compared by chi-square test. The MannWhitney U test, Student’s t-test, and analysis of variance were carried out for other continuous variables, depending on their distributions. The chi-square tests were carried out for other categorical variables. Statistical analyses were conducted using SPSS version 12.0J (SPSS Japan, Tokyo, Japan).

Results and Discussion Table 1 displays the characteristics of subjects with each oral status. Periodontal conditions of probing depth, AL, and BOP were significantly higher in subjects with periodontitis than in subjects with healthy gingiva. In general, there were significant differences in the leukocyte count and neutrophil ratio among the three groups. As a whole, the serum levels of TNF-α in this study were lower than the levels of elderly people reported by Bredz et al. [13]. The circulating levels of TNF-α may differ greatly between races. Bredz et al. showed that plasma TNF-α levels tend to be higher with the extent of periodontitis. In this study, the median serum TNF-α levels in subjects with healthy gingiva, subjects with periodontitis, and edentulous subjects were 0.64 pg/mL (range

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0–16.36), 0.72 pg/mL (range 0–4.53), and 0.00 pg/mL (range 0–1.83), respectively. The frequencies for detectable TNF-α levels were 48/76 (63.2%), 53/85 (62.4%), and 6/37 (16.2%), respectively (Table 2). There were no significant differences between subjects with and without periodontitis. However, TNF-α levels and the frequency of subjects with detectable levels of TNF-α in edentulous subjects were significantly lower than in the other groups (P < 0.001). No significant difference in serum TNF-α levels were observed between male and female, or between nonsmoker and past/current smoker groups. There was no significant relationship between serum TNF-α levels with BMI and fasting glucose levels in this study (data not shown). Salvi et al. reported that the secretion of inflammatory mediators from LPS-stimulated peripheral blood monocytes (PBMCs) was observed in patients with severe periodontitis who needed tooth extraction [15]. The TNF-α secretion of LPS-stimulated PBMCs from patients with severe, hopeless periodontitis was significantly elevated, whereas, that of subjects with moderate periodontitis were nearly zero. Several studies have demonstrated that monocyte/macrophage tolerance to LPS by repeated stimulation of LPS from oral pathogen resulted in a reduction of TNF-α secretion from monocyte/macrophage [16, 17]. The tolerance in responding to periodontal pathogens may be one reason why no significant difference between subjects with and without periodontitis was observed. In other words, long-lasting and repeated exposure to a periodontal pathogen may lead inflammatory cells to reduce the secretion of TNF-α in elderly people. Another reason is that few subjects with hopeless, severe periodontitis needing tooth extraction for severe periodontitis were included as subjects with periodontitis, as defined in this study. Recently, it has been reported that a higher number of teeth is associated with pyrexia in elderly dentate subjects, suggesting that teeth give habitat to oral pathogens leading to systemic inflammation such as pneumonia(18). Iwamoto et al. reported that periodontal treatment with the application of antibiotics and mechanical debridement of calculus once a week for 1 month decreased the serum levels of TNF-α and CRP[9]. A previous intervention study by Taylor et al. demonstrated that full-mouth tooth extraction reduced the serum CRP, which is a known indicator of coronary vascular disease, in subjects with advanced periodontitis [19]. Similarly, our present results demonstrate that the circulating TNF-α levels in edentulous subjects were lower than in the other two dentate conditions. Because the edentulous condition deprives habitat of periodontal pathogens, the inflammatory response releasing TNF-α in edentate subjects might be drastically attenuated when compared to that of subjects with teeth.

Conclusion In this study, the circulating TNF-α level was not affected by current periodontal status in elderly Japanese subjects. However, circulating TNF-α levels were significantly lower in edentulous subjects than in the other groups, suggesting that the oral cavity with teeth could be a source of systemic subclinical inflammation, which can have ill effects on general health. The oral health condition, especially the edentulous condition, may be a modifiable factor leading to the attenuation of circulating levels of inflammatory markers.

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Table 1. Characteristics of Subjects. Characteristics

Healthy gingiva

Periodontitis

Edentulous

Number of subjects

Mean ± SD 76

Mean ± SD 85

Mean ± SD 37

1.78 ± 0.21 2.81 ± 0.70 5.84 ± 7.58

2.52 ± 0.44 3.46 ± 0.87 15.79 ± 12.70

50.0 44.0 22.51 ± 2.68 118.07 ± 29.51 5.44 ± 1.27 20.03 ± 4.83 6.27 ± 1.73 52.92 ± 9.90

51.8 46.4 22.76 ± 2.67 122.74 ± 39.55 6.00 ± 1.45 20.23 ± 4.14 6.19 ± 1.98 57.78 ± 8.73

Periodontal condition Probing pocket depth (mm) Attachment loss (mm) BOP (%) General Condition† Male (%) Smoking BMI (kg/m2) Fasting glucose (mg/mL) Leukocyte count (× 103/µL) Platelet count (× 104/µL) Monocyte (%) Neutrophil (%)

P*

< 0.001 < 0.001 < 0.001 59.4 61.1 23.04 ± 3.39 136.81 ± 66.27 5.50 ± 0.94 20.35 ± 4.79 5.84 ± 1.78 52.47 ± 8.50

0.283 0.125 0.640 0.092 0.017 0.936 0.463 0.001

*The p-values were calculated by Student’s t-test, Mann-Whitney U-test, analysis of variance for continuous variables depending on their distributions, and by the chi-square tests for categorical variables. † One subject with healthy gingiva, one with periodontitis, and two edentulous had missing data on smoking habit. One subject with healthy gingiva had missing data on fasting glucose, leukocyte count, platelet count, monocyte, and neutrophil.

Table 2. Serum TNF-α concentrations and frequencies of detectable TNF-α levels in subjects with healthy gingiva, periodontitis, or edentulous.

Oral Health status healthy gingiva periodontitis edentulous

Serum TNF-α concentration Number of (pg/mL) subjects Median (range) P-value 76 0.64 (0-16.36) < 0.001 85 0.72 (0-4.53) 37 0.00 (0-1.83)

Frequency of detectable TNFα levels Frequency (%) P-value 48/76 (63.2) < 0.001 53/85 (62.4) 6/37 (16.2)

The Kruskal-Wallis test was used for the comparison of serum TNF concentrations, and the chi-square test for frequencies of detectable TNF-α levels.

Acknowledgments We thank Yumiko Yoshii for expert technical assistance. This study was supported by a Grant-in-aid from the Ministry of Health and Welfare of Japan (H10-Iryo02) and partially supported by a Grant-in-aid for Scientific Research (B) 19390542 (T.S.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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[12]

[13]

Rylev, M; Kilian, M. Prevalence and distribution of principal periodontal pathogens worldwide. J Clin Periodontol., 2008 Sep, 35(8 Suppl), 346-61. Slots, J. The predominant cultivable organisms in juvenile periodontitis. Scand J Dent Res., 1976 Jan, 84(1), 1-10. Socransky, SS; Haffajee, AD; Cugini, MA; Smith, C; Kent, RL Jr. Microbial complexes in subgingival plaque. J Clin Periodontol., 1998 Feb, 25(2), 134-44. Slots, J; Ting, M. Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in human periodontal disease: occurrence and treatment. Periodontol, 2000. 1999 Jun, 20, 82-121. Wilson, M; Reddi, K; Henderson, B. Cytokine-inducing components of periodontopathogenic bacteria. J Periodontal Res., 1996 Aug, 31(6), 393-407. Hotokezaka, H; Sakai, E; Ohara, N; Hotokezaka, Y; Gonzales, C; Matsuo, K; Fujimura, Y; Yoshida, N; Nakayama, K. Molecular analysis of RANKL-independent cell fusion of osteoclast-like cells induced by TNF-alpha, lipopolysaccharide, or peptidoglycan. J Cell Biochem., 2007 May 1, 101(1), 122-34. Iacopino, AM; Cutler, CW. Pathophysiological relationships between periodontitis and systemic disease: recent concepts involving serum lipids. J Periodontol., 2000 Aug, 71(8), 1375-84. Stashenko, P; Jandinski, JJ; Fujiyoshi, P; Rynar, J; Socransky, SS. Tissue levels of bone resorptive cytokines in periodontal disease. J Periodontol., 1991 Aug, 62(8), 5049. Iwamoto, Y; Nishimura, F; Soga, Y; Takeuchi, K; Kurihara, M; Takashiba, S; Murayama, Y. Antimicrobial periodontal treatment decreases serum C-reactive protein, tumor necrosis factor-alpha, but not adiponectin levels in patients with chronic periodontitis. J Periodontol., 2003 Aug, 74(8), 1231-6. Gorska, R; Gregorek, H; Kowalski, J; Laskus-Perendyk, A; Syczewska, M; Madalinski, K. Relationship between clinical parameters and cytokine profiles in inflamed gingival tissue and serum samples from patients with chronic periodontitis. J Clin Periodontol., 2003 Dec, 30(12), 1046-52. Yamazaki, K; Honda, T; Oda, T; Ueki-Maruyama, K; Nakajima, T; Yoshie, H; Seymour, GJ. Effect of periodontal treatment on the C-reactive protein and proinflammatory cytokine levels in Japanese periodontitis patients. J Periodontal Res., 2005 Feb, 40(1), 53-8. Ide, M; McPartlin, D; Coward, PY; Crook, M; Lumb, P; Wilson, RF. Effect of treatment of chronic periodontitis on levels of serum markers of acute-phase inflammatory and vascular responses. J Clin Periodontol., 2003 Apr, 30(4), 334-40. Bretz, WA; Weyant, RJ; Corby, PM; Ren, D; Weissfeld, L; Kritchevsky, SB; Harris, T; Kurella, M; Satterfield, S; Visser, M; Newman, AB. Systemic inflammatory markers, periodontal diseases, and periodontal infections in an elderly population. J Am Geriatr Soc., 2005 Sep, 53(9), 1532-7.

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[14] Furugen, R; Hayashida, H; Yamaguchi, N; Yoshihara, A; Ogawa, H; Miyazaki, H; Saito, T. The relationship between periodontal condition and serum levels of resistin and adiponectin in elderly Japanese. J Periodontal Res., 2008 Oct, 43(5), 556-62. [15] Salvi, GE; Brown, CE; Fujihashi, K; Kiyono, H; Smith, FW; Beck, JD; Offenbacher, S. Inflammatory mediators of the terminal dentition in adult and early onset periodontitis. J Periodontal Res., 1998 May, 33(4), 212-25. [16] Muthukuru, M; Jotwani, R; Cutler, CW. Oral mucosal endotoxin tolerance induction in chronic periodontitis. Infect Immun., 2005 Feb, 73(2), 687-94. [17] Tanabe, SI; Grenier, D. Macrophage tolerance response to Aggregatibacter actinomycetemcomitans lipopolysaccharide induces differential regulation of tumor necrosis factor-alpha, interleukin-1 beta and matrix metalloproteinase 9 secretion. J Periodontal Res., 2008 Jun, 43(3), 372-7. [18] Shimazaki, Y; Tomioka, M; Saito, T; Nabeshima, F; Ikematsu, H; Koyano, K; Yamashita, Y. Influence of oral health on febrile status in long-term hospitalized elderly patients. Arch Gerontol Geriatr., in press. [19] Taylor, BA; Tofler, GH; Carey, HM; Morel-Kopp, MC; Philcox, S; Carter, TR; Elliott, MJ; Kull, AD; Ward, C; Schenck, K. Full-mouth tooth extraction lowers systemic inflammatory and thrombotic markers of cardiovascular risk. J Dent Res., 2006 Jan, 85(1), 74-8.

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ISBN: 978-1-60741-708-8 © 2009 Nova Science Publishers, Inc.

Chapter VIII

Tumor Necrotic Factor in T Cell Disorder: Hypothesis and Proof of Idea Viroj Wiwanitkit Wiwanitkit House, Bangkhae, Bangkok Thailand 10160.

Abstract Tumor necrotic factor is a specific cytokine resulting from the cellular immunity. In the process of cellular immunity, lymphocytes, especially the T lymphocyte, acts mainly in an immune response. The author hereby uses the systomics approach to formulate a hypothesis on tumor necrotic factor expression in important T cell disorders. In addition, further proof of the idea was done by matching with previously published reports on the corresponding proposed items. The models of testing include three important T cell disorders, human immunodeficiency virus (HIV) infection, T cell leukemia and Hodgkin’s lymphoma.

Introduction Tumor necrotic factor is a specific cytokine resulting from cellular immunity. In the process of cellular immunity, lymphocytes, especially the T lymphocyte, are the main actors in the immune response. The key substance in the action is cytokine. Of several cytokines, the tumor necrotic factor is a widely studied cytokine. This cytokine gets its name from the observation of its property that can lead malignant tumor cells to apoptosis and further necrosis. In pathophysiology, the tumor necrotic factor is the main cytokine leading to fever [1-2], similar to interleukin. However, excessive imbalance expression of tumor necrotic factor can lead to pathology. A good example is the condition of cerebral malaria [3-7], which is affected by hyperactivity of the tumor necrotic factor that leads to a defect in the blood brain barrier function and anatomy.

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Here, it can be easily said that the process of tumor necrotic factor is directly related to T cell function. Therefore, any disorder of the T cell can be the condition that permits abnormal expression of tumor necrotic factor. The author hereby uses the systomics approach to formulate a hypothesis on tumor necrotic factor expression in important T cell disorders. In addition, further proof of the idea was done by matching with previously published reports on the corresponding proposed items. The models of testing include three important T cell disorders, human immunodeficiency virus (HIV) infection, T cell leukemia and Hodgkin’s lymphoma.

Condition 1: Human Immunodeficiency Virus Infection Model HIV infection is a well-known acquired immunodeficiency condition [8–2]. This infection is due to a retrovirus, namely the HIV virus. This virus mainly affects the T cell during the course of infection. CD4+ T cell will be affected and this brings several complications, the expression of immune deficit. HIV is a T cell disorder in which the T cell is suppressed by the HIV virus. Therefore, the hypothesis of this model is that tumor necrotic factor suppression must be seen and the expression or level of tumor necrotic factor must be reduced owing to T cell suppression. Further implication can also be made. It can be imagined that the tumor can easily appear in HIV infection, especially those with severe T cell suppression. In addition, there should be no condition that relates to hyperactivity of increased tumor necrotic factor level. To verify this hypothesis, it can be searched from PubMed, the standard basic medical database tool. It can be seen that there are a heap of published papers confirming the already proposed hypothesis. There are many reports mentioning the decreased level of tumor necrotic factor in HIV infection [13-14]. Ino et al. noted that HIV RNA amounts in HIV-1infected patients could be predicted by serum tumor necrotic factor level [15]. Morlat et al. also reported that tumor necrotic factor-alpha could potentially be an additional surrogate marker to CD4+ lymphocyte count for monitoring of HIV-infected cases [16]. Domingo et al. also noted that tumour necrosis factor alpha in fat redistribution syndromes was associated with combination antiretroviral therapy in HIV-1-infected patients [17]. However, some reports are discordant with these findings. For example, Trotti et al. reported on increased erythrocyte glutathione peroxidase activity and serum tumor necrosis factor-alpha in HIVinfected patients and proposed for relationship to on-going prothrombotic state [18]. In addition, there are also several reports on increased prevalence of cancer in HIV-infected patients [19-23]. It is presently confirmed that opportunistic cancer is the main problem inHIV infected patients.

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Condition 2: T Cell Leukemia Model T cell leukemia is a specific kind of leukemia. Hairy cell leukemia is the best described T cell leukemia in hematology [22-26]. Although leukemia is a condition with numerous multiplication of leukocytes as well as lymphocytes, the resulting cells are immature and non functional. Therefore, T cell leukemia has numerous poor functional immature T cells. This can formulate the hypothesis that the tumor necrotic factor should be down expressed in T cell leukemia. In addition, cellular immunity must be defected in T cell leukemia. The verification of this model can be done similar to previously described in condition 1. There are many supportive evidences [27-29]. Barak et al. reported that the tumor necrosis factor has a strong correlation with response to treatment in hairy cell leukemia [28]. Wetzler et al. found the correlation between leukemia inhibitory factor and increased expression of tumor necrotic factor [29]. However, some reports are discordant with these findings. For example, Buck et al. said that tumor necrosis factor-alpha could stimulate growth of tumor cells in hairy cell leukemia [30].

Condition 3: Hodgkin’s Lymphoma Model Hodgkin’s lymphoma is a specific kind of lymphoma. Hodgkin’s lymphoma is the wellknown lymphoma in hematology [31-32]. Although lymphoma is a condition with numerous multiplication of leukocytes as well as lymphocytes, the resulting cells are immature and non functional. Therefore, Hodgkin’s lymphoma has numerous poor functional immature T cells. This can formulate the hypothesis that the tumor necrotic factor should be down expressed in Hodgkin’s lymphoma, similar to T cell leukemia. In addition, cellular immunity must be defected in Hodgkin’s lymphoma. The verification of this model can be done similar to previously described in condition 1. Of interest, some reports confirm the hypothesis [33] while the other did not [34-35].

Conclusion There is some evidence on the alteration of tumor necrotic factor in the studied T cell disorders models. Of interest, many published reports confirming the hypothesis that T cell disorders are diseases with down expression in tumor necrotic factor. However, there are also some discordant reports. This brings us to further studies in this area.

References [1] [2] [3]

Warren, JS. Interleukins and tumor necrosis factor in inflammation. Crit Rev Clin Lab Sci., 1990, 28(1), 37-59. Warren, JS; Ward, PA; Johnson, KJ. Tumor necrosis factor: a plurifunctional mediator of acute inflammation. Mod Pathol., 1988 May, 1(3), 242-7. Combes, V; Coltel, N; Faille, D; Wassmer, SC; Grau, GE. Cerebral malaria: role of

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Viroj Wiwanitkit microparticles and platelets in alterations of the blood-brain barrier. Int J Parasitol., 2006 May 1, 36(5), 541-6. Coltel, N; Combes, V; Hunt, NH; Grau, GE. Cerebral malaria -- a neurovascular pathology with many riddles still to be solved. Curr Neurovasc Res., 2004 Apr, 1(2), 91-110. Nyangoto, EO. Cell-mediated effector molecules and complicated malaria. Int Arch Allergy Immunol., 2005 Aug, 137(4), 326-42. Giminez, F; Barraud de Lagerie, S; Fernandez, C; Pino, P; Mazier, D. Tumor necrosis factor alpha in the pathogenesis of cerebral malaria. Cell Mol Life Sci., 2003 Aug, 60(8), 1623-35. Wassmer, SC; Combes, V; Grau, GE. Pathophysiology of cerebral malaria: role of host cells in the modulation of cytoadhesion. Ann N Y Acad Sci., 2003 May, 992, 30-8. Castro, BA; Cheng-Mayer, C; Evans, LA; Levy, JA. HIV heterogeneity and viral pathogenesis. AIDS, 1988, 2 Suppl 1, S17-27. Jackson, JB; Balfour, HH Jr. Practical diagnostic testing for human immunodeficiency virus. Clin Microbiol Rev., 1988 Jan, 1(1), 124-38. Wells, KH; Poiesz, BJ. Biology of retroviruses. Detection, molecular biology, and treatment of retroviral infection. Obstet Gynecol Clin North Am., 1990 Sep, 17(3), 489521. Schulz, TF; Larcher, C; Dierich, MP. Human immunodeficiency virus (HIV): a review. Z Hautkr., 1990 Jul, 65(7), 619-22, 625-6, 629-32. Boyd, JE; James, K. Human immunodeficiency virus: strategies for protection and therapy. Microbiol Sci., 1988 Oct, 5(10), 300-2. Aukrust, PP; Svardal, AM; Müller, F; Lunden, B; Nordøy, I; Frøland, SS. Markedly disturbed glutathione redox status in CD45RA+CD4+ lymphocytes in human immunodeficiency virus type 1 infection is associated with selective depletion of this lymphocyte subset. Blood, 1996 Oct 1, 88(7), 2626-33. Blackard, JT; Kang, M; St Clair, JB; Lin, W; Kamegaya, Y; Sherman, KE; Koziel, MJ; Peters, MG; Andersen, J; Chung, RT. Aids Clinical Trials Group A5071 Study Team.Viral factors associated with cytokine expression during HCV/HIV co-infection. J Interferon Cytokine Res., 2007 Apr, 27(4), 263-9. Ino, T; Tada, A; Tominaga, A; Komori, Y; Chiba, H; Senpuku, H. Role of salivary tumour necrosis factor alpha in HIV-positive patients with oral manifestations. Int J STD AIDS, 2007 Aug, 18(8), 565-9. Morlat, P. Evolution of tumor necrosis factor-alpha serum concentrations in HIV infected individuals treated with zidovudine. Pathol Biol, Paris, 1996 Oct, 44(8), 7169. Domingo, P; Vidal, F; Domingo, JC; Veloso, S; Sambeat, MA; Torres, F; Sirvent, JJ; Vendrell, J; Matias-Guiu, X; Richart, C. HIV-FRS Study Group. cEur J Clin Invest., 2005 Dec, 35(12), 771-80. Trotti, R; Rondanelli, M; Anesi, A; Gabanti, E; Brustia, R; Minoli, L. Increased erythrocyte glutathione peroxidase activity and serum tumor necrosis factor-alpha in HIV-infected patients: relationship to on-going prothrombotic state. J Hematolther Stem Cell Res., 2002 Apr, 11(2), 369-75.

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[19] Pothoff, A; Brockmeyer, NH. HIV-associated tumors. Hautarzt., 2006 Nov, 57(11), 988, 990-3. [20] Aoki, Y; Tosato, G. Neoplastic conditions in the context of HIV-1 infection. Curr HIV Res., 2004 Oct, 2(4), 343-9. [21] Albu, E; Reed, M; Pathak, R; Niazi, M; Sivakumar, M; Fernandes, E; Mailapur, RV; Parithivel, VS; Gerst, PH. Malignancy in HIV/AIDs: a single hospital experience. J Surg Oncol., 2000 Sep, 75(1), 11-8. [22] Gates, AE; Kaplan, LD. AIDS malignancies in the era of highly active antiretroviral therapy. Oncology, Williston Park, 2002 Apr, 16(4), 441-51, 456, 459. [23] Bonnet, F; Morlat, P. Cancer and HIV infection: any association? Rev Med Interne., 2006 Mar, 27(3), 227-35. [24] Sarin, PS; Gallo, RC. Tcell malignancies and human T cell leukemia (lymphotropic) retroviruses (HTLV). Prog Clin Biol Res., 1985, 184, 445-56. [25] Semenzato, G; Pandolfi, F. T-cell chronic lymphocytic leukemia. Haematologica, 1983 Mar-Apr, 68(2), 245-86. [26] Catovsky, D; Linch, DC; Beverley, PC. T cell disorders in haematological diseases. Clin Haematol., 1982 Oct, 11(3), 661-95. [27] Khalaf, W; Maina, C; Byers, J; Harvey, W. Interferon-alpha 2b and vesnarinone influence levels of tumor necrosis factor-alpha, apoptosis, or interleukin 6 in ESKOL, a hairy cell leukemic cell line. A potential cytokine and oncogene relationship regulating apoptosis is suggested. Leuk Res., 2002 Feb, 26(2), 169-77. [28] Barak, V; Nisman, B; Polliack, A. The tumor necrosis factor family and and correlation with disease activity and response to treatment in hairy cell leukemia. Eur J Haematol., 1999 Feb, 62(2), 71-5. [29] Wetzler, M; Estrov, Z; Talpaz, M; Kim, KJ; Alphonso, M; Srinivasan, R; Kurzrock, R. Leukemia inhibitory factor in long-term adherent layer cultures: increased levels of bioactive protein in leukemia and modulation by IL-4, IL-1 beta, and TNF-alpha. Cancer Res., 1994 Apr 1, 54(7), 1837-42. [30] Buck, C; Digel, W; Schöniger, W; Stefanic, M; Ragnavachar, A; Heimpel, H; Porzsolt, F. Tumor necrosis factor-alpha, but not lymphotoxin, stimulates growth of tumor cells in hairy cell leukemia. Leukemia., 1990 Jun, 4(6), 431-4. [31] Matutes, E. Adult T-cell leukaemia/lymphoma. J Clin Pathol. 2007 Dec, 60(12), 1373-7. [32] Aisenberg, EG. Malignant lymphoma. N Engl J Med., 1973 Apr 26, 288(17), 883-90. [33] Villani, F; Viola, G; Vismara, C; Laffranchi, A; Di Russo, A; Viviani, S; Bonfante, V. Lung function and serum concentrations of different cytokines in patients submitted to radiotherapy and intermediate/high dose chemotherapy for Hodgkin's disease. Anticancer Res., 2002 Jul-Aug, 22(4), 2403-8. Becovici, JP; Machelon, V; Gaudin-Nome, F; Roudaut, N; Conan-Charlet, V; Leroy, [34] JP; Sensebe, L; Kerlan, V. Hodgkin's disease masquerading as fibrous thyroiditis: potential role of cytokines in in vivo and in vitro studies. Clin Endocrinol, Oxf., 2002 Nov, 57(5), 691-7. [35] Passam, M; Alexandrakis, MG; Moschandrea, J; Sfiridaki, A; Roussou, PA; Siafakas, NM. Angiogenic molecules in Hodgkin's disease: results from sequential serum analysis. Int J Immunopathol Pharmacol., 2006 Jan-Mar, 19(1), 161-70.

In: Tumor Necrosis Factor Editor: Toma P. Rossard

ISBN: 978-1-60741-708-8 © 2009 Nova Science Publishers, Inc.

Chapter IX

Tumor Necrotic Factor in Malaria Viroj Wiwanitkit Wiwanitkit House, Bangkhae, Bangkok Thailand 10160.

Abstract Malaria is an important tropical-mosquito-borne blood infection. Malaria has a complex pathogenesis. Tumor necrotic factor, a cytokine, is widely mentioned for its correlation to malaria in various aspects. In this article, the author will briefly review the important reports on tumor necrotic factor in malaria.

Introduction Malaria is an important tropical-mosquito-borne blood infection. Malaria has its complex pathogenesis. The etiological pathogens of human malaria are the five species of Plasmodium parasites. The clinical signs and symptoms of malaria are high fever with chills. Several systemic complications of malaria are confirmed. Malaria can bring death in severe cases. This infection is still a public health threat to many endemic countries, especially for those poor developing countries in the tropical region. Researches are needed to get new knowledge on malaria aiming at success in control and treatment of this disease. As previously stated, the complex pathogenesis in malaria has been described in medical science for a long time. The cytokine response in malarial infection is widely studied. Briefly, cytokine is a cellular secretion that involves the immune process, especially for cellular types. There are many discovered cytokines in the present day. Tumor necrotic factor is a cytokine. By its name, this cytokine mainly affects the tumor and it brings the tumor to necrotic status [1-2]. Therefore, this cytokine is widely studied in the pathogenesis of cancers. However, this cytokine also plays roles in other disorders including malarial infection. This is accepted as the T cell immunity phenomenon to malaria. Clark et al. noted the important roles of tumour necrosis factor in the illness and pathology of

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malaria [3]. Clark mentioned that non-antibody-mediated immunity was important in immunity to malaria—especially through mediators such as gamma interferon and tumor necrosis factor; however, the host can suffer if this was too much [4]. Tumor necrotic factor, a cytokine, is widely mentioned for its correlation to malaria in various aspects. It is concluded that tumor necrotic factors bring both defense and pathology in malaria [4]. In this article, the author will briefly review the important reports on tumor necrotic factor in malaria.

Tumor Necrotic Factor in Plasmodium Vivax Infection There are many reports on tumor necrotic factor in Plasmodium vivax infection. The focus reports are on the liver stage of this parasite. Indeed, vivax malaria has its interesting liver stage that makes it act as a silent infection. Yeom et al. studied serum cytokine profiles in patients with vivax malaria by making a comparison between those who presented with and without hepatic dysfunction [5]. Yeom et al. concluded for involvement of tumor necrotic factor in the development of hepatic dysfunction [5]. In addition to the reports on the effect of tumor necrotic factor in the liver stage of vivax malaria, there are also other reports in other aspects. Those interesting reports will be further detailed. Of interest, Seoh et al. reported that the average concentration of serum tumor necrosis factor-alpha was significantly higher in patients who presented with rather than those without hyperpyrexia [6]. Seoh et al. proposed that the serum concentration of tumor necrotic factor might parallel the concentration at the tissue sites of its production and action [6]. In addition, Park et al. also reported that thrombocytopenia associated with vivax malaria was associated with elevated serum concentrations of both pro- and anti-inflammatory cytokines [7]. Mendis and Carter concluded that tumor necrotic factor was not a causative agent for fever in vivax malaria but other manifestations [8] (as previously quoted in this section).

Tumor Necrotic Factor in Plasmodium Falciparum Infection There are many reports on tumor necrotic factor in Plasmodium falciparum infection. The focus reports are on cerebral malaria caused by this parasite [8]. Indeed, cerebral malaria is one of the deadly complication of falciparum malaria. Cerebal malaria is characterized not only by the cytoadherence of parasite-infected erythrocytes, but also by morphological and functional alterations of brain microvascular endothelial cells owing to their interactions with circulating cells, such as platelets, monocytes, lymphocytes, and dendritic cells [9]. The blood brain barrier aberration due to the cytokine, especially for tumor necrotic factor is also noted [9]. Wassmer et al. proposed that TGF-beta1 released from activated platelets could induce TNF-stimulated human brain endothelium apoptosis and underlined cerebral malaria [10].

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Wassmer et al. also reported that platelets led to brain endothelial alterations induced by Plasmodium falciparum [11]. Molecularly, significant association between tumor necrotic factor alpha promoter allele (-1031C, -863C, and -857C) and cerebral malaria was documented [12]. On the other hand, Cabantous et al. noted that alleles 308A and 238A in the tumor necrosis factor alpha gene promoter did not increase the risk of severe malaria in children [13]. Since tumor necrotic factor is accepted as molecularly important with a role in induction of cerebral malaria, it is focused on as a target of treatment. Lu et al. said that experimental disruption of JNK2 reduced the cytokine response to Plasmodium falciparum glycosylphosphatidylinositol in vitro and could bring protection in a cerebral malaria model [14]. This confirms the report of John et al. that low levels of RANTES were associated with mortality in cerebral malaria [15]. New therapeutic agents, such as LMP-420, are developed based on these described facts [16-17]. In addition to the reports on the effect of tumor necrotic factor in cerebral malaria due to falciparum malaria, there are also other reports on other aspects. Those interesting reports will be further detailed. Awandare et al. noted increased levels of tumor necrotic factor in children with severe Plasmodium falciparum malaria with respiratory distress [18]. Indeed, the severity of falciparum malaria, many complications, is confirmed for the correlation to elevated tumor necrotic factor level [19].

Tumor Necrotic Factor in Plasmodium Ovale Infection There are only a few reports on tumor necrotic factor in Plasmodium ovale infection. It is believed that the tumor necrotic factor has similar roles to those described in vivax and falciparum malaria [20]. However, Hemmer et al. reported on stronger host response per parasitized erythrocyte in Plasmodium ovale than in Plasmodium falciparum malaria [21].

Tumor Necrotic Factor in Plasmodium Malariae Infection There are also only a few reports on tumor necrotic factor in Plasmodium malariae infection. It is also believed that the tumor necrotic factor has similar roles to those described in vivax and falciparum malaria [20]. Singh et al. said that serum concentration of tumor necrotic factor-alpha correlated well with the severity of malaria and it could be used as an important prognostic marker of the disease [20].

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Tumor Necrotic Factor in Plasmodium Knowlesi Infection Because Plasmodium knowlesi is the newest human infected species, discovered for a few decades, there has been no report on tumor necrotic factor in Plasmodium knowlesi infection in human beings.

References [1]

Bienengräber A. Progress in scientific oncology. Stomatol DDR. 1974 Oct, 24(10), 695-704. [2] Humphrey, LJ; Pierce, GE. The role of immunology in the diagnosis and treatment of cancer. Prog Clin Cancer, 1975, 6, 15-36. [3] Clark, IA; Chaudhri, G; Cowden, WB. Roles of tumour necrosis factor in the illness and pathology of malaria. Trans R Soc Trop Med Hyg., 1989 Jul-Aug, 83(4), 436-40 [4] Clark, IA. Cell-mediated immunity in protection and pathology of malaria. Parasitol Today, 1987 Oct, 3(10), 300-5. [5] Yeom, JS; Park, SH; Ryu, SH; Park, HK; Woo, SY; Ha, EH; Lee, BE; Yoo, K; Lee, JH; Kim, KH; Kim, S; Kim, YA; Ahn, SY; Oh, S; Park, HJ; Min, GS; Seoh, JY; Park, JW. Serum cytokine profiles in patients with Plasmodium vivax malaria: a comparison between those who presented with and without hepatic dysfunction. Trans R Soc Trop Med Hyg., 2003 Nov-Dec, 97(6), 687-91. [6] Seoh, JY; Khan, M; Park, SH; Park, HK; Shin, MH; Ha, EH; Lee, BE; Yoo, K; Han, HS; Oh, S; Wi, JH; Hong, CK; Oh, CH; Kim, YA; Park, JW. Serum cytokine profiles in patients with Plasmodium vivax malaria: a comparison between those who presented with and without hyperpyrexia. : Am J Trop Med Hyg., 2003 Jan, 68(1), 102-6. [7] Park, JW; Park, SH; Yeom, JS; Huh, AJ; Cho, YK; Ahn, JY; Min, GS; Song, GY; Kim, YA; Ahn, SY; Woo, SY; Lee, BE; Ha, EH; Han, HS; Yoo, K; Seoh, JY. Serum cytokine profiles in patients with Plasmodium vivax malaria: a comparison between those who presented with and without thrombocytopenia. Ann Trop Med Parasitol., 2003 Jun, 97(4), 339-44. [8] Mendis, KN; Carter, R. The role of cytokines in Plasmodium vivax malaria. Mem Inst Oswaldo Cruz., 1992, 87 Suppl 3, 51-5. [9] Coltel, N; Combes, V; Hunt, NH; Grau, GE. Cerebral malaria -- a neurovascular pathology with many riddles still to be solved. Curr Neurovasc Res., 2004 Apr, 1(2), 91-110. [10] Wassmer, SC; de Souza, JB; Frère, C; Candal, FJ; Juhan-Vague, I; Grau, GE. TGFbeta1 released from activated platelets can induce TNF-stimulated human brain endothelium apoptosis: a new mechanism for microvascular lesion during cerebral malaria. J Immunol., 2006 Jan 15, 176(2), 1180-4. [11] Wassmer, SC; Combes, V; Candal, FJ; Juhan-Vague, I; Grau, GE. Platelets potentiate brain endothelial alterations induced by Plasmodium falciparum. Infect Immun., 2006 Jan, 74(1), 645-53. [12] Hananantachai, H; Patarapotikul, J; Ohashi, J; Naka, I; Krudsood, S; Looareesuwan, S; Tokunaga, K. Significant association between TNF-alpha (TNF) promoter allele

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[14]

[15]

[16] [17] [18]

[19]

[20] [21]

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(-1031C, -863C, and -857C) and cerebral malaria in Thailand. Tissue Antigens., 2007 Mar, 69(3), 277-80. Cabantous, S; Doumbo, O; Ranque, S; Poudiougou, B; Traore, A; Hou, X; Keita, MM; Cisse, MB; Dessein, AJ; Marquet, S. Alleles 308A and 238A in the tumor necrosis factor alpha gene promoter do not increase the risk of severe malaria in children with Plasmodium falciparum infection in Mali. Infect Immun., 2006 Dec, 74(12), 7040-2. Epub 2006 Sep 18. Lu, Z; Serghides, L; Patel, SN; Degousee, N; Rubin, BB; Krishnegowda, G; Gowda, DC; Karin, M; Kain, KC. Disruption of JNK2 decreases the cytokine response to Plasmodium falciparum glycosylphosphatidylinositol in vitro and confers protection in a cerebral malaria model. J Immunol., 2006 Nov 1, 177(9), 6344-52. John, CC; Opika-Opoka, R; Byarugaba, J; Idro, R; Boivin, MJ. Low levels of RANTES are associated with mortality in children with cerebral malaria. J Infect Dis., 2006 Sep 15, 194(6), 837-45. Epub 2006 Aug 16. Wassmer, SC; Cianciolo, GJ; Combes, V; Grau, GE. LMP-420, a new therapeutic approach for cerebral malaria? Med Sci., Paris, 2006 Apr, 22(4), 343-5. Burton, A. New hope for cerebral malaria treatment? Lancet Neurol., 2005 Oct, 4(10), 600 Awandare, GA; Goka, B; Boeuf, P; Tetteh, JK; Kurtzhals, JA; Behr, C; Akanmori, BD. Increased levels of inflammatory mediators in children with severe Plasmodium falciparum malaria with respiratory distress. J Infect Dis., 2006 Nov 15, 194(10), 143846. Prakash, D; Fesel, C; Jain, R; Cazenave, PA; Mishra, GC; Pied, S. Clusters of cytokines determine malaria severity in Plasmodium falciparum-infected patients from endemic areas of Central India. J Infect Dis., 2006 Jul 15, 194(2), 198-207. Singh, S; Singh, N; Handa, R. Tumor necrosis factor-alpha in patients with malaria. Indian J Malariol., 2000 Mar-Jun, 37(1-2), 27-33. Hemmer, CJ; Holst, FG; Kern, P; Chiwakata, CB; Dietrich, M; Reisinger, EC. Stronger host response per parasitized erythrocyte in Plasmodium vivax or ovale than in Plasmodium falciparum malaria. Trop Med Int Health, 2006 Jun, 11(6), 817-23.

In: Tumor Necrosis Factor Editor: Toma P. Rossard

ISBN: 978-1-60741-708-8 © 2009 Nova Science Publishers, Inc.

Chapter X

Tumor Necrosis with Special Reference to Autophagic Cell Death (SelfCannibalism) and Xeno- Cannibalism in Gastric Cancer: Our Experience and Review of the Literature* Rosario A. Caruso Department of Human Pathology, University of Messina, Messina, Italy.

Abstract Cell death is a field that has attracted much attention in recent years, leading to several new and important insights in cell biology, development, and pathology, but the recruitment of many new researchers to the field has led to some confusion in terms. According to the Recommendations of the Nomenclature Committee on Cell Death, the definition of cell death must be based on precise terms of the parameters that describe the presumed cell death pathway involved. Since the precise characterization of biochemical checkpoints controlling cell death is still awaiting, different cell death types are defined by morphological criteria. Four morphological entities, without a clear reference to precise biochemical mechanism, have been described: apoptosis, autophagy, necrosis and mitotic catastrophe. The most common and well-defined form of programmed cell death is apoptosis, which is a physiological “cell-suicide” programme that is essential for embryonic development, immune system function and the maintenance of tissue homeostasis in multicellular organisms. Apoptosis is characterized by the activation of a specific family of cysteine proteases, the caspases, followed by a series of morphological changes including cellular and nuclear shrinkage (pyknosis), chromatin condensation and

*

A version of this chapter was also published in Gastric Cancer Research Trends, edited by Marilyn B. Tompkins, Nova Science Publishers. It was submitted for appropriate modifications in an effort to encourage wider dissemination of research.

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Rosario A. Caruso nuclear fragmentation (karyorhexis) with formation of apoptotic bodies. Tumor growth involves two essential deviations from the normal state including the induction of proliferative stimuli, and simultaneous suppression of potentially compensatory cell death. It is well recognized that apoptosis is impaired in many cancers by mutations in genes such as p53, but nonapoptotic mechanisms have been largely overlooked in studies of cancer causation, progression and therapy. It has recently suggested that the development of an invasive cancer involves a progressive switch from predominantly apoptotic to necrotic tumor cell death. This disordered cell death is supported by the frequent observation in a large number of common tumors that the presence of microscopic necrosis predicts a poor prognosis. This review summarizes the recent discoveries on cell death and its role in neoplasms. In particular, our experience on the ultrastructural features of autophagic cell death and xeno-cannibalism in gastric carcinomas is reported. Profound knowledge of the morphology of cell death may be useful for inform and drive the development of more effective biologic therapies for patients with cancer.

Introduction Tumor necrosis has garnered increased attention over the last few years, in part because a number of studies have now shown that tumor necrotic tissue represents a significant prognostic marker with an independent influence on metastasis-free survival in patients with neoplasm [34,41,42,80,81,126,136]. In particular, recent studies suggest that surgical pathologic evaluation of tumor necrosis should routinely record its presence or absence [81,126]. Such an assessment is easily performed through routine histologic evaluation with reasonably high rate of reproducibility among pathologists. The death of a cell can be defined as an irreversible loss of plasma membrane integrity [47]. It may occur through different mechanisms leading to distinct morphologies. Consequently, different terms have been coined including apoptosis [70], necrosis [70], oncosis [98], autophagy [12,22], paraptosis [131], mitotic catastrophe [73,121], and the descriptive model of apoptosis-like and necrosis-like cell death [88]. At present, the identification of different cell death types is based on pathognomonic morphologic characteristics, without a clear reference to precise biochemical mechanism [75]. It is well recognized that apoptosis is impaired in many cancers by mutations in genes such as p53, but nonapoptotic mechanisms have been largely overlooked in studies of cancer causation, progression and therapy. This review summarizes the histopathologic features of apoptosis and necrosis in tumors, and describes the ultrastructural features of paraptosis, autophagic cell death, mitotic catastrophe. As necrotic phenomena have been most extensively investigated in thyroid and breast cancers, these tumors have been chosen as model for studying the relationship between tumor necrosis and hypoxia, and p53 status in solid tumors. Finally, the morphological features of autophagic cell death (self-cannibalism) and neutrophil-tumor cell xeno-cannibalism in gastric carcinoma are described on the basis of the author’s experience and the literature.

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Apoptosis Patterns of cell death have been divided into apoptosis and accidental necrosis. Apoptosis is characterized by the sequence of nuclear shrinkage (pyknosis), chromatin margination, nuclear fragmentation (karyorhexis), little or no ultrastructural modification of cytoplasmic organelles, plasma membrane blebbing, maintenance of plasma membrane until late stage of the process, and engulfment by neighboring cells [70,71,150]. All of these changes occur before plasma membrane integrity is lost. There are many different approaches to analysis of apoptosis, but none can exceed light and electron microscopy examination. The major limitation of electron microscopy is that the study of large number of cells is not feasible. Additional method helpful in analysis include the TUNEL assay for identification of DNA fragmentation [43]. It has been pointed out that this method was sensible but not absolutely specific for apoptosis because DNA fragmentation also occurs in necrotic processes [48]. Studies into the effects of fixation times have yielded variable results [28]. Prolonged fixation times can reduce the number of detectable apoptotic cells, while delayed fixation can increase their number [28]. Therefore, serious reservations have been expressed concerning the applicability of this method to evaluate apoptosis [144]. Recently, new antibodies directed against active caspase 3 and cleaved cytokeratin 18 have become available, enabling the determination of apoptotic cells with higher sensitivity and specificity than is possible using the TUNEL method [144].

Molecular Executioners of Apoptosis Cell death by apoptosis is usually committed by the activation of a specific family of cysteine proteases called caspases [134,138]. The caspase family can be divided into initiator caspases and effector caspases. Effector caspases are cleaved and activated by initiator caspases and, once active, are a self-destruction signal to the cell. Two main pathways have been described so far: the extrinsic pathway, also called the death receptor pathway, and the intrinsic pathway, or mitochondrial –mediated pathway [11,57,69,158]. In the extrinsic pathway, binding of ligand to the CD95 receptor (also known as FAS) or members of the tumor-necrosis factor (TNF) receptor superfamily recruits, through their intracellular domains, a death-inducing signalling complex (DISC), which activates the caspase-8 and this triggers the death of the cell. The intrinsic pathway integrates many death stimuli, including genotoxic stress, and is associated with the release of many proteins from the mitochondria, in particular cytochrome c [74]. Once released, cytochrome c stimulates the formation of another caspase-activating complex, called the apoptosome, which activates caspase-9 [11,46,57,69,158].

Deregulation of Apoptosis in Tumors Acquired resistance toward apoptosis is a hallmark of most and perhaps all types of cancer [10]. In many cancers, pro-apoptotic proteins have inactivating mutations (i.e. p53) or

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the expression of antiapoptotic proteins is upregulated (i.e. BCL2, Survivin, MUC1), leading to the unchecked growth of tumors and the development of resistance to chemotherapy [9].

BCL2 Originally cloned as the deregulated oncogene at the translocation breakpoint of t(14;18) follicular lymphomas, Bcl-2 enhances tumorigenesis by prolonging survival [25,143]. Bcl2 is also overexpressed in many other cancers [36]. For most cancers, the overexpression of Bcl2 correlates with poor survival and progression of disease [36]. However, contrary to what might be expected, in breast [84] and colorectal tumors [67] the overexpression of Bcl2 has been associated with low proliferative potential and better prognosis.

Survivin Survivin is a member of the inhibitor of apoptosis (IAP) gene family [124], which is expressed in mitosis and localized to components of the mitotic apparatus [40]. It is potentially involved in both the inhibition of apoptosis and control of cell division [110]. Survivin is found in most human cancers but is either undetectable or expressed at a very low level in differentiated adult tissues [130]. In most cancers, expression of survivin correlated with reduced apoptotic index, poor prognosis, and increased risk of recurrence [141]. Dysfunction of survivin leads to the cell being incapable of division and induces mitotic catastrophe [122].

MUC1 MUC1 functions in providing a protective barrier against damage to the apical borders of epithelial cells [148]. The MUC1 cytoplasmic tail also signals environmental stress at the apical membrane to the cell [148]. MUC1 interacts with members of the ErbB family [91] and is targeted to nucleus and mitochondria [91,119]. It suppress p53-dependent and p-53 independent apoptotic responses to DNA damage [148]. MUC1 is an oncoprotein aberrantly overexpressed in the cytosol and on the entire cell membrane of diverse human carcinomas [148]. Thus, human tumors that overexpress MUC1 may have a survival advantage to genotoxic and potentially other forms of stress by exploiting physiologic mechanisms that evolved for the repair of damaged epithelia [148].

p53 In response to a range of stresses, including DNA damage, hypoxia or proliferative signals, p53 becomes stabilized, causing cells to undergo either cell-cycle arrest or apoptosis. Thus, p53 acts as a “guardian of the genome” in protecting cells against cancer [3,89,151].

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According to this important function, p53 activity is controlled in a very complex manner, including several auto-regulatory loops, through the intervention of dozens of modulator proteins (the ‘p53 interactome’) [78,139]. It is fundamental that both researchers and clinicians are aware of the recent knowledge on the molecular mechanisms involved in p53 family regulation and on the pathways they regulate, both in physiological and pathological conditions.The most important normal function of p53 is probably to direct cell cycle arrest at the G1 or G2 phase of the cell cycle after certain types of DNA damage and to induce apoptosis when the damage is too severe [3]. In humans, inheritance of a TP53 mutant allele results in a rare familial autosomal disorder, the Li–Fraumeni syndrome [142]. It is characterized by a high incidence of multiple early cancers [142]. p53 mutations have been found in most types of tumours, with frequencies ranging from 5% (cervix) to 50% (lung). Basically, alterations in p53 can be analyzed by mutation analysis or immunohistochemical staining. Mutations in the TP53 gene can either result in the production of a stable p53 protein, which can be detected immunohistochemically, or production of truncated p53 protein. The latter type of mutations (null mutations) will result in false-negative immunohistochemical staining for the p53 protein and to the erroneous conclusion that no mutation is present [54]. On the other hand, wild-type p53 may accumulate in some tumours as a result of a response to DNA damage or by binding to other cellular proteins, giving a positive immunohistochemical result [116]. Therefore, it is questionable whether immunohistochemistry correlates with the actual TP53 mutation status. This is relevant because immunohistochemistry is frequently used as the sole method of analyzing dysregulation of p53. Aberrant overexpression of p53 detected by widespread immunohistochemical staining of tumour cell nuclei (>20% of cells) strongly concurs with the presence of p53 gene mutation determined by single strand conformational polymorphism (SSCP) and mutational analysis [52]. Therefore, the methods used to assess p53 status are immunohistochemistry, indicating abnormal accumulation of p53, and sequence analysis, indicating presence of p53 mutations. Recently, Nenutil et al. [106] investigated factors that might improve immunohistochemistry as a marker for p53 status by combining assays that relate p53 stabilization to transcriptional activation. Excessively high levels of p53 without increased MDM2 identify inactivating mutations that stabilize p53, whereas tumours in which high levels of p53 are caused by stabilization of wild-type protein are identified by concomitantly increased MDM2 staining. Measurement of staining for p53 and MDM2 accurately identifies the TP53 status of tumours. This simple and cost-effective method, applicable to automated staining and quantitation methods, improves the identification of TP53 status over standard methods for p53 immunostaining and provides information about tumour p53 phenotype that is complementary to genotyping data.

Morphology of Necrosis The factors contributing to necrosis are mostly extrinsic in nature, such as osmotic, thermal, toxic, hypoxic-ischemic, and traumatic insults. The morphology of a necrotic cell is very distinct from that of a cell undergoing classic apoptosis, with ultrastructural changes occurring in both the cytoplasm and the nucleus. The main characteristic features are

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chromatin flocculation, swelling and degeneration of the entire cytoplasm and the mitochondrial matrix, blebbing of the plasma membrane, and eventual shedding of the cytoplasmic contents into the extracellular space with subsequent inflammation [71]. Unlike in apoptosis, the chromatin is not packed into discrete membrane-bound particles, but it forms many unevenly textured and irregularly shaped clumps, a feature that is being used for differentiating between the two modes of cell death [71]. The mitochondria undergo inner membrane swelling, cristeolysis, and disintegration. Polyribosomes are dissociated and dispersed throughout the cytoplasm, giving the cytoplasmic matrix a dense and granular appearance [12,22]. Dilation and fragmentation of the cisterns of rough endoplasmic reticulum and Golgi apparatus are frequently observed [79]. The common usage of the term “necrosis” is somewhat problematic because 1) dead cells are so severely degraded by the final stage that it cannot be morphologically determined whether they died via apoptosis or necrosis; and 2) necrosis refers only to an irreversible stage of cell death, even though dying cells generally progress from a reversible to an irreversible stage. To address this issue, Majno and Joris [98] revived an old term, “oncosis”, which refers to cell death accompained by swelling. They proposed to substitute oncosis for necrosis in cells dying via a process involving cellular, and contrasted oncosis with apoptosis, which is accompained by cellular shrinkage. They then proposed that necrosis be used to refer to the final stage of either apoptosis or oncosis. However, the Nomenclature Committee on Cell Death recommends limiting the use of the expression “oncosis”, as it overlaps with necrosis, and with partial apoptosis evolving into necrosis [75]. Although the name “oncosis” corresponds well to the morphological appearance of this type of cell death, “necrosis” should be maintained for historical reasons [75].

Molecular Mechanisms of Necrosis Even though great progress has been made in the last decade in understanding the molecular mechanisms of apoptosis, the biochemical pathways leading to necrotic cell death remain poorly understood. Necrosis is long thought to be a "passive" process occurring as a consequence of acute ATP depletion. Several ATP-dependent ion channels become ineffective, leading rapid influx of Na+, Ca2+, and water, progressive loss of cytoplasmic membrane integrity, disruption of the actin cytoskeleton, cytoplasmic swelling, and eventual collapse of the cell [14]. However, recent reports suggest a sequence of events specific to necrotic cell death [37,154,161]. Such a sequence includes mitochondrial dysfunction; namely, production of reactive oxygen species by mitochondria and swelling of mitochondria; ATP depletion; failure of Ca2+ homeostasis; perinuclear clustering of organelles; activation of a few proteases, in particular calpains and cathepsins; lysosomal rupture; and ultimely plasma membrane rupture [47].

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The Apoptosis-Necrosis Continuum Despite the numerous models proposed to categorize cell death, exclusive definitions are difficult to make and are probably artificial due to the overlap and shared signaling pathways between the different programs. Both necrotic and apoptotic morphologies occur in the hepatic ischemia model originally used by Kerr [70] to introduce the term apoptosis. Heterogeneous toxic [118], ischemic [33], degenerative [115] and immunologic stimuli [83] that are typically associated with necrosis can induce the apoptotic phenotype. Stimuli that may under some circumstances result in apoptosis, can under other circumstances induce a necrotic phenotype [107]. Two clearly identifiable factors that will convert an ongoing apoptotic process to a necrotic process are the availability of intracellular ATP and the availability of caspases [47,107]. Typical apoptotic morphology is caspase-dependent and elimination or inhibition of caspases does not prevent cell death but results in an prevalence of cells with nonapoptotic morphology [88]. In vitro, in the absence of phagocytosis, apoptotic bodies ultimately will swell and lyse, and this terminal process of cell death has been termed "secondary necrosis." (so-called because of cell swelling and membrane rupture) [29]. Secondary necrosis may occur in vivo in autoimmune disorders associated with impaired clearance of apoptotic cells [58]. In recent years, it has become evident that necrosis and apoptosis cannot be considered conceptually distinct forms of cell death [159,47] but they represent only the extreme ends of a wide range of possible morphological and biochemical deaths [47].

Patterns of Tumor Necrosis Invasive tumors may show several patterns of necrosis, including coagulative necrosis, colliquative necrosis, comedo-type necrosis, peritheliomatous necrosis, dirty necrosis, intraglandular necrotic debris, crypt lumen apoptosis.

Figure 1. Coagulative necrosis is present adjacent to tumor tissue showing a solid growth pattern.

Coagulative necrosis may be found in tumors characterized by a solid growth pattern (Figure 1). In this case, the autolytic enzymes are inactive and the cells within an area of coagulative necrosis retain their shape but are highly eosinophilic “ghosts” of their previous

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form. After a period of time, this pattern is replaced by colliquative necrosis, in which the cellular structures are broken down by proteolitic enzymes released from ruptured lysosomes and similar enzymes released by infiltrating inflammatory cells. Microvascular hyperplasia is a form of angiogenesis and is usually present in regions adjacent to necrosis [7]. Degenerating and thrombosed blood vessels may be found within necrotic areas. Coagulative necrosis may be replaced by a scar-like area that is defined as “fibrotic focus” [55,109,146]. It appears as a radially expanding fibrosclerotic core and consists of loose, dense or hyalinized collagen bundles and a variable number of fibroblasts. Elastic tissue may be abundant. The arrangement of fibroblasts or collagen fibers forming fibrotic foci differs from that of the surrounding tumor stroma, which is more orderly [55]. In solid tumors, necrosis may follow a peritheliomatous pattern, in which a cord or sheath of viable tumor cells surrounds or clings to a centrally disposed blood vessel (Figure 2) [149]. The sheath of viable cells appears to remain as thick as diffusion allows exchange of nutrients, oxygen, and waste products by the crowded cell population (Figure 2). Peritheliomatous necrosis is probably related to the comedo-type necrosis. The term “comedo” is derived from the gross appearance of compressed ducts exuding cheesy necrotic material often seen in comedo ductal carcinoma in situ (DCIS) of the breast (Figure 3). In general, the pattern of comedo-type necrosis is characterized by the presence of well-circumscribed epithelial nests containing central necrotic material [1]. In addition to DCIS of the breast, comedo-type necrosis may be found in the following entities: carcinoma arising in pleomorphic adenoma and duct carcinoma of salivary glands; cervical carcinoma in situ with features of impending invasion; basaloid squamous carcinoma of the lung, salivary glands, esophagus, anal canal and sinonasal tract [1].

Figure 2. Peritheliomatous pattern of necrosis in a gastric carcinoma. Sheath of viable tumor cells surrounds a centrally disposed blood vessel. Ki-67 immunoreactivity in many viable tumor cells.

In ordinary adenocarcinomas, necrotic phenomena may involve both neoplastic glands and intervening stroma. In some cases, they may remain confined within the lumen of neoplastic glands, as seen in patterns defined “dirty necrosis”, “intraglandular necrotic debris” and “crypt lumen apoptosis”. (Figures 4A,B). When not filled with secretory mucin, malignant lumina in histologic sections may either appear empty or contain deeply eosinophilic material that is frequently admixed with necrotic cell debris and neutrophils [67]. This eosinophililic material (dirty necrosis) is strongly positive with periodic acidSchiff and expresses transmembrane glycoprotein MUC1 [67]. Such dirty necrosis has been

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described in colorectal adenocarcinomas [67] and is often accompanied by segmental necrosis of the glandular lining [30,49,155,156]. The presence of a garland pattern with cribriform areas and "dirty" necrosis is the most distinctive features that are helpful in correctly differentiating metastatic colorectal adenocarcinomas from primary endometrioid ovarian carcinoma, with which they are often confused [30,82]. Foci of "dirty necrosis" are also common in pulmonary metastases of colonic carcinomas, whereas are rarely observed in primary lung adenocarcinomas [39]. Well-differentiated and focally mucinous tumors and the absence of dirty necrosis correlate with phenotype of microsatellite unstable colorectal carcinomas [49]. Intraglandular necrotic debris is defined as eosinophilic material with necrotic epithelial fragments within the lumen of a dilated atypical gland (Figure 4A). There is segmental necrosis of the glandular lining characterized by cytoplasmic vacuolization and dark nuclei [147]. Our ultrastructural studies clarified the nature of the cell death, showing the presence of necrosis-like cell death characterized by extensive cytoplasmic vacuolization, loss of microvilli, swelling of nuclear envelope and chromatin condensation in small masses into the nucleus (Figure 4B) [16]. The pattern of intraglandular necrotic debris has been observed in gastric adenomas and adenocarcinomas, and it is similar to that described as dirty necrosis in colorectal adenocarcinomas. There are several intraglandular contents that must be distinguished from intraglandular necrotic debris [147]. These are: 1) intraglandular fibrinous exudate with or without infiltrating neutrophils; 2) intraglandular foam cell aggregation and 3)intraglandular aggregation of red blood cells. Intraglandular fibrinous exudate with or without infiltrating neutrophils is sometimes encountered in biopsy specimens taken from patients with chronic active gastritis or gastric ulcer. The presence of necrotic cells suggest intraglandular necrotic debris rather than any type of the other types of contents described above. Concerning the relationship between intraglandular necrotic debris and differentiation (or grade) of gastric adenocarcinoma, moderately differentiated adenocarcinoma showed the highest incidence of intraglandular necrotic debris compared to well-differentiated and poorly differentiated adenocarcinomas [147]. “Crypt lumen apoptosis” is a pattern of tumor necrosis described in colorectal adenomas and adenocarcinomas by Brodie et al. [8]. Differently from intraglandular necrotic debris and dirty necrosis, it consists of single apoptotic adenoma cells or small clusters of apoptotic cells, not associated with neutrophils, within lumina of neoplastic glands. In conclusion, there is a spectrum of apoptotic-necrotic phenomena in ordinary (non-mucinous) adenocarcinomas of the gastrointestinal tract, ranging from crypt lumen apoptosis (mainly due to apoptosis of adenocarcinoma cells) to dirty necrosis/intraglandular necrotic debris (mainly due to necrosis-like cell death) up to tissue necrosis (due to necrosis) (Table 1). Table 1. Necrotic phenomena in ordinary (non-mucinous) gastrointestinal adenocarcinoma Histopathologic Pattern Crypt lumen apoptosis Dirty necrosis/intraglandular necrotic debris Coagulative necrosis, involving neoplastic glands and intervening stroma

Type of cell death Apoptosis Necrosis-like cell death Necrosis

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Figure 3. High-grade DCIS. The feature peculiar to this variety of carcinoma is the substantial central core of necrotic tumor cells in the affected ducts. Moreover, the tumor itself- within the ducts and surrounded by the basement membrane – is avascular.

A

B

Figure 4. Intraglandular necrotic debris/dirty necrosis. A) Large clusters of dead cells, associated with neutrophils, are contained in atypical glands, lined by epithelium with extensive phenomena of cytoplasmic vacuolization. B) At the electron microscope, necrosis–like cell death is characterized by extensive cytoplasmic vacuolization, loss of microvilli, swelling of nuclear envelope and chromatin condensation in small masses into the nucleus. Note the presence of a neutrophil in the gland lumen.

Pathogenesis of Tumor Necrosis

It is thought that tumor necrosis is caused by chronic ischemia (i.e. hypoxia, low pH, low glucose, high lactate) within tumors, due to vascular collapse, high interstitial pressure and/or rapid tumor growth outstripping its blood supply. Anemia, the most common cancerassociated morbidity, further reduces the blood capacity to transport O2 [133], and it is an adverse prognostic factor for survival, independent of tumor type [15]. Methods to detect hypoxic areas more precisely are only applicable in a research setting. These include the immunohistochemical labelling of pimonidazole, an intravenously injected drug that is reductively activated in hypoxic cells [127], and the immunohistochemical staining of HIF1α [85] and carbonic anhydrase IX [113]. The contiguous or sheet-like nature of the necrosis indicates that the cause of death is likely ischemic injury, affecting a field or group of tumor

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cells, fed or drained by a single vessel [149]. In other words, this confluent necrosis suggests that the large feeding artery or exit vein became obstructed, leading to either an arterial or a venous infarct [149]. It has been postulated that coagulative necrosis within the primary tumor may compromise the tumor vasculature, thereby facilitating the systemic dissemination [126]. A strong relationship between abnormal blood clotting and human malignancy is well established. Cancer patients are highly susceptible to thromboembolic complications, which some have estimated accounts for a significant percentage of the morbidity and mortality of the disease. In particular, patients with tumors may manifest deep vein thrombosis and pulmonary thromboembolism [123]. Not all of the mechanisms for the production of the hypercoagulable state characteristic of cancer are entirely understood [7,120] One of these mechanisms is likely related to microscopic thrombotic vascular occlusion present in some human tumor specimens. Jain et al [66] have presented convincing evidence that intratumoral mechanical stresses, caused by tumor cell proliferation, can create focal large-vessel obstruction, leading to ischemic intratumoral infarcts. This stress, which greatly exceeds blood pressure in tumor vessels, is sufficient to induce the collapse of vascular structures, and, like the intratumoral vascular supply, these intratumoral mechanical stresses are likely to be heterogeneous, both spatially and temporally. This would explain the patchy nature of intratumoral infarct-like necrosis. Moreover, the high intratumoral interstitial pressures, which may approximate intravascular pressures, as well as the increased blood flow resistance, combine to create a permissive environment for vascular stasis and necrosis. In contrast with this hypothesis, recent studies show that tumor necrosis frequently occurs within regions that display relatively increased microvessel density [86]. On the other hand, there are tumors in which coagulative necrosis is rare, although the tumor stage is advanced. For example, the lowest frequency of tumor necrosis is seen in mucinous adenocarcinomas of the gastrointestinal tract [49,147]. Furthermore, primary pulmonary adenocarcinomas rarely demonstrate tumor necrosis unless the tumors are very large or poorly differentiated [39,156]. Instead, necrosis is frequently found in secondary adenocarcinomas of the lung [39].

p53 Mutation and Necrosis in Thyroid Carcinomas

An alternative explanation is that coagulative necrosis in tumor is related to p53 mutation. This relationship is evident in thyroid carcinomas, which are broadly divided into well-differentiated, poorly differentiated and undifferentiated types on the basis of histological and clinical parameters [45]. Well-differentiated thyroid carcinoma includes papillary and follicolar types. Papillary thyroid adenocarcinomas are histologically characterized by well differentiated papillary and follicular structures, distinctive nuclear features, absence of necrosis, and infrequent mitotic figures. Follicular carcinoma is composed of well-differentiated follicular epithelial cells and lacks necrosis. Poorly differentiated carcinomas show loss of structural and functional differentiation. Caratteristically, these lesions show widely infiltrative growth, coagulative necrosis, vascular invasion and numerous mitotic figures. Undifferentiated carcinomas are composed of cells without structural follicular cell differentiation, and usually exhibit extensive areas of

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coagulative necrosis [59]. There are three main morphological patterns: squamoid, spindle cell and pleomorphic giant cells. Gene p53 mutations are restricted to aggressive tumors (1738% of poorly differentiated thyroid carcinomas and 67-88% of undifferentitiated thyroid carcinomas versus 0-9% of well-differentiated thyroid carcinomas) [45,157]. These data suggest that p53 mutation in thyroid tumor cells is associated with coagulative necrosis, whether provoked by the progressive imbalance between growth of the tumor and its supporting blood supply or by more acute vascular events (compression, thrombosis or spasm).

p53 Mutation and Necrosis in Invasive Breast Carcinomas

Tissue necrosis has also been extensively investigated in breast tumors. In invasive carcinoma of the breast tumor, necrosis has been shown to correlate with increased tumor size, high-grade disease, microvessel density, macrophage infiltrates that express vascular endothelial growth factor (VEGF), negative ER status, decreased relapse free survival and a worse prognosis [86,87]. The following data suggest that the presence of apoptosis and/or necrosis is a complex function that is in part related to the histologic type, grade of differentiation, growth pattern of the tumors, p53 status and acute/chronic hypoxia.

3D Cell-Culture Model

In vitro 3D cell-culture models employing the human mammary epithelial cell line MCF10A recapitulate numerous features of the glandular epithelium in vivo [31]. Lumen formation in MCF-10A spheroids is associated with the death of centrally located cells [32]. Furthermore, caspase-3 activation is observed in these dying cells, indicating that classical apoptosis is involved [32]. In addition, overexpression of Bcl-2, which abrogates caspase-3 activation, delays lumen formation [31]. Ultrastructural studies revealed abundant autophagic vacuoles within cells in the lumen of MCA-10A acini, suggesting that autophagy and apoptosis represent two distinct but complementary processes involved in the cell elimination during morphogenesis of lumen formation [100]. A hallmark of poorly differentiated adenocarcinomas is the absence of a hollow lumen. Even within the carcinoma of a single individual, the invasive component is comprised of diverse morphologies that include clusters, cords, sheets, poorly formed glands and isolated single cells. When proliferation is increased within MCF-10A acini, a hollow architecture is maintained by the increased apoptosis of excess cells which occupy the lumens of these structures [31,32]. However, luminal filling does occur when increases in proliferation are combined with the inhibition of apoptosis via the overexpression of anti-apoptotic Bcl2 proteins or expression of oncoproteins with anti-apoptotic activities, including ERBB2, SRC, IGF1R [31].

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DCIS

DCIS of the breast is a neoplastic expansion of ductal lining cells confined by the basement lamina of the breast [1]. Blood vessels are present in the stromal compartment and, therefore, early carcinogenesis occurs in an avascular environment. DCIS will inevitably develop hypoxic regions near the oxygen diffusion limit, as persistent proliferation leads to a thickening of the epithelial layer, pushing cells ever more distant from their blood supply, which remains on the other side of the basement membrane (Figure 3).There are two categories of DCIS: non-comedo and comedo [1]. Pathologists are able to easily distinguish between the two categories of DCIS, because comedo type DCIS tends to plug the center of the breast ducts with necrosis. Necrosis is often seen with microcalcifications. On mammography, the calcifications have a characteristic branched, linear, casting pattern, diagnostic of malignancy. Recent ultrastructural studies indicate that the comedonecrosis in DCIS is due to apoptotic cell death with subsequent secondary necrosis [102]. The level of comedonecrosis is positively correlated with p53, but inversely correlated with Bcl2 in DCIS [62]. These data suggest that disregulation of p53, due to interactome component, may be related to comedo-type necrosis of DCIS.

Luminal Subtype, HER2+/Estrogen Receptor (ER)- , and Basal-like Breast Cancers

Breast cancer is a very common but extremely heterogeneous disease that exhibits a full range of biological behaviour from relatively indolent or innocuous disease to fast–growing, aggressive malignancy with short survival. Despite this range of behavior, most tumors (7580%) are categorized within a single histological diagnostic category of invasive ductal carcinoma of no special type. Recent technological advances have allowed the simultaneous evaluation of multiple RNAs (micro-arrays) or proteins (tissue arrays) in tumour samples or breast cancer cell lines. These studies have revealed that the breast tumours could be sorted into a very few classes characterized by the high level of expression of specific groups of genes/proteins [108]. The number of classes that have been defined in most micro-arraybased or tissue array-based studies is three including luminal subtype, HER2+/(ER)-, and basal-like cancers. About two-thirds of tumours express features characteristic of luminal cells. These lesions are often well differentiated, have a low grade and demonstrate relatively high levels of cytokeratins 8/18/19, ER, PgR, BCL2, CDH1 (E-cadherin) [130]. Basal-like carcinomas constitute about 15% of invasive breast cancer, are characterized by lack of expression of ER, and progesteron receptor and absence of HER2 protein overexpression (the so-called “triple negative” phenotype) and have a poor prognosis. They express relatively high levels of cytokeratins 8/18 and cytokeratin 5/6, vimentin (Figure 5), EGF receptor (EGFR), c-kit , cyclin E, MIB1, MCM2, and other proliferation markers [108]. These tumors are often seen in women with BRCA1 mutations, but also occur among sporadic breast cancers. Morphologic features strongly associated with the basal-like subtype include elevated mitotic rate, coagulative tumor necrosis (74%), pushing margin of invasion and a stromal lymphocytic response [42]. Some tumors (22%) show regions of ribbon-like

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architecture with associated tumor necrosis [93]. Breast carcinomas described prior to the era of the gene expression profiling studies as poorly differentiated invasive ductal carcinomas with central acellular zones or with fibrotic foci most likely belong in basal-like group [55,68]. The presence of necrosis could potentially be used to alert pathologists to the diagnosis of basal or HER2+/ER- tumors and guide them to consider ancillary investigation with immunohistochemistry for basal markers [93]. It appears that p53 mutation is much more frequent in the ‘basal/myoepithelial-like’ and ERBB2 classes than in the ‘luminal-like’ one: 82%, 71%, and 31% respectively, according to Sorlie [129]. Coagulative necrosis is not found in luminal carcinomas, but is frequently seen in HER2+ER- and basal-like tumors [42]. Thus, there is a relationship between p53 mutation and presence of coagulative necrosis in breast carcinomas.

Figure 5. Basal-like carcinoma of the breast. Strong immunoreactivity for vimentin in carcinoma cells adjacent to coagulative necrosis.

Micropapillary Carcinomas

Invasive micropapillary carcinoma, originally described as a distinctive type of invasive carcinoma in the breast, is being increasingly recognized as a separate entity in many other organs. Histologically, it is defined by predominant or focal papillary cell clusters devoid of a fibrovascular core, surrounded by empty lacunar spaces [105] (Figure 6). In the organs in which it is best characterized, invasive micropapillary carcinoma has been shown to have a highly infiltrative nature, often presenting at advanced stages, and showing high degrees of lymphotrophism. It is characterized by an abnormality in cell polarity that occurs in a fashion that is not seen in conventional carcinoma. In fact, in invasive micropapillary carcinoma the surface of the tumor cells that faces the stroma acquires apical secretory properties. This was evidenced earlier by electron microscopy and by immunohistochemical staining for MUC1 [105].

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Mucinous Carcinomas

Mucinous carcinoma of the breast is a rare histologic type characterized by production of abundant extracellular mucin [1]. Similar histologic types have been described in the gastrointestinal tract, pancreas, lung, prostate, ovary, and uterus [1]. Extracellular mucin accumulation may be due to inversion of polarity in cell, with mucin secretion directed towards base of cell/stroma, rather than luminal border. A major molecular difference between invasive micropapillary carcinoma (a tumor with a high propensity for early dissemination) and mucinous carcinoma (a tumor with a much better prognosis than conventional carcinoma), however, is the positivity of the latter for MUC2, the gel-forming intestinal mucin, and the total lack of MUC2 expression in invasive micropapillary carcinoma. It has been postulated that MUC2 plays a major role in the indolent behavior of mucinous carcinomas mainly by 'containing' the tumor cells and acting as a physical barrier against their spread. Therefore, Nassar et al [105] speculate that the reverse polarization in invasive micropapillary carcinoma coupled with the absence of the gel-forming mucin MUC2 facilitates the secretion towards the stroma by the tumor cells of products responsible for stromal and vascular invasion, namely metalloproteinases permitting 'easier' dissemination of the neoplastic cell clusters. This may explain the fact that the morphology of the tumor is retained in lymphovascular spaces as well as in lymph nodes and distant metastatic sites. It could also explain why tumors with this particular morphology, even when small in size, display a higher propensity for lymph node metastases. Interestingly, intraglandular necrotic debris and other forms of tumor necrosis are rarely found in mucinous adenocarcinomas which are characterized by absence of p53 mutation [78,147].

Figure 6. Micropapapillary carcinoma of the stomach. Numerous apoptotic neutrophils with compaction of chromatin are present within large, confluent cytoplasmic vacuoles of tumor cells. Immunohistochemical reactivity for caspase-3 in some neutrophils observed in large cytoplasmic vacuoles of tumor cell.

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Tumor Cells can die in Several Ways

In addition to apoptosis and necrosis, various models of cell death have been proposed, including autophagy, paraptosis, mitotic catastrophe, and the descriptive model of apoptosislike and necrosis-like cell death. According to the recommendations of Nomenclature Committee on Cell Death, the definition of cell death must be based on precise terms of the parameters that describe the presumed cell death pathway involved [75]. Electron microscopy is the “gold standard” for detection of these types of cell death. It is now agreed upon that a rational combination of at least two techniques should be utilized, one to visualize morphological changes and the second to determine biochemical changes, when the modes of cell death are asserted. Since the precise characterization of biochemical checkpoints controlling caspase-independent programmed cell death is still awaiting, Leist and Jaattela [88] proposed a descriptive model, which classifies cell death into four subclasses, according to their nuclear morphology. Classical apoptosis is characterized by chromatin condensed to compact and almost geometric figures. Apoptosis-like programmed cell death is defined by less compact, lumpy chromatin masses. In contrast, no chromatin condensation, but sometimes chromatin clustering to loose speckles and dissolution of nucleoli is characteristic of necrosis-like programmed cell death, whereas necrosis is characterized by cytoplasmic swelling and cell membrane rupture.

Paraptosis

Paraptosis has recently been characterized by cytoplasmic vacuolization that begins with progressive swelling of mitochondria and the endoplasmic reticulum [131,132]. In some cases, cytoplasmic vacuoles are so large that the cytoplasm seems dramatically reduced and the nucleus is deformed, and occupies a peripheral position. The plasma membrane, however, remains well preserved [132,145]. These vacuoles are distinct from autophagic vacuoles, because they are devoid of intracellular content [13,21,125]. According to morphologic classification of Leist and Jaattela [88], paraptosis is considered as a particular form of necrosis-like cell death.

Figure 7. Mitotic catastrophe. Gastric carcinoma cell with multiple micronuclei showing uncondensed chromatin.

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Mitotic Catastrophe

There is still no broadly accepted definition of the term “mitotic catastrophe”, presumably because the major processes involved have not been described in molecular and genetic terms. For example, some researchers have argued that mitotic catastrophe is fundamentally different from apoptosis, because overexpression of anti-apoptotic genes including Bcl-2 and MDR1 can actually enhance the frequency of catastrophic mitosis [121]. In contrast, a recent study showed that mitotic catastrophe induced by DNA damage is dependent on caspase activation, suggesting that it constitutes a special case of apoptosis [20]. Moreover, it should be pointed that mitotic catastrophe is not considered a form of death, but rather an irreversible trigger for cell death [75]. Almost all tumors are completely or partially deficient in a least some cell cycle checkpoint, including G1 checkpoints (which are regulated primarily by p53), G2 checkpoint, prophase checkpoint and mitotic spindle checkpoint [27,53]. The G2 checkpoint is important for preventing mitotic catastrophe in cells treated with DNA-damaging agents [27,65]. This checkpoint includes p53-independent and p53-dependent mechanisms, with p53 playing a critical role in the maintenance of G2 checkpoint arrest [89]. The G2 checkpoint of the cell cycle is responsible for blocking mitosis when a cell has sustained an insult to its DNA. DNA damage activates a number of molecules that promote cellular activities such as cell-cycle arrest, DNA repair or apoptosis, if the damage cannot be repaired [35,65]. However, if the G2 checkpoint is defective, a cell can enter mitosis prematurely, before DNA replication is complete, or DNA damage has been repaired [63,64]. This aberrant mitosis causes the cell to undergo death by mitotic catastrophe. The primary function of survivin seems to be the regulation of mitotic progression [122]. It is likely that loss of survivin coupled with p53 inactivation might have cumulative devastating effects on mitosis [111]. Loss of survivin induces cell-cycle arrest and cell death by mitotic catastrophe. From a morphological viewpoint, mitotic catastrophe results from aberrant mitosis and is characterized by supernumerary centrosomes, failure of cytokinesis and a significant increase in the percentage of abnormal nuclei, the appearance of which includes multiple multilobated nuclei, micronuclei and abnormally large-size (giant) nuclei (Figure 7) [121]. Micronucleated cells are nonviable and arise through the formation of nuclear envelopes around clusters of chromosomes or chromosome fragments during catastrophe mitosis [121]. They can be easily distinguished from apoptotic cells by their morphology (Figure 7). Apoptotic cells are characterized by shrunken cytoplasm and condensed chromatin, whereas cells that underwent mitotic catastrophe are large and contain uncondensed chromatin (Figure 7) [121]. Cells that die through mitotic catastrophe usually do not show DNA breaks that are detectable by TUNEL staining in apoptotic cells [121]. At present, detection methods of mitotic catastrophe include light and electron microscopy, as well as assays for mitotic markers (survivin, MPM2) [110]. Mitotic catastrophe has been characterized as the main form of cell death induced by ionizing radiation and occurs in response to several anticancer drugs [96,97,103,135]. Because preoperative chemotherapy is being used more frequently in the management of advanced tumors, pathologists should be aware of the resultant morphological effects, which may result in difficulties in tumor typing and grading and in the identification of residual neoplasia [95]. The morphologic features of lung, breast and ovarian cancer treated with chemotherapy include both nuclear and

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cytoplasmic alterations and pronounced stromal changes [13,61]. Nuclei show extreme enlargement with very irregular outlines, and, sometimes, are similar to multinucleated giant cell [95]: a feature compatible with mitotic catastrophe. Nuclear size has been shown to be a useful prognostic indicator in ovarian and breast cancer, and therefore an increased nuclear size post-chemotherapy may influence the results if this measurement is used as a predictor of outcome [95]. Post-chemotherapy tumor cells are usually arranged singly or in small groups, often without glandular formation, and mitotic figures are inconspicuous [95]. This means that tumor grading, which has important prognostic implications and which depends on the assessment of both cytological and architectural features, including mitotic activity, is not reliable after chemotherapy. Autophagic Cell Death

Autophagy is a cellular degradation process responsible for the turnover of unnecessary or dysfunctional organelles and cytoplasmic proteins, and has been suggested to be an essential function for cell homeostasis and adaptation to an adverse environment [153]. It is typically activated by starvation, when the cytoplasmic proteins or organelles are delivered to the lysosome and degraded [153]. In autophagy, cytoplasmic proteins or dysfunctional organelles are sequestrated in a double-membrane-bound vesicle, termed autophagosome, delivered to the lysosome by fusion, and then degraded [90]. The autophagosome is a double membrane structure containing indigested cytoplasmic material including organelles, while the autolysosome is a single membrane structure containing cytoplasmic components at various stages of degradation [101]. Since clear differentiation between autophagosomes and autolysosomes is sometimes difficult, these structures are often generalized as “autophagic vacuoles” [101]. Autophagy is a process commonly induced by hypoxia and representing the 'ultimate' nutritional source for tumor cells to survive low-nutrient conditions.An in vitro model demonstrated that the onset of the mitochondrial permeability transition within a cell leads to mitochondrial degradation through autophagy [33]. According to this model, elimination of damaged mitochondria by autophagy would prevent the release of proapoptotic substances from mitochondria, thus preventing apoptosis. These data suggest that autophagy may be involved in the protection against apoptosis in the setting of chronic ischemia [153]. Autophagy triggered by ischemia could be a homeostatic mechanism, by which apoptosis is inhibited and the deleterious effects of chronic ischemia are limited. Other recent reports indicate that dysregulation of autophagy can result in pathological states such as Parkinson's, Huntington's, and Alzheimer's diseases, myocardial hypertrophy, cardiomyopathies, and ischemic heart disease [128,137]. Formation of autophagosomes and degradation of the bulk of cytoplasm are also observed in cells undergoing cell death. Thus, programmed cell death could be another general function of autophagy, namely the inducible mechanism for massive degradation of cytoplasmic components leading to caspaseindependent cell death. This type of cell death is designated type II programmed cell death or autophagic cell death, in contrast to apoptosis, which is referred to type I programmed cell death [12]. According to Leist and Jaattela [88] autophagic cell death is considered as a particular variant of necrosis-like cell death. However, the mechanism of the autophagic cell death program is unclear. To date, the most convincing and standard method to detect

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autophagy is to examine the ultrastructure of the cells by transmission electron microscopy [101]. A review of the literature revealed only few reports showing in vivo morphologic features of autophagic cell death in human tumors [2,16]. The precise role of autophagy in cancer development, progression and response to therapy is not well understood. Autophagy could be in advanced stages of cancer to guarantee survival of cancer cells under extreme conditions, such as the restricted access of cells located in the inner areas of solid tumors to nutrients [26]. Beclin 1 – a mammalian gene capable of inducing autophagy- was found to be monoallelically deleted in a high percentage of ovarian, breast, and prostate cancer [92]. Furthermore, animals with reduced levels of Beclin 1 display a pronounced increase in epithelial and hematopoietic malignancies [117]. Bcl-2 promotes the survival of cancer cells by inhibiting apoptosis. In addition to antiapoptotic function, Bcl-2 prevents also autophagic cell death [114]. Binding of the Bcl-2 protein to the protein product of beclin-1 inhibits autophagy. If Bcl-2 activity is excessive, as it is in some cancers, the consequent suppression of autophagy could allow damaged cells to complete a cancerous transformation [114]. In other words, as autophagy is a form of cell death, reduced autophagic activity might simply promote the survival of tumor cells [76]. Therefore, like apoptosis, autophagic cell death may be found to be suppressed in malignant tumors.

Immunogenic Versus Non-immunogenic Cell Death

It seems that clearance of apoptotic cells operate differently from that of necrotic cells. Whereas apoptotic cells (which shrink) are engulfed completely, necrotic cells are phagocytosed only after loss of membrane integrity by a macropinocytotic mechanism involving formation of multiple ruffles directed towards necrotic debris [77]. This means that uptake is delayed and less efficient. The late uptake of necrotic cells allows the dying cells to activate pro-inflammatory and immuno-stimulatory responses [160], whereas apoptotic cell death is immunologically and inflammatorily silent. This difference was thought to result from an intrinsic capacity of cells dying from non-apoptotic cell death to stimulate the immune response: for example, by stimulating local inflammatory responses and/or by triggering the maturation of dendritic cells. Spillage of the contents of necrotic cells into the surrounding tissue activates inflammatory signalling pathways. Depending on molecular signals from necrotic cells, diverse types of immune cells (neutrophils, macrophages, dendritic cells) become involved in the immune response. In contrast, apoptotic cells induce antigen presenting cells (APCs) to secrete cytokines that inhibit Th1 responses. Immature dendritic cells efficiently phagocytose a variety of apoptotic and necrotic tumour cells, but only the latter induce maturation and optimal presentation of tumor antigens [160]. In vivo, apoptotic cells, which fail to be taken up by phagocytes, undergo secondary necrosis with its attendant risk of pro-inflammatory consequences [29] .

Phagocytosis of Apoptotic Cells

Removal of apoptotic cells by neighboring viable cells or professional phagocytes is

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required to prevent tissue injury [58]. Macrophages are known to remove dying cells and release anti-inflammatory mediators in response; however, many cells traditionally thought of as poor phagocytes can mediate this function as well [112]. These include epithelial cells, mesangial cells, vascular smooth muscle cells, hepatic cells and endothelial cells [112]. For many years the significance of the removal of apoptotic cells has been underestimated. New data indicate that phagocytic clearance of cells dying by apoptosis is much more than mere waste disposal.

Autophagy and Xeno-Cannibalism

Malorni et al. [99] showed that cells that are able to exert intense autophagic activity are also able to engulf and digest entire sibling cells. This phenomenon represents a sort of cannibalism. Recently, cells with cannibalistic behavior have been detected in tumors of different histology and their presence was related to a poor prognosis. Tumor cell cannibalism may involve engulfment of neutrophils and erythrocytes, implying that cannibal tumor cells do not distinguish or select between normal (including stromal or tumorinfiltrating immune cells) and sibling neoplastic cells. Malorni et al [99] suggest the hypothesis that these two phenomena, autophagy and cannibalism, could be related, the latter being an exacerbation of the first and providing a further survival option to the cells. The phenomenon of neutrophil-tumor cell emperipolesis or phagocytosis (cannibalism) has been documented by the light microscopy in pleomorphic (giant) cell carcinomas of the lung, gall bladder, pancreas, and intestine [4,19,38,50,51,140]. Pleomorphic (giant) cell carcinoma is defined as a tumor lacking any identifiable glandular, squamous or other differentiation [140]. The characteristic histologic findings include marked pleomorphism, lack of cohesiveness of tumor cells, aggregates or sheets of mononucleated and multinucleated giant cells, and extensive necrosis. A particular finding reported by Mosnier et al. [104] in a case of pleomorphic giant cell carcinoma of the esophagus was the strong expression of CD68 by nearly all the tumor cells. This was also associated with the phenomenon neutrophil-tumor cell phagocytosis. CD68 - a glycoprotein expressed by granulocytes, monocytes and macrophages -. may have a role in lysosomal function. Therefore, expression of CD68 might be related to the phenomenon of neutrophil-tumor cell emperipolesis or phagocytosis (cannibalism). Expression of CD68 by tumor cells has also been observed in squamous cell carcinoma and melanomas associated with erythrophagocytotic phenomenon. One peculiar finding in invasive micropapillary carcinoma of ampullo-pancreatobiliary region noted by Khayyata et al [72] is the presence of tumor-infiltrating neutrophils. Neutrophils were abundant and could be identified both within the carcinoma cells ('cannibalism') as well as in the stroma adjacent to the tumor cells. Furthermore, the neutrophils showed striking tumoralcentric distribution, decreasing in numbers away from the tumor cells. We also observed a case of gastric micropapillary carcinoma characterized by neutrophil-tumor cell cannibalism (Figure 6). Thus, the phenomenon of neutrophil-tumor cell emperipolesis or phagocytosis (cannibalism) has been reported in pleomorphic giant cell carcinoma and in micropapillary carcinomas: two histologic types characterized by a poor prognosis. Recently, Lugini et al [94] investigated the occurrence, the in vivo relevance, and the underlying mechanisms of

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cannibalism in human melanoma. As first evidence, they observed that tumor cannibalism was clearly detectable in vivo in metastatic lesions of melanoma and often involved T cells, which could be found in a degraded state within tumor cells. Then, in vitro experiments confirmed that cannibalism of T cells was a property of metastatic melanoma cells but not of primary melanoma cells. In particular, morphologic analyses, including time-lapse cinematography and electron microscopy, revealed a sequence of events, in which metastatic melanoma cells were able to engulf and digest live autologous melanoma-specific CD8(+) T cells. Importantly, this cannibalistic activity significantly increased metastatic melanoma cell survival, particularly under starvation condition, supporting the evidence that tumor cells may eat live lymphocytes as a way to "feed" in condition of low nutrient supply. The mechanism underlying cannibalism involved a complex framework, including lysosomal protease cathepsin B activity, caveolae formation, and ezrin cytoskeleton integrity and function. In conclusion, Lugini et al. [94] suggest that cannibalism may represent a sort of "feeding" activity aimed at sustaining survival and progression of malignant tumor cells in an unfavorable microenvironment. It has been generally considered that DNA from dying cells is degraded after apoptosis and, thus, inactivated. However, recent studies have demonstrated that horizontal DNA transfer between mammalian cells can occur through the uptake of apoptotic bodies, where genes from the apoptotic cells were transferred to neighbouring cells phagocytosing the apoptotic bodies [6,60,152]. The regulation of this process is poorly understood. p53, via the activation of p21, blocks normal cells from replicating transferred DNA from engulfed apoptotic bodies [5]. This may be one protection level against the propagation of potentially pathological DNA. It was shown that the ability of cells as recipients of horizontally transferred DNA was enhanced by deficiency of p53 or p21. These findings indicate that horizontal transfer of DNA from apoptotic bodies could be one explanation to the chromosomal instability observed in cancer cells [5].

Our Experience Looking at the problem from this new prospective, so different from the traditional one, we have tried to evaluate the gastric neoplastic lesions on file in the Department of Human Pathology of the University of Messina (Italy) with particular reference to the phenomenon of autophagic cell death (self-cannibalism) and neutrophil-tumor cell xeno-cannibalism in gastric carcinomas. In our Department, gastric tumours are routinely processed for both light and electron microscopic observations [16-18]. Briefly, the fragments of fresh tumor tissue were divided into two portions with a sharp razor blade. The first member of the pair was processed for routine paraffin-embedding together with additional tissue samples taken from the tumour as well as from the surgical borders of the specimens. These sections were stained with hematoxylin-eosin. The second piece of the paired samples was minced into smaller pieces and destined for electron microscopy. This material was fixed in 3% glutaraldeyde in phosphate-buffered solution, postfixed in 1% osmium tetroxide, and subsequently dehydrated in graded ethanol and embedded in Araldite. Semi-thin Giemsa-stained sections were reviewed. Ultrathin sections were stained with uranyl acetate and lead citrate and examined under an electron microscope (Zeiss EM 109).

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We recently described the ultrastructural features of autophagic cell death in one case of gastric adenocarcinoma [16]. At light microscopy, tumor celle were arranged in solid sheets and showed hepatoid differentiation. At the electron microscopy, the neoplastic cells were arranged in tightly packed clusters, with little intercellular space. Nuclei had irregular contour and showed moderated to marked chromatin clustering. These nuclear alterations were associated with the occurrence of large autophagic vacuoles (figure 8). There were also reticulum-bound lipidic vacuoles which appear as pale gray inclusions in the cytoplasm. Chromatin margination at the nuclear membrane as well as apoptotic bodies were not seen. Electron microscopy disclosed nuclear chromatin condensation in tumor cells suggestive of necrosis-like cell death. In contrast to classical apoptosis, the chromatin is not condensed into compact and almost geometric figures under the nuclear membrane, but small patches of condensed chromatin are spread throughout the nucleus [16]. The nuclear and cytoplasmic changes do not seem to fit typical description of classical necrosis that is characterized by cytoplasmic swelling and cell membrane rupture. These nuclear changes were often found in association with autophagy including endoplasmic reticulum-bound lipidic vacuoles, lipofuscin granules and autophagic vacuoles. In conclusion, the constellation of ultrastructural findings such as cell shrinkage, autophagic vacuoles, reticulum-bound lipidic vacuoles, lipofuscin granules, and moderate to marked chromatin clustering are compatible with autophagic cell death in tumor cells.

Figure 8. Large autophagic vacuole is noted in the paranuclear cytoplasmic area of a gastric adenocarcinoma cell. The rest of the cytoplasm contains a few organelles.

Emperipolesis has been defined as the random passage of different types of cells through the cytoplasm of another cell without any important effects on either the host or invading cells [44]. It differs from phagocytosis (cannibalism) as an engulfed cell only temporarily exists within another cell. From a morphological viewpoint, phagocytosis (cannibalism) is

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suggested by the presence of degenerate or necrotic cell within a vacuole of host cell. In order to study the phenomenon of neutrophil-tumor cell emperipolesis or phagocytosis (cannibalism) in gastric tumor, the ultrastructural findings observed in 9 cases of advanced gastric carcinomas were reviewed and analyzed. An interepithelial localization of neutrophils was seen in 2 out of 9 cases. They showed the characteristic equipment of discrete primary and secondary granules, glycogen and lipid bodies. Other neutrophils were present within vacuoles of adenocarcinoma cells and showed various phases of apoptotic changes. Ultrastructural signs of early apoptosis included nuclear chromatin separation into dense and electron lucent areas, rounded nuclear profiles, preservation of cytoplasmic granules, and maintenance of cell membrane integrity (Figure 9). Late apoptotic morphology was characterized by cell shrinkage, tightly packed cytoplasmic granules, and uniform collapsed nucleus [23,56]. Secondary degeneration of apoptotic neutrophils within the phagocytic vacuoles of tumor cells included cellular swelling, electron-lucent cytoplasm, vacuolization and indiscernible cell membrane. To the author’s knowledge, there are no ultrastructural studies of neutrophil phagocytosis (cannibalism) in human carcinomas. Ultrastructural findings compatible with neutrophil cannibalism were described in experimental tumors by Constantinides et al [24]. Thus, this study provide morphologic details on apoptotic neutrophils phagocytosed by adenocarcinoma cells. In summary, the ultrastructural features described above characterize the primary process of neutrophil death in 2 out of 9 cases of gastric cancer as apoptosis. Therefore, the phagocytic nature (cannibalism) of this interaction was demonstrated, and the possibility of emperipolesis was excluded.

Figure 9. Neutrophil-tumor cell cannibalism in a gastric adenocarcinoma. The phagocytized neutrophil shows early apoptotic changes, including condensed nuclear chromatin, rounded nuclear profiles, glycogen granules depletion, and preservation of the cytoplasmic granules.

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Conclusion Most of the studies that have been carried out so far have concentrated on the dichotomy between apoptosis and necrosis. Recent research suggests, however, that apoptosis and necrosis represent only the extreme ends of a wide range of possible morphological and biochemical deaths. Electron microscopy is the “gold standard” for detection of different cell death types, including apoptosis, mitotic catastrophe, auophagic cell death, and necrosis. It is, however, agreed upon that a combination of at least two techniques should be utilized, one to visualize morphological changes and the second to determine biochemical changes, when the modes of cell death are asserted. It has recently suggested that the development of an invasive cancer involves a progressive switch from predominantly apoptotic to necrotic cell death. However, the presence of tumor necrosis is a complex function that is in part related to the histologic type, grade of differentiation, growth pattern of the tumors, p53 mutation and acute/chronic hypoxia. Mitotic catastrophe has been characterized as the main form of cell death induced by ionizing radiation and occurs in response to several anticancer drugs. Because preoperative chemotherapy is being used more frequently in the management of advanced tumors, pathologists should be aware of the resultant morphological effects, (i.e. the presence of multinucleated giant cells due to mitotic catastrophe) which may result in difficulties in tumor typing and grading. Based on recent literature and our own data, autophagy (self-cannibalism) and xeno-cannibalism provide a further survival option to the tumor cells in an unfavorable microenvironment characterized by low nutrient supply. Where horizontal DNA transfer from apoptotic cells to neoplastic cells, phagocytosing the apoptotic bodies, determine their chromosomal instability, will require further exploration.

Acknowledgments The Author wishes to acknowledge the useful discussion/comments by members of his laboratory and his collaborators. The pletora of literature related to tumor necrosis makes a complete and extensive review extremely challenging and I apologizes in advance for any inadvertent omission. I thank my wife Luciana and my son Valerio for their enthusiastic support of this study.

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In: Tumor Necrosis Factor Editor: Toma P. Rossard

ISBN: 978-1-60741-708-8 © 2009 Nova Science Publishers, Inc.

Chapter XI

Tumor Necrosis Factor and Carcinoma by Hepatitis B and C Virus Infection* Kazuya Shirato1 and Tetsuya Mizutani2† 1

Laboratory of Acute Respiratory Viral Diseases and Cytokines, Department of Virology III, 2 Laboratory of Special Pathogens, Department of Virology I, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashimurayama, Tokyo 208-0011 Japan

Abstract Hepatitis B virus (HBV) and hepatitis C virus (HCV) are global public health problems. The clinical courses of both HBV and HCV infection vary from acute hepatitis to chronic persistent infection that may progress to cirrhosis and carcinoma. Acute inflammation is a defense response and chronic inflammation can lead to cancer. Several proinflammatory gene products have been identified in both HBV- and HCV-infected patients. The expression of these genes is mainly regulated by the transcription factor NF-κB, which is constitutively active in most tumors and is induced by tumor promoters and carcinogenic viral proteins. Anti-inflammatory agents that suppress NF-κB may be useful in both the prevention and treatment of cancer. Tumor necrosis factor alpha (TNFα) is thought to be an important factor underlying the mechanisms of action of these types of viral hepatitis and carcinoma because TNFα plays an important role in the host immune response to HBV and HCV infection. Recent studies indicated that TNFα promoter polymorphisms are significantly associated with viral clearance. Neutralization of TNFα(anti-TNF therapy) has been shown to be associated with activation of HBV infection, but not HCV infection. However, TNF has been reported to induce clearance of HBV. One of the inhibitory mechanisms of anti-TNF therapy is that TNF blocks HBV replication by promoting destabilization of viral nucleocapsids. HCV proteins are able to trigger production of TNFα and modulate nuclear factor *

A version of this chapter was also published in Oncogene Proteins: New Research Gastric, edited by Artur H. Malloy and Earl C. Carson, Nova Science Publishers. It was submitted for appropriate modifications in an effort to encourage wider dissemination of research † Corresponding author. Telephone +81-42-561-0771, Facsimile +81-42-565-3315, Email [email protected]

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kappa B activation and apoptosis stimulated by this cytokine. This review highlights the importance of TNFα production for hepatitis and carcinoma by HBV and HCV infection.

Abbreviations CAH: HBV: HCC: HCV: IFN: IL: MHV: NF-κB: NS: PBMC: pDC: TNFα:

Chronic active hepatitis Human hepatitis B virus Hepatocellular carcinoma Human hepatitis C virus Interferon Interleukin Mouse hepatitis virus Nuclear factor kappa B Nonstructural protein Peripheral blood mononuclear cell Plasmacytoid dendritic cells Tumor necrosis factor alpha

Introduction Hepatitis B virus (HBV) and hepatitis C virus (HCV) are global public health problems. The clinical courses of both HBV and HCV infection vary from acute hepatitis to chronic persistent infection that may progress to cirrhosis and carcinoma. Several inflammatory mediators are up-regulated during virus-induced hepatic diseases and these proinflammatory cytokines are regulated by the nuclear factor kappa B (NF-κB) pathway. Among these proinflammatory cytokines, tumor necrosis factor alpha (TNFα) is thought to be important for viral hepatitis progression or remission. TNFα is a multifunctional cytokine, mainly produced by activated monocytes and macrophages, which is closely related to the development and regulation of the immune system and host defense. In addition, it was reported that TNFα plays an important role in acute and chronic liver injury induced by various stimuli, such as severe alcoholic hepatitis and hepatotoxic drugs. This chapter presents an overview of the importance of TNFα expression during HBVand HCV-induced hepatic injury.

Hepatitis B and C Viruses 1. Hepatitis B Virus

Human hepatitis B virus (HBV) is a member of the family Hepadnaviridae and has a circular DNA consisting of a longer complete negative-sense strand, with a piece missing at a nick site, and an incomplete positive-sense strand. HBV causes acute and chronic hepatocyte

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injury and is associated with liver cancer (Beasley et al., 1981). Although geographic correlations have been reported, chronic infection by HBV is a major risk factor for hepatocellular carcinoma (HCC) (Block et al., 2003) and occult HBV infection, which is characterized by HBV DNA persistence in HBV surface antigen-negative patients, and increased risk of HCC (Squadrito et al., 2006). HBV vaccine consisting of purified HBV s antigen is now commercially available, and the incidence rates of chronic HBV infection have begun to decrease (Kane, 1995; Liu and Kao, 2007).

2. Hepatitis C Virus

Human hepatitis C virus (HCV) belongs to the family Flaviviridae and causes acute, and in many cases chronic, hepatitis. In 1989, the cloning of part of the genome of non-A and non-B hepatitis virus was first reported and this virus was HCV, a major cause of non-A and non-B hepatitis (Choo et al., 1989; Kuo et al., 1989). It was reported that the core (C) protein of HCV can transform HCV-infected cells (Kato, 2001; Ray and Ray, 2001) and chronic HCV infection appears to be a major factor involved in chronic hepatitis, liver cirrhosis, and hepatic cancer (Erhardt et al., 2002; Kato, 2001). About 150 to 200 million people worldwide are infected with HCV. The prevalence rate of HCV is generally less than 3% in developed nations. It has been reported that the major reason for the spread of HCV in Japan and Italy is medical treatment, while that in the USA is drug abuse (Guadagnino et al., 1997; Kelen et al., 1992; Noguchi et al., 1997). Several commercial therapeutic agents based on IFNα are available and IFNα treatment in combination with ribavirin has been shown to reduce the HCV genome copy number, although the effect is dependent on the HCV genotype and various side effects have been reported (Fried et al., 2002; Manns et al., 2001; Manns, Wedemeyer, and Cornberg, 2006; McHutchison et al., 1998; Poynard et al., 1998).

Cytokine Induction During Hepatic Inflammation Various inflammatory mediators, such as cytokines and chemokines, are secreted during hepatic inflammation and are correlated with disease progression or host protection. In the murine model of hepatic inflammation induced by concanavalin A, Dgalactosamine/lipopolysaccharide injection, TNFα, IFNγ, IL-2, IL-4, IL-10, and IL-12 are highly up-regulated and it has been shown that TNFα induces liver injury while IL-10 has a protective role (Sass et al., 2002) . Mouse hepatitis virus (MHV) is a positive-strand RNA virus that causes a variety of clinical diseases, such as encephalomyelitis, respiratory diseases, and hepatitis in susceptible mice (De Albuquerque et al., 2006; Lai and Cavanagh, 1997; Levy, Leibowitz, and Edgington, 1981; Li et al., 1992). The MHV3 infection model is a commonly used murine model of virus-induced hepatitis. In MHV-3-induced fulminant hepatitis, it was reported that TNFα, IL-1, and TGFβ were up-regulated in susceptible mouse strains but not in resistant mice, and TNFα is considered one of the pathogenic determinants (Ning et al., 1998; Pope et al., 1995).

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Serum levels of proinflammatory cytokines, such as IL-1β, IL-6, and TNFα have been reported to be up-regulated in patients with hepatitis A-, B-, and C-induced hepatitis (Torre et al., 1994). In HBV-induced chronic active hepatitis (CAH), high levels of TNFα production were seen in plasma, and the TNFα level was reduced in advanced cirrhosis (Kiki et al., 2006). Barnaba et al. also reported that cytotoxic T cells isolated from HBV-induced CAH patients secreted TNFα and IL-2 (Barnaba et al., 1994). In hepatitis C virus infection, it was reported that IFNα levels were diminished by reducing the number of plasmacytoid dendritic cells (pDC) via the monocyte-derived TNFα and IL-10, which were induced by HCV core antigen stimulation, and neutralization of TNFα and IL-10 prevented HCV core-induced inhibition of IFNα production (Dolganiuc et al., 2006). In addition, peripheral blood mononuclear cells (PBMC) from patients with chronic HCV infection cultured with HCV core antigen were shown to secrete IFNγ and IL-10 (Rigopoulou et al., 2007). As described above, TNFα is considered a key mediator of liver injury.

Relations between TNFα and Cancer and Liver Diseases 1. TNFα and Cancer

Inflammation is an important host reaction to various stimuli, such as injury or infection; however, inflammation may be either protective or immunopathological for the host. Cytokines are bioactive molecules that have signaling activities, and many cytokines are secreted during acute and chronic inflammation and build complicated networks. Chronic inflammation is a risk factor for various diseases, such as cardiovascular diseases, diabetes, arthritis, Alzheimer’s disease, pulmonary diseases, autoimmune diseases, and cancer (Aggarwal, 2004; Aggarwal et al., 2006). Some proinflammatory cytokines, such as TNFα, IL-1β, and IL-6, are associated with these inflammatory diseases, and these proinflammatory cytokines are regulated by the transcription factor NF-κB. Among the proinflammatory cytokines, it was reported that TNFα induced cellular transformation, proliferation, and tumor promotion (Balkwill and Coussens, 2004; Balkwill and Mantovani, 2001; Mantovani, 2005). TNFα is a multifunctional cytokine, mainly produced by activated monocytes and macrophages, which is intricately involved in the development and regulation of the immune system and host defense (Tracey and Cerami, 1993). TNFα was first identified as an antitumor cytokine (Aggarwal, 2003) and has cytolytic effects on tumor cells (Sugarman et al., 1985) and blocks the growth of cancer cells (Shen et al., 2002). On the other hand, TNFα is also secreted by various tumor cells (Aggarwal et al., 2006), and TNFα can promote cancer progression (Balkwill, 2006; Mocellin et al., 2005). In addition, TNFα can contribute to various steps of tumorigenesis, such as cellular transformation, promotion, proliferation, invasion, angiogenesis, and metastasis (Aggarwal et al., 2006).

2. TNFα and Liver Diseases

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TNFα is secreted at the early stages of many types of liver injury and TNFα induces other cytokines production and hepatic curing responses. TNFα promotes hepatocellular proliferation rather than cell death (Tilg and Diehl, 2000), and hepatectomy liver regeneration is induced through TNF signaling (Akerman et al., 1992). However, it was reported that TNFα plays a detrimental role in acute and chronic liver injury induced by severe alcoholic hepatitis, human hepatitis C virus infection, and hepatotoxic drugs (Kusumoto et al., 2006; Muto et al., 1988; Rothe et al., 1993; Tilg, Kaser, and Moschen, 2006; Winwood and Arthur, 1993). Plasma levels of TNFα and soluble TNF receptor are correlated with liver disease progression, and neutralization of TNFα successfully prevents liver injury in alcohol-fed rats (Iimuro et al., 1997). In addition, TNF receptor p55-deficient mice (TNFRp55-/-) resisted lipopolysaccharide-, acetaminophen-, or dimethylnitrosamine-induced liver injury (Ishida et al., 2004; Kitamura et al., 2002; Tsuji et al., 1997). On the other hand, TNFRp55-/- mice exhibited high susceptibility to Listeria monocytogenes infection (Pfeffer et al., 1993; Rothe et al., 1993), and Puro et al. reported that TNF blocked hepatitis B virus replication by promoting destabilization of viral nucleocapsids (Puro and Schneider, 2007). These reports suggest that TNFα plays an important role in liver injury or protection. Apoptosis is thought to be one of the processes involved in TNFα-induced liver injury. Fas, which belongs to the TNF receptor family, can induce cell apoptosis via its ligand, FasL (Nagata and Golstein, 1995), and the up-regulation of Fas/FasL-induced apoptosis was seen in human liver failure caused by human hepatitis B and C infection (Galle et al., 1995; Nagata and Golstein, 1995; Ryo et al., 2000). TNFα induces cell apoptosis through TNFR (Bradham et al., 1998; Nagata, 1997) and TNF is also able to induce Fas expression by upregulating NF-κB expression (Streetz et al., 2000). Therefore, it was suggested that TNFα contributes to the apoptosis seen in liver injury.

TNFα and HBV and HCV Infection 1. Role of TNFα in HBV and HCV Infection

The roles of TNFα in HBV and HCV infection are summarized in figure 1. Many reports have suggested that TNFα plays a protective role during HBV infection. Generally, plasma level of TNFα in patients with acute HBV infection are higher than those in non-HBV-infected individuals (Koulentaki et al., 2004), and TNFα down-regulates the expression and replication of HBV in the liver (Chen et al., 2005) and the expression of HBV antigen was shown to be inhibited by TNFα in HBV transgenic mice (Cavanaugh et al., 1998). In addition, TNFα and IFNγ induced activated immunoeffector cells in lesions and TNFα and IFNγ can induce noncytolytic HBV suppression (Kim et al., 2003). Moreover, Puro et al. reported that TNFα destabilizes HBV nucleocapsids by inhibiting accumulation of transcription template without ubiquitination or methylation of protein (Puro and Schneider, 2007). On the other hand, Dunn et al. reported that natural killer cells may contribute to liver inflammation by increasing TNF-related apoptosis-inducing ligand-mediated hepatocellular death during active HBV infection (Dunn et al., 2007).

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In HCV infection, the role of TNFα is not understood in detail. HCV core protein, which is a 20-kDa structural protein, induces TNFα production in monocytes. It was also reported that HCV core protein inhibits TNF-induced apoptosis of human breast carcinoma cells (Ray et al., 1998). In chronic hepatitis C patients, plasma levels of TNFα and TNF receptor are upregulated and increased TNFα and TNF receptor levels lead to aggravation of HCV infection (Kallinowski et al., 1998; Nelson et al., 1997). TNFα is also secreted by infiltrating mononuclear cells in HCV infection, and these infiltrating lymphocytes are usually not specific for HCV antigen (Ando et al., 1997; Kinnman, Andersson, and Hultcrantz, 2000). Therefore, TNFα is considered a critical mediator in nonspecific tissue destruction by infiltrates (Tilg, Kaser, and Moschen, 2006). TNFα expression is regulated by the NF-κB signaling pathway (Aggarwal, 2004). It has been reported that the HCV core proteins can induce NF-κB pathway activation (Marusawa et al., 1999; You, Chen, and Lee, 1999). Dolganiuc et al. reported that core protein or nonstructural protein (NS) 3 of HCV reduced cytoplasmic Iκ-Bα protein levels, which is the initiation step of the NF-κB signal cascade (Dolganiuc et al., 2003). In addition, Choi et al. reported that NS5B protein of HCV inhibited TNFα-induced NF-κB activation and NS5B protein also activated TNFα-mediated JNK activity in hepatic cells (Choi et al., 2006). Moreover, NS5A protein of HCV negatively regulates TNFα-induced NF-κB activation via TNF receptor-associated factor 2 signaling (Park et al., 2002).

Figure 1. The role of TNFa in HBV and HCV infection.

2. Association of TNFα Promoter Polymorphism with the Outcome of Hepatic Virus Infection

As described above, TNFα plays a critical role in the host immune response against HBV infection, and the capacity for TNFα production in an individual is a major genetic determinant of susceptibility to HBV. A number of recent reports described the relation

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between TNFα promoter polymorphism and the outcome of HBV infection (Cheong et al., 2006; Du et al., 2006; Kim et al., 2003; Li et al., 2005; Niro et al., 2005). More than 10 SNPs have been reported in the promoter region of the TNFα gene (e.g., –238G/A, –244A/G, – 308G/A, –376A/G, –575A/G, –857C/T, –863C/A, –1031T/C, –1125G/C, –1196C/T), and these SNPs influence TNFα production (Baena et al., 2002; Du et al., 2006; Heesen et al., 2004; Higuchi et al., 1998; Jaber et al., 2004; Negoro et al., 1999). Among these SNPs, TNFα promoter haplotypes (–238/–308/–857/–863/–1031) are associated with the outcome of HBV infection (Cheong et al., 2006; Du et al., 2006; Kim et al., 2003; Li et al., 2005; Niro et al., 2005). Several reports have suggested that the frequencies of –238GG, –308GG, and – 857CC are significantly associated with persistent HBV infection, and the frequencies of – 308AA or AG, –857TT, and –863CC are significantly associated with spontaneous clearance of HBV (Du et al., 2006; Li et al., 2005; Niro et al., 2005). In addition, it was reported that TNFα haplotypes (–238G, –308G, –857C, –863A, –1031T) and (–238G, –308G, –857T, – 863A, –1031T) are strongly associated with chronic HBV infection, while haplotypes (– 238G, –308G, –857C, –863C, –1031T) and (–238G, –308G, –857C, –863A, –1031C) are associated with HBV clearance (Du et al., 2006; Kim et al., 2003). On the other hand, although some reports suggested that TNFα promoter polymorphisms are associated with response to IFNα therapy, the contribution of TNFα promoter polymorphism to HCV infection remains controversial. Dai et al. reported that the TNF-308A allele was associated with the failure of high-dose IFNα treatment in HCV patients, and the failure was particularly remarkable in HCV genotype 1b infection and in cases with high serum HCV RNA levels (Dai et al., 2006a). They also reported that the TNF-308A allele appeared to be associated with variability in severity of fibrosis and viral load in chronic HCV infection (Dai et al., 2006b). On the other hand, Rosen et al. and Yu et al. reported that TNF-308 polymorphism did not play a direct role in susceptibility or pathogenesis of HCV infection or response to antiviral IFNα therapy (Rosen et al., 2002; Yu et al., 2003). However, Kusumoto et al. reported that TNFα promoter variations were not associated with alterations in HCV clearance or alanine aminotransferase release but may be associated with hepatic fibrosis (Kusumoto et al., 2006).

Anti-TNFα Therapy During HBV and HCV Infection A number of anti-TNFα agents, such as infliximab, which is a human-mouse chimeric anti-TNFα monoclonal antibody, and etanercept, which is a soluble p75 TNFα receptorconjugated Fc of IgG, are commercially available and have been used successfully in chronic inflammatory diseases, such as rheumatic disease and Crohn’s disease. TNFα has been suggested to be involved in the pathogenesis of liver diseases. Therefore, treatments capable of neutralizing TNFα may be useful to ameliorate liver injury. In alcoholic steatohepatitis, anti-TNFα therapy combined with steroid medicine was useful in a rat model (Iimuro et al., 1997) and was significantly effective in disease recovery in some clinical trials (Spahr et al., 2002; Tilg et al., 2003), although another report suggested that anti-TNFα therapy had detrimental effects in patient with severe alcoholic hepatitis (Naveau et al., 2004).

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In HBV infection, TNFα plays a protective role as described above and several reports suggested that anti-TNFα therapy induces reactivation of HBV and fulminant hepatitis (Esteve et al., 2004; Michel et al., 2003; Ostuni et al., 2003; Wendling et al., 2005). AntiTNFα treatment in HBV surface antigen carrier and Crohn’s disease patients leads to severe reactivation of HBV (Millonig et al., 2006; Tilg, Kaser, and Moschen, 2006). In addition, it was reported that HBV reactivation occurred in HBV antigen-negative patient with Crohn’s disease treated with anti-TNFα therapy (Madonia et al., 2007). Anti-TNFα therapy is commonly applied in the treatment of Crohn’s disease. Therefore, it is important to check the HBV status of patients scheduled to receive anti-TNFα therapy. On the other hand, as described above, serum levels of both TNFα and TNFα receptor were up-regulated in HCV-infected patients, and it has been suggested that TNFα enhances liver injury during HCV infection. However, the effects of anti-TNFα therapy in HCV infection remain unclear. Some reports indicated that rheumatic disease patients infected with HCV and receiving long-term treatment with anti-TNFα agents did not show reactivation of hepatitis C infection or aggravation of hepatic inflammation (Aslanidis et al., 2007; Linardaki et al., 2007; Parke and Reveille, 2004). Zein et al. reported that anti-TNFα therapy was directly effective against HCV infection. They reported that IFNα-2b and ribavirin treatment with etanercept significantly improved virological response in HCV patients (Zein, 2005) and this is the first evidence that an anti-TNFα strategy is beneficial for HCV infection. However, further studies are needed to confirm the effectiveness of anti-TNFα therapy in the treatment of HCV infection.

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Index A aab, 44 AAC, 58 abdomen, 103 absorption, 55, 97 access, 188 accessibility, 26 accidental, 172 ACE, 2 acetaminophen, 209, 215 acetate, 40, 191 achievement, 67 acid, 3, 5, 7, 8, 12, 21, 26, 51, 54, 87, 92, 98, 99, 123, 146, 178, 199 acidic, 116 acidification, 202 actin, 117, 176 activation, vii, viii, xi, xii, 1, 2, 3, 13, 14, 15, 16, 17, 19, 20, 21, 22, 27, 29, 30, 31, 32, 33, 37, 38, 39, 45, 47, 49, 53, 54, 55, 56, 58, 59, 61, 63, 67, 72, 75, 77, 80, 82, 83, 84, 88, 89, 91, 92, 99, 100, 110, 113, 115, 121, 128, 135, 145, 147, 149, 171, 173, 175, 176, 182, 186, 191, 195, 205, 210, 216, 217, 219 activation state, 54 activators, 12 acute, xi, 2, 20, 24, 31, 35, 39, 42, 43, 54, 75, 110, 115, 135, 136, 157, 161, 176, 182, 194, 205, 206, 207, 208, 209, 213, 215, 217, 219 acute alcoholic hepatitis, 217

acute rejection, 42 acute renal failure, 24, 215 adalimumab, viii, 25, 26, 27, 29, 30, 36, 38, 49, 51, 66, 70, 76, 134, 137, 139, 141 adaptation, 188 ADCC, 29 adducts, ix, 96, 107 adenine, 2, 23 adenocarcinoma, 92, 120, 179, 180, 191, 192, 193, 195, 196, 204 adenocarcinomas, 68, 178, 181, 182, 185, 195, 199 adenoma, 178, 179 adenomas, 179, 194 adenovirus, 121, 132, 213 adenylyl cyclase, 145 adherens junction, 116, 117, 131, 132 adhesion, 2, 16, 17, 20, 47, 55, 58, 60, 94, 110, 111, 113, 116, 131 adhesions, 82 adipocyte, 59 adiponectin, 157, 158 adipose, x, 3, 74, 151 adipose tissue, x, 3, 74, 151 adjustment, 144, 153 administration, x, 24, 25, 42, 46, 66, 68, 89, 101, 103, 106, 109, 115, 120, 121, 143, 146 adrenal cortex, 145 adult, 100, 125, 158, 174, 202, 216 adult respiratory distress syndrome, 100, 125 adult tissues, 174, 202

222

Index

adults, 65, 137 adverse event, 69 aetiology, 23, 50, 57 AFMK, 144 African American, 56 Ag, 20 age, 51, 56, 134, 145, 146 agent, vii, ix, 1, 27, 61, 62, 67, 68, 95, 96, 98, 99, 101, 103, 111, 125, 135, 136, 148, 149, 166 agents, vii, viii, xi, 1, 12, 26, 27, 29, 30, 49, 51, 67, 96, 97, 102, 106, 135, 137, 138, 141, 147, 167, 187, 201, 205, 207, 211, 212, 217 aggregates, 53, 59, 190 aggregation, 112, 179 aggressiveness, 202 aging, 203 agonist, 140 aid, 156 AIDS, 99, 162, 163 AIP, 202 Air Force, 133 AKT, 17 alanine, 100, 211 alanine aminotransferase, 211 albumin, 118 alcohol, 209 algae, 144 algorithm, 136 alkylating agents, 110 allele, 37, 56, 167, 168, 175, 211 alleles, 3, 23, 25, 56, 167 allograft, 75, 100 alopecia, 22 alopecia areata, 22 alpha, viii, ix, xi, 2, 3, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 58, 73, 74, 75, 76, 77, 90, 93, 95, 96, 98, 99, 100, 101, 102, 103, 104, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 124, 125, 126, 127, 128, 129, 130, 132, 137, 138, 140, 141, 148, 157, 160, 161, 162, 163, 167, 168, 169, 205, 206, 212, 213, 214, 215, 216, 217, 218, 219 alternative, 19, 103, 106, 112, 136, 181, 199, 202, 203 Alzheimer’s disease, 188, 208 amelioration, 138 amino acid, vii, 3, 5, 6, 7, 8, 11, 14, 26, 98, 99, 100, 101, 123 amino acids, vii, 3, 6, 7, 11, 14, 26, 100, 101 amputation, 105, 106, 107, 126

anaemia, 202 analysis of variance, 154, 156 anastomosis, 52 anatomy, 159, 196 androgen, 145, 149 anemia, 180, 195 anger, 41 angiogenesis, x, 30, 87, 93, 108, 109, 113, 114, 115, 117, 128, 129, 130, 131, 144, 177, 196, 203, 208 angiogenic, 48, 112, 113, 114, 115 angiogenic process, 115 angiosarcoma, 107 angiotensin-converting enzyme, 47 anhydrase, 180 animal models, 24, 108, 109, 112, 121, 145, 148, 219 animal studies, 97, 101 animals, 97, 144, 146, 148, 188 ankylosing spondylitis, 21, 22, 51, 134, 137 anorexia, 40, 101 antagonism, 29, 134, 135 antagonist, ix, 26, 27, 29, 46, 89, 133, 134, 135, 136, 139, 140, 141 antagonists, 25, 27, 28, 29, 30, 38, 44, 102, 133, 134, 135, 136, 137, 138, 139, 140 anti-angiogenic, 112, 116 antiapoptotic, 173, 188, 201 anti-apoptotic, 16, 22, 58, 147, 182, 186 antibacterial, 34 antibiotic, 52, 74, 96 antibiotics, 155 antibodies, 32, 212, 214 antibody, viii, 24, 26, 28, 36, 40, 41, 42, 44, 46, 48, 49, 51, 61, 62, 64, 67, 68, 69, 70, 74, 75, 76, 77, 82, 89, 99, 113, 130, 131, 139, 141, 166, 202, 211, 219 anti-cancer, ix, x, 30, 41, 95, 96, 97, 99, 101, 125, 144, 149, 187, 194, 201, 216 anticancer drug, x, 144, 187, 194 anticoagulation, 202 antigen, 26, 39, 44, 53, 54, 55, 56, 60, 62, 63, 71, 73, 99, 115, 146, 189, 196, 207, 208, 209, 210, 211, 217, 218 antigen presenting cells, 54, 189 antigen presenting cells (APCs), 189 antigens, 37, 169, 214 anti-inflammatory, 189, 214 anti-inflammatory agents, 51 anti-mitochondrial antibodies, 81 antineoplastic, 102, 103

Index antioxidant, ix, 143, 144, 146, 147, 148 antisense, 130 anti-tumor, ix, x, 37, 40, 41, 43, 47, 74, 76, 77, 95, 99, 101, 102, 107, 108, 112, 116, 119, 120, 121, 124, 126, 127, 128, 129, 137, 138, 139, 144, 147, 208, 217, 218 antiviral, 42, 43, 211, 218 antiviral therapy, 218 anus, 50, 141 APC, 54, 55, 59 APCs, 56 APO, 9, 113, 214 apoptosis, viii, xi, xii, 1, 2, 14, 15, 16, 17, 19, 21, 22, 27, 31, 32, 34, 35, 36, 37, 38, 41, 43, 45, 46, 47, 55, 59, 60, 61, 67, 72, 74, 75, 79, 82, 90, 91, 113, 121, 129, 147, 149, 159, 163, 166, 168, 171, 172, 173, 174, 175, 176, 177, 178, 180, 182, 185, 186, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 203, 204, 205, 209, 216, 218, 219 apoptosis pathways, 19 apoptotic, xi, 14, 16, 17, 21, 22, 41, 58, 60, 68, 75, 82, 171, 173, 174, 176, 179, 182, 183, 185, 186, 187, 189, 191, 192, 193, 196, 197, 198, 199, 201, 202, 203 apoptotic cells, 173, 177, 179, 187, 189, 191, 194, 196, 201 apoptotic effect, 22 apoptotic pathway, 22 appetite, 20 application, 63, 65, 104, 138, 149, 155 APRIL, 5, 6 arachidonic acid, 87 arginine, 118, 119 argument, 115 Army, 133 arrest, 174, 187, 201 arteries, 113, 145 artery, 47, 58, 103, 113, 180 arthralgia, 68 arthritis, vii, 1, 134, 138, 140, 208, 216, 217 ascites, 199 Asian, 56 aspartate, 16 aspiration, 197 assessment, 51, 172, 187 asthma, 21, 22, 23, 31, 41 astrocytes, 3, 99 ATM, 83, 92 ATP, 176, 177, 201 attachment, 75, 153

223

attention, xi, 171, 172 autoantibody, 70 autocrine, 55, 59, 81 autoimmune disease, vii, ix, 1, 20, 22, 25, 35, 36, 57, 81, 133, 135, 136, 140, 208 autoimmune diseases, vii, ix, 1, 20, 22, 25, 35, 133, 135, 136, 140, 208 autoimmune disorders, 21, 177 autoimmunity, 36 autophagic cell death, xi, 172, 188, 191 autophagic vacuoles, 182, 186, 188, 191 autophagy, xi, 171, 172, 182, 185, 188, 190, 192, 194, 195, 196, 199, 200, 201 autosomal dominant, 22 availability, 80, 153, 177 axilla, 103

B B cell, 2, 19, 23, 26, 134, 141, 203 B cells, 19, 26, 134, 203 B lymphocytes, 75 bacillus, 97 Bacillus, 122 bacillus Calmette-Guerin, 97 bacteria, x, 6, 53, 56, 57, 96, 97, 98, 144, 151, 157 bacterial, 3, 19, 24, 29, 53, 55, 56, 57, 83, 84, 90, 91, 92, 96, 97, 98, 99, 101, 113, 122, 136, 152 bacterial infection, 24, 29, 55, 83, 84, 96, 97, 98, 122 bacterium, 97, 219 BAFF, 5, 6, 9, 12 barrier, 53, 57, 58, 60, 71, 72, 74, 118, 119, 132, 159, 162, 166, 174, 185, 194 barriers, 145 basement membrane, 179, 182 Bax, 59, 74, 198 B-cell lymphoma, 2 BCG, 97, 98 bcl-2, 147, 199 Bcl-2, 59, 174, 182, 186, 188, 198, 201, 203 BDNF, 10 behavior, 183, 185, 190, 198 Belgium, 56, 110, 111 bell-shaped, 6 benchmark, 103 beneficial effect, x, 19, 99, 122, 143 benefits, 30, 106, 122 benign, 51 bile, viii, 79, 80, 81, 82, 83, 84, 85, 86, 88, 90, 91, 92

224

Index

bile acids, 82 bile duct, viii, 79, 80, 81, 82, 83, 84, 85, 86, 88, 90, 91, 92 biliary cirrhosis, 81, 91 biliary obstruction, 92 biliary tract, 90, 93 binding, 3, 4, 6, 7, 12, 13, 14, 15, 21, 26, 27, 29, 35, 38, 39, 42, 43, 48, 56, 89, 101, 112, 117, 123, 130, 145, 146, 147, 149, 173, 175, 215 bioassays, 36 biochemistry, 197 biologic agents, 137 biological activity, 7, 100, 101 biopsies, 60, 110, 111, 117, 134 biopsy, 59, 134, 179, 203 biosynthesis, x, 37, 143, 147 biotechnology, 99 biotin, 2 birth, 57 bladder, 25, 61, 190 bladder cancer, 25 bleeding, 108, 153 blocks, x, xii, 44, 130, 143, 149, 191, 205, 208 blood, vii, xi, 29, 40, 50, 55, 90, 98, 99, 103, 105, 108, 113, 115, 116, 117, 119, 121, 123, 128, 130, 131, 132, 147, 149, 155, 159, 162, 165, 166, 177, 178, 179, 180, 182, 206, 208, 213 blood clot, 180 blood flow, 113, 116, 117, 119, 128, 181 blood pressure, 181 blood supply, 90, 108, 180, 182 blood vessels, vii, 103, 108, 115, 116, 177 blood-brain barrier, 162 B-lymphocytes, 62 body mass index (BMI), 154, 155, 156 bonds, 10 bone loss, 152 bone marrow, 102, 144 BOP, 153, 154, 156 Bose, 38 Boston, 197 bowel, 50, 51, 52, 61, 67, 70, 71, 73 brain, x, 15, 45, 47, 102, 105, 118, 143, 144, 159, 166, 167, 168, 196, 201 brain microvascular endothelial cell, 166 branching, 93 breakdown, 117 breast, 183, 209, 218 breast cancer, 43, 93, 149, 172, 183, 187, 195, 198, 199, 200, 218

breast carcinoma, 25, 41, 182, 184, 199, 200, 201, 202, 209 broad spectrum, 52 Brussels, 110 burn, 24, 32, 33 burns, 24, 33, 40 bypass, 24, 47, 102, 103, 131

C Ca2+, 112, 116, 176 cachexia, 3, 19, 20, 25, 40, 99, 101 CAD, 2, 21 cadherin, 116, 117, 118, 131, 132, 183, 198 calcium, 12 calculus, 155 calibration, 153, 154 calmodulin, 145, 148 cAMP, 12, 43 Canada, 45, 125 cancer, vii, x, xi, 1, 3, 22, 25, 30, 39, 40, 41, 47, 67, 86, 93, 96, 98, 99, 102, 104, 105, 108, 110, 122, 125, 126, 130, 131, 132, 143, 145, 146, 147, 148, 149, 160, 168, 171, 172, 173, 174, 180, 183, 187, 188, 191, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 205, 206, 207, 208, 212, 213, 216, 218 cancer cells, x, 38, 93, 98, 99, 108, 143, 145, 146, 147, 148, 149, 188, 191, 200, 208, 218 cancer progression, 30, 208 cancer treatment, x, 30, 93, 96, 125, 144, 146 cancerous cells, 108 capillary, 24, 55, 58, 101, 110 carcinoembryonic antigen, 196 carcinogenesis, x, 144, 182 carcinogenic, xi, 205 carcinogens, 146 carcinoma, xi, 25, 37, 40, 41, 42, 45, 87, 88, 90, 97, 112, 120, 125, 131, 172, 178, 179, 181, 182, 183, 184, 185, 186, 190, 194, 195, 196, 197, 199, 200, 201, 202, 204, 205, 206, 209, 213, 216, 218 carcinomas, xi, 92, 97, 172, 174, 178, 181, 182, 183, 184, 185, 190, 191, 192, 196, 197, 198, 199, 202, 204 CARD15, 56, 73 cardiac myocytes, 3, 12 cardiac surgery, 103 cardiopulmonary, 102, 131 cardiopulmonary bypass, 102, 131 cardiovascular disease, 208

Index cardiovascular risk, 158 carrier, 211, 217 casein, 32 caspase, 16, 21, 22, 47, 56, 60, 61, 75, 173, 177, 182, 185, 186, 188, 195, 202 caspase-dependent, 21, 177 caspases, xi, 2, 14, 17, 21, 22, 171, 173, 177, 195, 199 casting, 183 catalytic activity, 86 cathepsin B, 36, 191 cation, 44 Caucasian, 31 Caucasian population, 31 Caucasians, 23 causation, xi, 171, 172 C-C, 2, 19 CCL19, 40 CCL21, 40 CD26, 9 CD30, 5, 9, 10, 17 CD95, 8, 17, 113, 173, 214 CDC, 29 cDNA, 11, 36, 43, 100, 101, 123, 213 cell adhesion, 2, 17, 20, 58, 60, 111, 114, 116 cell culture, 12, 61, 117 cell cycle, 16, 175, 187, 202 cell death, vii, ix, xi, 1, 2, 8, 16, 17, 18, 21, 22, 32, 37, 38, 44, 45, 95, 123, 147, 148, 149, 171, 172, 176, 179, 180, 183, 185, 186, 188, 189, 191, 192, 193, 195, 197, 198, 199, 200, 201, 202, 203, 204, 209 cell differentiation, 181, 214 cell division, 174 cell fate, 204 cell fusion, 152, 157 cell growth, 108, 145, 149 cell invasion, 86, 87, 110, 128 cell line, viii, 2, 19, 34, 45, 80, 87, 88, 89, 93, 94, 108, 112, 123, 152, 163, 182, 183, 196, 214 cell lines, 45, 87, 89, 93, 108, 112, 123, 183, 196 cell membranes, 84, 145 cell signaling, 17, 83 cell surface, 7, 12, 35, 39, 47, 61, 82, 101, 108, 116, 123 cellular adhesion, 58 cellular homeostasis, 21 cellular immunity, x, 159, 161 central nervous system, 100 cervical carcinoma, 178

225

cervix, 175 c-fos, 20 channels, 145, 176 chemoattractant, 2, 20, 82 chemokine, 2, 19, 30, 46, 82, 93, 135, 141 chemokine receptor, 93, 135, 141 chemokines, 16, 29, 40, 82, 207 chemotaxis, 112 chemotherapeutic agent, 102, 106 chemotherapeutic drugs, 147 chemotherapy, ix, 95, 102, 103, 106, 126, 146, 163, 173, 187, 194, 195, 198, 200 childhood, 73, 105 children, 59, 71, 167, 169 China, 57, 73 Chinese, 198, 200, 214 chloride, 6 cholangiocarcinoma, viii, 79, 86, 87, 88, 89, 90, 93, 94 cholangitis, viii, 51, 79, 81, 82, 83, 84, 85, 90, 92, 93 cholestatic liver disease, 92 chromatin, xi, 21, 171, 172, 175, 179, 180, 185, 186, 187, 191, 192, 193, 203 chromatography, 7, 44, 45 chromosomal instability, 191, 194 chromosome, ix, 3, 4, 7, 56, 95, 100, 187, 198, 200, 202 chromosomes, 5, 56, 187 chronic active hepatitis, 207, 215 chronic hypoxia, 182, 194 chronic lymphocytic leukemia, 131, 163 ciprofloxacin, 52 circadian, 144, 145, 149 circadian rhythms, 144, 145 circulation, 24, 53, 102, 103, 135 cirrhosis, xi, 205, 206, 207, 208, 215 c-Jun kinase, 37 CK, viii, 7, 79, 84, 85, 168 classes, 183 classical, 6, 21, 29, 55, 182, 192 classification, 11, 51, 56, 70, 71, 104, 186 cleavage, 6, 12, 21, 22, 45, 80, 100, 203 clinical symptoms, 40 clinical trial, 25, 30, 43, 44, 62, 125, 133, 211 clinical trials, 25, 30, 43, 44, 62, 64, 125, 133, 211 clinicopathologic correlation, 196 clone, 213 cloning, 3, 44, 99, 123, 124, 207 closure, 63, 65 cluster of differentiation, 54

226

Index

clustering, 101, 131, 140, 176, 186, 191 clusters, 179, 180, 182, 184, 185, 187, 191 c-myc, 21, 199, 203 Co, 82, 92 coagulation, vii, 202 codes, 100 coding, 23, 51, 56, 124 cohesiveness, 190 cohort, 50, 55, 61, 72, 73, 134, 137, 153 colitis, 51, 71, 72 collagen, 75, 177 collateral, 147 collateral damage, 147 colon, viii, 49, 50, 53, 60, 74, 94, 131 colon cancer, 94 colonization, 53, 58 colony-stimulating factor, 6, 195 colorectal adenocarcinoma, 178 colorectal cancer, 145, 198 combination therapy, 109, 120, 213 commercial, 207 community, 23, 47, 73, 214 co-morbidities, 70 compaction, 185 competence, 13 competitor, 12 complement, 6, 26, 29, 99, 149 complementarity, 26 complementarity determining region, 26 complementary DNA, 124 complete remission, 64, 67 complexity, 17, 50, 53, 116 complications, 24, 50, 102, 137, 160, 165, 167, 180, 216 components, 16, 27, 84, 86, 90, 91, 92, 97, 112, 144, 157, 174, 188 composition, 27 compounds, 145 concentrates, 121 concentration, ix, 12, 19, 20, 21, 29, 89, 95, 102, 107, 119, 127, 146, 156, 166, 167 concordance, 56 condensation, xi, 21, 171, 179, 180, 186, 191, 198, 200, 202 confidence, 96 confusion, xi, 171 connective tissue, 61, 112 connectivity, 11 consensus, 109, 137 consent, 153

conservation, 11 contact dermatitis, 133 control, 21, 37, 52, 62, 69, 70, 73, 102, 113, 116, 144, 165, 174, 194, 196, 197, 199, 215, 216 conversion, 87 cooling, 24, 47 coronary artery bypass graft, 47 coronary heart disease, 37, 153 corpus luteum, 115 correlation, xi, 23, 25, 73, 101, 113, 114, 134, 161, 163, 165, 166, 167, 198, 203 correlations, 92, 196, 199, 206 cortex, 145 corticosteroids, 6, 51, 136 corticotrophin releasing hormone, 2 corticotropin, 20 cost-effective, 175 covering, 136 COX-1, 88, 89 COX-2, viii, 80, 87, 88, 89 CRD, 2, 11, 13 C-reactive protein, 2, 20, 67, 153, 157 CRH, 2, 20 critical analysis, 122 critically ill, 40 Crohn’s disease, 211 crosslinking, 7, 27, 29 cross-linking, 82 crosstalk, x, 17, 143 CRP, 2, 67, 153, 155 crystal structure, 12, 14, 38, 101 crystal structures, 12, 14 C-terminal, 3, 7, 13, 22, 26, 116 C-terminus, 2, 8 cultivation, 93 culture, 21, 57, 61, 85, 88, 89, 91, 92, 121, 122, 182 curcumin, 147 curing, 99, 208 cycling, 108 cycloheximide, 108 cyclooxygenase-2, viii, 80 cyclosporine, 51, 136 cysteine, xi, 2, 6, 7, 8, 10, 11, 14, 16, 18, 100, 101, 171, 173 cysteine proteases, xi, 171, 173 cysteine residues, 7, 8, 10, 11, 18, 100, 101 cystic fibrosis, 22 cytochrome, 22, 173, 197 cytokeratins, 183

Index cytokine, vii, viii, ix, x, xi, xii, 2, 6, 13, 19, 22, 25, 27, 29, 30, 40, 46, 54, 59, 61, 71, 72, 79, 85, 86, 92, 93, 95, 99, 112, 113, 124, 128, 129, 135, 140, 143, 146, 147, 149, 157, 159, 162, 163, 165, 166, 167, 168, 169, 205, 206, 208, 213, 217 cytokine response, 165, 167, 169, 217 cytokines, vii, viii, 1, 6, 12, 16, 19, 22, 29, 43, 48, 54, 55, 72, 79, 80, 82, 83, 90, 92, 99, 108, 127, 128, 146, 147, 149, 152, 157, 159, 163, 165, 166, 168, 169, 189, 206, 207, 208, 214, 215, 218, 219 cytokinesis, 187 cytology, 197 cytomegalovirus, 213 cytoplasm, 81, 83, 84, 85, 87, 175, 186, 187, 188, 191, 192 cytoplasmic membrane, 85, 176 cytoplasmic tail, 14, 16, 116, 174 cytoskeleton, 117, 176, 191 cytosol, 174, 198 cytosolic, 112, 145 cytotoxic, 2, 18, 21, 39, 47, 54, 82, 99, 108, 113, 123, 124, 126, 208, 213 cytotoxic effects, 21 cytotoxicity, 29, 36, 43, 108, 124, 125 cytotoxins, 124

D database, 160 de novo, 68, 72, 108, 133 death, vii, xi, 1, 2, 3, 8, 10, 14, 15, 16, 19, 21, 23, 25, 30, 31, 38, 41, 44, 45, 61, 68, 80, 82, 97, 101, 145, 165, 171, 172, 173, 176, 177, 179, 180, 182, 183, 185, 186, 188, 189, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 209, 216 deaths, 105, 177, 193, 199 debridement, 155 decay, 152 defense, x, xi, 19, 21, 22, 29, 34, 137, 143, 166, 205, 206, 208 defense mechanisms, 19 deficiency, 191 deficit, 160 definition, xi, 3, 57, 105, 171, 185, 186, 195, 197 degenerate, 192 degradation, 6, 16, 30, 118, 188 degradation process, 188 dehydration, 101 dehydrogenase, 81 delivery, 25, 116, 121, 132

227

demyelinating disease, ix, 136 denaturation, 101 dendritic cell, 19, 28, 36, 40, 53, 54, 58, 71, 135, 140, 166, 189, 206, 208, 214 Denmark, 56 density, 12, 13, 27, 29, 31, 181, 182, 199 dentists, 153 Department of Defense, 133 deposits, 112 depressed, 145 derivatives, 82, 148 dermatitis, 136, 138 dermatitis herpetiformis, 136 dermatologist, 133, 136 dermatology, 136 dermis, 135 destruction, 19, 54, 100, 109, 110, 111, 121, 148, 173, 195, 210 destructive process, 112 detachment, 113 detection, 59, 73, 104, 185, 187, 193, 196, 197 developed nations, 207 developing countries, 165 diabetes, vii, x, 1, 22, 39, 100, 151, 153, 208 diabetes mellitus, 153 diacylglycerol, 20 diagnostic criteria, 72 diapedesis, 58, 60 diarrhoea, 50, 55, 57 dichotomy, 22, 193 dietary, 57, 146 differentiation, vii, 1, 21, 54, 72, 145, 148, 149, 179, 181, 182, 188, 190, 191, 194, 200, 204, 214 diffusion, 116, 148, 178, 182 digestion, 200 dimer, 26 disease activity, 31, 51, 58, 70, 163 disease progression, 52, 54, 207, 209 disease rate, 57 disease-free survival, 105 diseases, vii, viii, ix, 1, 19, 22, 23, 25, 29, 30, 79, 80, 81, 82, 90, 136, 138, 140, 161, 206 disintegrin, 6, 32 disorder, 160, 175 displacement, 3, 111 dissociation, 13, 15, 27, 145 distress, 167, 169 distribution, 19, 21, 30, 92, 111, 157, 190, 214 disulfide, 6, 7, 11, 42 diversity, 195

Index

228

division, 103, 174, 199 dizygotic, 72 dizygotic twins, 72 DNA, ix, 20, 21, 57, 61, 68, 96, 107, 136, 144, 149, 173, 174, 186, 191, 194, 196, 197, 198, 203, 206, 217 DNA damage, 174, 186, 194 DNA repair, 187 DNase, 2, 21, 194 donors, 23 dopamine, 145 Doppler, 119 dosage, 68 dosing, 63, 64 down-regulation, 29, 132 drug abuse, 207 drug discovery, 196 drug exposure, 103, 140 drug induced lupus, ix, 136 drugs, 27, 43, 70, 102, 146, 147, 187, 194, 206, 209 duality, 19 duodenum, 53 duration, 21, 30, 64, 67 dysplasia, 194 dysregulation, 175, 188

E E-cadherin, 60, 183, 198 ECM, 86 eczema, 136 elderly, x, 151, 152, 153, 154, 155, 157, 158 elderly population, 157 electrical resistance, 60 electrolyte, 24 electrolyte imbalance, 24 electron, 111, 117, 128, 173, 180, 184, 187, 188, 190, 191, 192, 194, 195, 197, 200 electron microscopy, 111, 173, 184, 187, 188, 190, 191 ELISA, 84, 152, 154 e-mail, 151 embryo, 130 embryonic development, xi, 171 employment, x, 144, 146 encephalomyelitis, 207 encoding, 3, 5, 84, 129 endocrine, 84, 145, 149 endoplasmic reticulum, 176, 186, 192 endoscopy, 50

endothelial cell, vii, 3, 12, 20, 26, 27, 34, 36, 60, 72, 93, 108, 111, 112, 113, 114, 115, 117, 118, 128, 129, 130, 132, 146, 149, 189 endothelial cells, vii, 3, 12, 20, 26, 60, 72, 111, 112, 113, 114, 117, 128, 130, 146, 149, 166, 189 endothelium, 55, 99, 108, 109, 110, 112, 113, 116, 117, 128, 129, 166, 168 energy, 198, 199 engagement, 27 enlargement, 187 enteritis, 71 enthusiasm, 101 environment, 53, 73, 181, 182, 188 enzymatic, 7, 14 enzymatic activity, 7, 14 enzymes, 21, 144, 177 eosinophils, 99, 134 epidemiology, 72, 216 epidermis, 135 epithelia, 84, 174 epithelial cell, viii, 19, 43, 55, 58, 60, 79, 81, 82, 84, 85, 86, 91, 92, 93, 174, 181, 182, 189 epithelial cells, viii, 19, 55, 58, 60, 79, 81, 82, 84, 85, 86, 91, 92, 93, 174, 181, 189 epithelium, 53, 55, 58, 60, 61, 83, 91, 92, 144, 180, 182 epitopes, 27 equipment, 192 erysipelas, 96 erythema multiforme, 136 erythematous, 134, 135 erythrocyte, 160, 162, 167, 169 erythrocytes, 101, 166, 190 Escherichia coli, ix, 43, 45, 74, 95, 101, 123, 124 esophagus, 84, 92, 178, 190, 200 ester, 119 esters, 12, 47 estrogen, 145, 148, 149, 183 etanercept, viii, 25, 26, 27, 29, 30, 35, 38, 39, 49, 134, 137, 138, 139, 141, 211, 212, 216 ethanol, 191, 214 Europe, 64, 65 European Union, 25 evidence, 181, 190, 195, 199, 203, 212, 218 evolution, 204 examinations, 152, 153 excision, 24, 52 exclusion, 44, 50, 70, 149 excretion, 103 execution, 36

Index exons, 3, 7, 100 exposure, 20, 53, 67, 73, 101, 103, 108, 109, 110, 113, 128, 140, 155, 198, 214 extracellular matrix, viii, 30, 49, 58, 61, 86 extraction, 155, 158 extravasation, 109, 110, 113, 129 exudate, 179

F faecal, 50, 55, 74 faecal bacteria, 74 failure, 24, 39, 47, 101, 102, 105, 176, 187, 209, 211, 215, 216, 218 falciparum malaria, 35, 166, 167, 169 familial, 23, 56, 175 family, vii, xi, 1, 2, 5, 6, 11, 13, 16, 17, 18, 19, 22, 26, 30, 40, 41, 42, 47, 48, 72, 86, 124, 133, 134, 136, 163, 171, 173, 174, 175, 195, 206, 207, 209 family history, 133, 134, 136 family members, 5, 11, 13, 16, 17, 22, 26, 47, 72 Far East, 83 Fas, 2, 4, 6, 8, 10, 12, 15, 16, 43, 45, 82, 91, 113, 129, 209, 212, 214, 216, 218 fascia, 105 FasL, 5, 6, 13, 82, 113, 209 fasting, 154, 155, 156 fasting glucose, 154, 155, 156 fat, ix, 95, 107, 160 FDA, viii, 49, 62, 64, 65, 66, 67, 76 FDA approval, viii, 49, 64, 67 fear, 96 feedback, 6 feeding, 180, 191 females, 134 fetal, 131 fever, 20, 50, 90, 159, 165, 166 fibers, 178 fibrin, 111 fibrinogen, 75 fibroblast, viii, 5, 49, 58, 61, 89, 198 fibroblasts, 3, 19, 46, 61, 75, 89, 93, 99, 178 fibrosarcoma, 3, 11, 109, 112, 120, 128 fibrosis, 22, 68, 70, 83, 93, 211, 215 fibrous tissue, 105 fire, 19 first degree relative, 56 fistulas, 76 fixation, 26, 67, 173, 196 flocculation, 175

229

flora, 53 flow, 52, 103, 113, 116, 117, 119, 128, 181 flow rate, 103 fluid, 12, 24, 34 fluorescence, 85 focal adhesion kinase, 94 follicular, 41, 174, 181 follicular lymphoma, 174 food, 57, 146 fragmentation, xi, 20, 21, 171, 172, 176, 197, 200, 203 France, 56 free radical, 118, 120, 146, 147, 148 free radicals, 146, 147, 148 Friedmann, 28, 36 fulminant hepatitis, 207, 211, 219 functional aspects, 55 fungal, 136 fungi, 99 fusion, 26, 28, 29, 62, 152, 157, 188 fusion proteins, 26, 29

G gall bladder, 81, 91, 190, 195, 197 games, 90 gas, 118 gastric, vi, xi, 25, 40, 92, 130, 145, 171, 172, 178, 179, 190, 191, 192, 193, 195, 203 gastric ulcer, 179 gastritis, 40, 179 gastrointestinal, viii, 50, 51, 71, 79, 90, 94, 102, 179, 180, 181, 184 gastrointestinal tract, viii, 50, 79, 90, 102, 179, 181, 184 gel, 53, 55, 60, 184 gender, 154 gene, ix, xi, 3, 4, 6, 7, 16, 21, 23, 26, 31, 34, 35, 36, 37, 40, 41, 42, 44, 45, 47, 54, 56, 61, 71, 72, 73, 75, 84, 91, 92, 95, 98, 99, 100, 114, 121, 124, 129, 132, 148, 167, 169, 174, 175, 183, 188, 201, 203, 205, 210, 213, 214, 215, 216, 217 gene expression, 44, 72, 92, 121, 132, 148, 183 gene promoter, 34, 41, 167, 169, 213, 215, 216 gene therapy, 34 gene transfer, 203 generalization, 29 generation, 22, 35, 47, 62, 68, 69, 70, 83, 146

230

Index

genes, viii, xi, 3, 5, 7, 16, 19, 21, 22, 23, 25, 31, 33, 42, 49, 55, 56, 75, 100, 123, 124, 171, 172, 183, 186, 191, 199, 205 genetics, 56, 57 genome, 73, 124, 174, 207, 213 genotoxic, 173, 174, 201, 203 genotype, 42, 73, 207, 211, 214 Georgia, 1 Germany, 56 gingival, 157 gland, x, 84, 135, 143, 144, 179, 180 glioblastoma, 194 glioma, 130, 145, 195 gliomas, 195 glucocorticoids, 12 glucose, 154, 155, 156, 180 glutathione, 149, 160, 162 glutathione peroxidase, 160, 162 glycogen, 192, 193 glycol, 26 glycoprotein, 10, 18, 111, 116, 178, 190, 200 glycoproteins, 7 glycosylated, 7, 26, 101 glycosylation, 7 glycosylphosphatidylinositol, 167, 169 goblet cells, 84, 85 gold standard, 185, 193 G-protein, 145 grades, 52, 105 grading, 187, 194 grafting, 24 gram negative, 98 gram-negative, 152 gram-negative bacteria, 97 granules, 192, 193 granulocyte, 6 granulomas, 55, 72, 73, 74, 82 granzyme, 82 gravity, 104 groups, 17, 38, 57, 64, 65, 67, 74, 99, 120, 144, 152, 154, 155, 183, 187 growth, x, xi, 2, 10, 43, 48, 50, 54, 58, 61, 86, 87, 94, 97, 98, 99, 102, 108, 114, 115, 116, 117, 119, 121, 130, 131, 132, 143, 145, 146, 149, 161, 163, 171, 173, 177, 180, 181, 182, 194, 199, 201, 208, 215, 218 growth factor, 2, 10, 48, 54, 58, 61, 116, 130, 131, 132, 146, 182, 199, 215, 218 growth factors, 146 growth inhibition, 131

guanine, 23 guardian, 174, 194 guidelines, 139, 140 gut, 42, 53, 55, 60, 74, 135, 144

H habitat, 155 haemostasis, 112 hairy cell leukemia, 161, 163 half-life, 26, 99, 115 hanging, 71 haplotype, 23, 25, 31 haplotypes, 3, 31, 41, 211 harmful effects, 19 HBV, xi, 205, 206, 207, 209, 210, 211, 216, 217 HBV infection, xii, 205, 206, 209, 210, 211 health, x, xi, 143, 144, 151, 152, 155, 202, 205, 206 health problems, xi, 205, 206 heart, 29, 37, 72, 102, 130, 153 heart disease, 37, 188 heart failure, 29 heat, 20, 196, 200 heating, 103 height, 6 hematological, 102 hematology, 161 hematopoietic, 103, 188 hematopoietic cells, 12, 80 hematoxylin-eosin, 191 hemorrhage, 109, 111, 122 hemostasis, 118 hepatic failure, 216, 218 hepatic fibrosis, 93, 211 hepatic injury, 206 hepatic necrosis, 214 hepatitis, xi, 22, 91, 100, 205, 206, 207, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219 hepatitis a, xii, 205, 206, 215, 216 hepatitis B, vi, xi, 22, 205, 206, 209, 213, 214, 215, 216, 217, 218, 219 hepatitis C, xi, 91, 205, 206, 207, 208, 209, 210, 212, 213, 214, 215, 216, 217, 218, 219 hepatitis d, 219 hepatocellular, 25, 37, 206, 208, 209, 213, 216, 218, 219 hepatocellular carcinoma, 25, 37, 206, 213, 216, 218 hepatocyte, 39, 90, 206 hepatocytes, 195 hepatoma, 146

Index hepatotoxic drugs, 206, 209 HER2, 183 heritability, 72 herpes simplex virus type 1, 44 heterogeneity, 162 heterogeneous, 71, 181, 183 heterotrimeric, 47, 145 high risk, 24, 25, 104, 126 histamine, 118 histochemical, 197 histologic type, 182, 184, 190, 194 histological, 65, 105, 136, 181, 183, 201 histology, 190 HIV, x, 99, 159, 160, 162, 163 HIV infection, 160, 163 HIV-1, 160, 163 HLA, 20, 23, 37, 51 HLA-B, 51 HLA-B27, 51 homeostasis, vii, xi, 1, 29, 171, 176, 188, 203 homocysteine, 72 homogeneity, 35 homolog, 75 homology, 2, 3, 6, 11, 14, 42, 43, 101, 123 homotrimers, 3 Hong Kong, 57 horizontal gene transfer, 203 hormone, 2, 3, 20, 125, 145 hospital, 96, 163 hospitalized, 158 host, xi, 19, 22, 29, 32, 34, 53, 98, 100, 137, 162, 166, 167, 169, 192, 205, 206, 207, 208, 210 House, 159, 165 HPV, 46 HRM, 138 HTLV, 163 human brain, 166, 168, 196 human genome, 124 human immunodeficiency virus, x, 159, 160, 162, 216 human leukocyte antigen, 39 human neutrophils, 197 humans, 30, 46, 62, 90, 100, 102, 144, 175 hybrid, 121 hybridization, 92 hybridoma, 62 hydro, 100 hydrogen, 11, 12 hydrogen peroxide, 12 hydrophilic, 100

231

hydrophobic, 4, 13, 100 hydroxyl, 144, 148 hygiene, 57, 73 hyperactivity, 159, 160 hypercoagulable, 181 hyperplasia, 134, 177 hyperpyrexia, 166, 168 hypersensitivity, 67 hypersensitivity reactions, 67 hyperthermia, 102, 103, 118, 125, 126, 132, 200 hypertrophy, 94, 188 hypothalamic, 20 hypothalamic-pituitary-adrenal axis, 20 hypothalamus, 20 hypothesis, x, 57, 74, 115, 159, 160, 161, 181, 190 hypoxia, 6, 101, 114, 115, 117, 130, 172, 174, 180, 182, 188, 194, 195, 199, 202 hypoxia-inducible factor, 199 hypoxic, 114, 116, 175, 180, 182 hypoxic cells, 180 hypoxic-ischemic, 175

I IAP, 2, 22, 174, 202 IBD, 53, 73 ICAM, 2, 47, 58, 111 ICC, 87 ICD, 2, 7, 8, 11, 14 ice, 39, 44 ICE, 15 id, 111, 167, 198 identification, 56, 59, 91, 98, 99, 103, 172, 173, 175, 187 identity, 5, 98 idiopathic, 134, 138 IgG, 26, 211 IL-1, 3, 6, 19, 20, 27, 32, 33, 36, 43, 47, 53, 54, 55, 59, 71, 72, 86, 146, 147, 149, 152, 163, 207, 208, 217 IL-10, 6, 43, 53, 54, 207, 208 IL-17, 6, 19, 55, 71, 72 IL-2, 6, 12, 20, 54, 55, 56, 58, 71, 146, 147, 149, 207, 208 IL-4, 12, 19, 54, 55, 71, 163, 207 IL-6, 7, 12, 20, 24, 41, 43, 54, 55, 58, 85, 86, 146, 152, 153, 207, 208 IL-8, 12, 24, 25, 43, 46 ileum, 50, 53, 57, 58, 75 immune cells, x, 54, 101, 143, 145, 189, 190

232

Index

immune function, x, 29, 30, 143, 146 immune reaction, 29 immune regulation, 30 immune response, ix, x, xi, 19, 29, 53, 54, 62, 80, 91, 95, 145, 159, 189, 196, 205, 210, 213 immune system, x, xi, 12, 29, 30, 35, 53, 71, 81, 143, 147, 148, 171, 195, 206, 208 immunity, x, 29, 30, 53, 54, 71, 73, 99, 127, 148, 159, 165, 168, 195 immunization, 62 immunobiology, 42 immunocytochemistry, 197 immunodeficiency, 160, 162, 216 immunohistochemical, 59, 118, 128, 175, 180, 184, 194, 196, 197, 204 immunohistochemistry, 74, 111, 175, 183, 201 immunological, 35, 56 immunology, 168 immunomodulatory, 218 immunopathology, 91 immunoprecipitation, 39 immunoreactivity, 59, 178, 184 immunostimulatory, 30, 135 immunosuppression, 68, 69, 146 immunosuppressive, 29, 70, 75, 134, 135 immunosurveillance, 204 immunotherapy, 36, 147 Immunotherapy, 24 in situ, 91, 178, 196, 197 in transition, 199 in vitro, viii, 29, 38, 46, 61, 80, 84, 88, 101, 108, 111, 113, 114, 117, 118, 121, 125, 148, 152, 163, 167, 169, 188, 190, 200, 203, 218 in vivo, x, 12, 13, 23, 26, 29, 32, 46, 61, 75, 91, 92, 101, 108, 112, 113, 114, 117, 121, 124, 129, 131, 132, 143, 147, 163, 177, 182, 188, 190, 203 inactivation, 21, 187 inactive, 177 incidence, 23, 29, 55, 56, 57, 61, 68, 104, 134, 146, 175, 179, 207 inclusion, 65 incubation, 89 India, 169 Indian, 169 indicators, 73 indices, 60 indirect effect, 61 indirect measure, 119 indole, 145, 146, 148 indolent, 183, 185

indomethacin, 88 inducer, 82, 114 induction, viii, ix, xi, 19, 21, 22, 25, 29, 36, 49, 54, 60, 61, 64, 66, 67, 72, 79, 82, 83, 88, 112, 121, 127, 128, 135, 136, 146, 158, 167, 171, 200 industrialization, 57 industry, 69 infancy, 73 infarction, 115 infection, x, xi, 22, 24, 33, 40, 55, 68, 73, 83, 96, 97, 98, 101, 122, 134, 143, 151, 159, 160, 162, 163, 165, 166, 167, 168, 169, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219 infections, 19, 29, 69, 84, 96, 99, 133, 134, 149, 153, 157, 213 infectious, 33, 53, 96, 97 infiltration, 54 inflammation, vii, viii, ix, x, xi, 1, 16, 17, 19, 20, 25, 29, 30, 39, 46, 49, 50, 53, 55, 56, 57, 59, 73, 79, 80, 95, 99, 109, 110, 129, 135, 139, 143, 151, 152, 153, 155, 161, 175, 205, 207, 208, 209, 212, 214, 216 inflammatory, vii, viii, ix, xi, 1, 12, 13, 16, 19, 20, 21, 22, 23, 24, 25, 29, 30, 33, 40, 44, 49, 50, 51, 53, 54, 56, 59, 71, 72, 73, 74, 76, 77, 79, 80, 81, 83, 84, 85, 90, 92, 93, 100, 102, 112, 118, 125, 133, 134, 135, 136, 139, 140, 141, 143, 146, 147, 152, 153, 155, 157, 158, 166, 169, 177, 189, 205, 206, 207, 208, 211, 212, 214, 215, 219 inflammatory bowel disease, vii, viii, ix, 1, 22, 49, 50, 59, 71, 72, 73, 74, 76, 134, 136, 139, 140 inflammatory cells, viii, 79, 81, 84, 85, 155, 177 inflammatory disease, vii, 13, 19, 25, 30, 33, 50, 140, 152, 208, 211 inflammatory mediators, x, 24, 29, 118, 144, 153, 155, 169, 189, 206, 207 inflammatory response, 16, 20, 23, 24, 40, 50, 53, 74, 102, 125, 153, 155, 189 inflammatory responses, 16, 24, 53, 74, 189 infliximab, viii, 25, 26, 27, 30, 36, 38, 39, 43, 49, 51, 60, 62, 64, 65, 68, 69, 70, 76, 133, 134, 137, 138, 139, 140, 141, 211, 214, 216, 217, 218, 219 influenza, 99 informed consent, 153 infusions, 64, 68, 133 inguinal, 103, 134 inheritance, 175 inherited, 12, 41

Index inhibition, 16, 27, 36, 39, 45, 54, 109, 114, 115, 119, 121, 130, 131, 132, 135, 141, 146, 174, 177, 182, 200, 202, 208 inhibitor, 2, 15, 17, 21, 44, 45, 60, 61, 88, 90, 108, 119, 140, 146, 148, 153, 174 inhibitors, 12, 22, 37, 38, 43, 45, 54, 88, 89, 94, 138, 141, 146 inhibitory, xii, 2, 15, 21, 22, 92, 145, 161, 163, 205 initiation, 14, 41, 133, 135, 136, 146, 210 injection, 97, 103, 112, 133, 134, 136, 146, 207 injections, 96 injuries, 24, 45, 82 injury, 19, 24, 25, 32, 36, 39, 42, 43, 45, 46, 47, 48, 54, 59, 82, 90, 100, 101, 125, 180, 189, 206, 207, 208, 209, 211, 212, 213, 215, 219 innate immunity, 29, 30, 91 iNOS, 118, 119 inositol, 2, 17 insertion, 33 insight, 202 instability, 191, 194, 197 insulin, vii, 2, 20, 39, 61, 100 insulin resistance, vii, 20, 100 insulin signaling, 20 insults, 21, 175 integrin, 55, 75, 113, 129, 141 integrity, 21, 116, 117, 119, 132, 172, 173, 176, 189, 191, 193 interaction, viii, 7, 13, 19, 21, 55, 79, 89, 90, 91, 123, 145, 148, 193, 195 interactions, 14, 27, 29, 166 intercellular adhesion molecule, 2, 58 interface, 13, 87, 89 interferon (IFN), 2, 6, 12, 21, 31, 30, 44, 54, 55, 58, 59, 60, 74, 83, 85, 86, 93, 102, 111, 113, 123, 124, 129, 134, 135, 140, 141, 146, 147, 166, 206, 207, 208, 209, 211, 212, 214, 216, 217, 219 interferon gamma, 54, 58, 111, 129 interferon-γ, 2, 6 interleukin, 2, 3, 6, 42, 44, 47, 53, 54, 58, 71, 72, 85, 86, 91, 141, 146, 152, 158, 159, 163, 213, 216, 219 interleukin-1, 3, 20, 42, 44, 47, 53, 54, 71, 141, 152, 158, 213, 215, 216, 219 interleukin-17 (IL-17), 54 interleukin-2, 54 interleukin-6, 42, 72, 91, 152, 219 internalization, 21, 34, 37, 43, 44, 60, 199 interrelations, 22 interstitial, 55, 72, 180

233

intervention, 24, 50, 52, 155, 174 intestinal obstruction, 51, 67, 68 intestinal tract, 73 intestinal villi, 51 intestine, viii, 52, 53, 60, 74, 79, 84, 90, 190, 194 intracellular signaling, 15, 17, 36 intracranial, 195 intraocular, 139 intraperitoneal, 109 intravascular, 103, 106, 111, 116, 128, 181 intravenous, 62, 125 intravenously, 180 intrinsic, 7, 22, 173, 189 intron, 7 introns, 7, 100 invasive, xi, 33, 53, 57, 74, 86, 172, 182, 183, 184, 190, 193, 197, 198, 199, 200, 201 invasive cancer, xi, 172, 193 invasive species, 53 inversion, 184 involution, 146 ion channels, 176 ionizing radiation, 149, 187, 194 Ireland, 49, 56 irradiation, 6, 147, 198 IRS, 2, 20 ischaemia, 45 ischemia, 6, 24, 36, 37, 43, 47, 48, 100, 176, 180, 188, 204 ischemia reperfusion injury, 24 ischemic, 24, 47, 114, 176, 180, 188 ischemic heart disease, 188 Islam, 202 island formation, 130 isolation, 99, 102, 103 Israel, 64, 65, 72 Italy, 56, 171, 191, 207, 214

J JAMA, 70 Japan, 72, 79, 92, 93, 151, 152, 153, 154, 156, 205, 207, 215, 217 Japanese, x, 56, 73, 151, 152, 153, 155, 157, 158, 214 JNK, 2, 14, 21, 22, 38, 43, 46, 210, 214 joint destruction, 100 joints, 46 Jordan, 213 Jun, 2, 17, 73, 74, 157, 158, 163, 168, 169

Index

234 Jung, 213 juvenile idiopathic arthritis, 134, 138

K kappa, xii, 2, 17, 31, 32, 33, 37, 39, 43, 45, 154, 205, 206, 212, 217 kappa B, xii, 31, 33, 37, 43, 205, 206, 217 keratinocytes, 3 Ki-67, 178, 198 kidney, viii, x, 24, 79, 84, 85, 93, 102, 103, 143, 145 killer cells, 149, 209 killing, 212 kinase, 2, 12, 15, 17, 20, 22, 32, 34, 35, 37, 48, 60, 89, 90, 130, 131, 213, 214 kinases, vii, 1, 14, 17, 46, 146, 202, 214 kinetic parameters, 27 kinetics, 198 knockout, 53

L L1, 51 L2, 51 labeling, 2, 197 Lactobacillus, 71 LAK, 20 lamina, 53, 58, 59, 71, 74, 182 Langerhans cells, 99 laser, 73 lead, xi, 205, 210 leakage, 126 leishmaniasis, 23, 33 leprosy, 23, 44 lesions, viii, 50, 79, 83, 91, 103, 104, 105, 111, 133, 134, 135, 136, 137, 138, 139, 140, 181, 183, 190, 191, 209 leucocyte, 24 leukaemia, 163 leukemia, x, 2, 32, 131, 159, 160, 161, 163 leukemic, 149, 163 leukocyte, 29, 39, 40, 46, 47, 55, 58, 109, 110, 112, 113, 129, 154, 156 leukocytes, 54, 60, 99, 114, 161, 195 licensing, 63, 65 life span, 149 lifestyle, 57 lifestyle changes, 57 Li-Fraumeni syndrome, 203

ligand, vii, 1, 2, 4, 5, 6, 7, 8, 11, 12, 13, 17, 18, 27, 33, 37, 38, 43, 47, 48, 68, 80, 82, 89, 91, 101, 113, 129, 147, 173, 200, 209, 212, 214, 219 ligands, 6, 11, 13, 14, 17, 18, 28, 30, 121 limitation, 173 linear, 183 linkage, 56, 134, 138 links, 6, 57 linoleic acid, 146 lipid, vii, 20, 21, 39, 97, 191, 192 lipid metabolism, vii lipid rafts, 21, 39 lipids, 101, 157 lipofuscin, 192 lipopolysaccharide, 2, 3, 42, 84, 97, 152, 157, 158, 207, 209 lipopolysaccharides, 19 Listeria monocytogenes, 209, 218 literature, 172, 188, 194 liver, 20, 81, 82, 84, 85, 87, 90, 91, 92, 102, 103, 105, 131, 166, 201, 206, 207, 208, 209, 211, 212, 213, 214, 215, 219 liver cancer, 206 liver cirrhosis, 207, 215 liver damage, 214 liver disease, 92, 209, 211, 219 liver failure, 81, 90, 209 liver transplant, 81, 90 liver transplantation, 81 living conditions, 57 localization, 42, 116, 123, 192 locus, 3, 46 London, 194 long period, 19 longevity, 59 low power, 58 low-grade inflammation, 153 LPS, 2, 27, 32, 84, 85, 97, 98, 99, 152, 155 LTA, 23, 25 luciferase, 23 lumen, 53, 58, 85, 110, 177, 178, 180, 182, 200 luminal, 53, 56, 61, 83, 84, 182, 183, 184, 196 lung, x, 24, 34, 38, 105, 119, 143, 145, 175, 178, 181, 184, 187, 190, 196, 197, 203 lung cancer, 38, 203 lungs, 24 lupus, 64, 68, 69, 70, 90, 135, 136, 140 lupus erythematosus, 22, 31, 136, 140 lymph node, 53, 103, 104, 105, 185 lymphatic, 104, 105

Index lymphocyte, viii, 20, 40, 49, 53, 54, 55, 58, 59, 67, 146, 160, 162 lymphocytes, x, 2, 3, 12, 19, 53, 54, 55, 58, 59, 62, 68, 75, 81, 82, 91, 99, 135, 144, 159, 161, 162, 166, 191, 200, 210, 213 lymphoid, vii, 1, 12, 18, 19, 29, 36, 40, 41, 53, 99 lymphoid cells, 18, 19, 99 lymphoid tissue, 19, 41 lymphoma, x, 2, 33, 34, 64, 68, 141, 159, 160, 161, 163 lymphomas, 69, 174 lysis, 29, 199 lysophosphatidic acid, 199 lysosome, 188 lysosomes, 177

M mAb, 26 macromolecules, 110 macrophage, viii, 2, 6, 32, 49, 54, 58, 59, 61, 74, 119, 124, 128, 152, 155, 182, 195, 199, 217 macrophages, 2, 3, 6, 12, 19, 20, 42, 54, 59, 87, 89, 93, 98, 99, 109, 111, 112, 119, 134, 146, 147, 152, 189, 190, 195, 206, 208 maintenance, viii, xi, 49, 52, 53, 64, 65, 66, 67, 69, 70, 76, 171, 172, 187, 193 major histocompatibility complex, 2, 3, 4, 33, 124 malabsorption, 55 malaria, xi, 22, 23, 38, 41, 100, 159, 161, 162, 165, 166, 167, 168, 169 males, 56 malignancy, 96, 180, 183 malignant, x, 86, 96, 105, 108, 122, 126, 144, 159, 178, 189, 191, 196 malignant cells, 86, 108 malignant melanoma, 126 malignant mesothelioma, 196 malignant tumors, 86, 122, 189 malnutrition, 50 mammalian cells, 191 mammals, 148 mammography, 183 management, viii, 49, 50, 51, 52, 59, 93, 139, 140, 141, 187, 194 manufacturer, 154 MAPK, 2, 43 MAPKs, 17, 146 marrow, 103 mast cells, 3, 12, 19

235

matrix, viii, 2, 30, 45, 49, 58, 61, 86, 90, 93, 94, 99, 158, 175 matrix metalloproteinase, 2, 45, 61, 86, 90, 93, 94, 158 maturation, 54, 55, 115, 131, 189 maturation process, 131 MCA, 182 MCP, 2, 72 MCP-1, 2, 72 measurement, 126, 187 measures, 27, 52 mechanical stress, 181 mechanical ventilation, 47 media, 12 median, 64, 65, 152, 154 mediation, 33, 202 mediators, x, 19, 20, 22, 24, 29, 110, 118, 144, 153, 155, 158, 166, 169, 189, 196, 206, 207 medication, ix, 136 medications, 51, 54, 133, 134 medicine, 62, 139, 211 megakaryocytes, 111 MEK, 17 melanoma, ix, x, 25, 35, 43, 95, 102, 103, 104, 105, 106, 107, 110, 111, 112, 113, 114, 115, 117, 121, 126, 127, 128, 129, 130, 143, 145, 190, 200 melatonin, x, 143, 144, 145, 146, 147, 148, 149 membranes, 84 men, 213 meningitis, 100 mesangial cells, 189 mesentery, 51, 59 mesothelioma, 196 meta-analysis, 31 metabolic, 22, 145 metabolic pathways, 22 metabolism, vii, ix, 95, 103, 145 metabolite, 144, 146, 148 metabolites, 54 metalloproteinase, viii, 6, 32, 43, 79 metalloproteinases, 45, 86, 185 metaphase, 196 metastases, ix, 43, 95, 102, 104, 105, 106, 110, 113, 117, 129, 178, 185, 196 metastasis, viii, x, 80, 86, 104, 105, 125, 144, 172, 199, 201, 208 metastatic, 96, 99, 105, 178, 185, 190, 196, 199, 200 metastatic disease, 96 methotrexate, 136 methylation, 209

236

Index

MHC, 2, 23, 54, 55 mice, 3, 14, 31, 44, 45, 56, 62, 71, 76, 97, 98, 99, 102, 109, 114, 115, 117, 119, 121, 123, 127, 130, 146, 147, 149, 204, 207, 209, 213, 215, 216 microbes, 96 microbial, 29, 53, 71, 157 microbiota, 57 microenvironment, 36, 109, 116, 117, 191, 194 microflora, 53, 56 micronucleus, 202 microparticles, 162 microscope, 180, 191 microscopy, 111, 173, 184, 185, 187, 188, 190, 191, 193 microtubule, 12, 196, 202 microvascular, 47, 55, 60, 72, 111, 117, 131, 132, 168 microvasculature, 58, 118, 132 Middle East, 56 middle-aged, 81 migration, viii, 17, 20, 21, 80, 86, 87, 88, 89, 90, 93, 99, 114, 135 mimicking, 26 Ministry of Education, 156 minority, 133, 136 mirror, 120 mitochondria, 173, 174, 176, 186, 188 mitochondrial, 21, 22, 45, 81, 173, 175, 176, 188, 195, 198, 199 mitochondrial toxins, 195 mitogen, 2, 15, 54, 89, 90, 114, 149 mitogen-activated protein kinase, 2, 89, 90 mitogenesis, 146 mitogenic, 108 mitosis, 174, 186, 198 mitotic, xi, 171, 172, 174, 181, 183, 185, 186, 193, 195, 196, 198, 200, 201 mixing, 98 MMP, viii, 2, 79, 86, 87, 88, 89, 90, 93 MMP-9, viii, 79, 86, 87, 88, 89, 90, 93 MMPs, 86 mobility, 88 modality, 41 model system, 30 models, x, 24, 53, 102, 108, 112, 113, 117, 121, 127, 145, 147, 148, 159, 160, 161, 176, 182, 185, 218, 219 modulation, 31, 58, 162, 163 modules, 11 molecular biology, 162, 215

molecular changes, 116 molecular mass, 6, 7, 112 molecular mechanisms, 59, 90, 108, 149, 174, 176, 200 molecular structure, 30 molecules, 11, 13, 16, 18, 20, 22, 26, 29, 55, 60, 80, 84, 86, 88, 90, 91, 93, 98, 99, 110, 112, 113, 116, 117, 118, 121, 124, 144, 147, 162, 163, 187, 208 monoclonal, viii, 2, 25, 33, 36, 40, 44, 46, 47, 48, 49, 62, 67, 75, 76, 77, 82, 130, 131, 141, 202, 211, 219 monoclonal antibodies, 2, 26, 33, 47, 61, 62, 131 monoclonal antibody, viii, 36, 44, 46, 48, 49, 67, 75, 76, 77, 82, 131, 141, 202, 211, 219 monocyte, 128, 129, 152, 154, 155, 156, 208, 214, 216 monocytes, 3, 19, 33, 46, 72, 99, 112, 123, 152, 155, 166, 190, 206, 208, 209 monolayer, 118, 132 monomer, 6, 19, 27 monomers, 6, 12, 13, 15, 18 mononuclear cell, 42, 59, 84, 87, 146, 206, 208, 210 mononuclear cells, 42, 59, 84, 87, 146, 208, 210 monotherapy, 137 morbidity, 65, 105, 180 morning, 147, 154 morphogenesis, 93, 131, 182 morphological, xi, 166, 171, 172, 176, 177, 181, 185, 187, 192, 193, 195, 197, 198, 201 morphology, xi, 71, 134, 138, 172, 175, 177, 185, 187, 193, 203 mortality, 24, 40, 167, 169, 181, 215, 216 mouse, 3, 11, 26, 37, 47, 53, 71, 75, 97, 98, 99, 119, 120, 123, 131, 152, 198, 207, 211, 218 mouse model, 53, 119, 120, 218 mouth, 50, 155, 158 movement, 117 mRNA, 4, 6, 8, 20, 44, 84, 88, 89, 100, 115, 134 mucin, viii, 55, 72, 79, 83, 84, 85, 92, 178, 184 mucosa, viii, 50, 53, 57, 58, 59, 60, 79, 84, 85, 92 mucus, 93 multicellular organisms, xi, 171 multiple alleles, 3 multiple sclerosis, vii, 1, 22, 33, 38, 100 multiplication, 161 multiplicity, 134 multivariate, 61 murine model, 32, 114, 207, 219 muscle, ix, 3, 20, 36, 47, 48, 58, 72, 95, 99, 105, 107, 189

Index muscle atrophy, 20 muscle cells, 3, 72, 99, 189 mutagenesis, 7 mutant, 56, 175, 219 mutants, 40, 46 mutation, 26, 56, 61, 73, 175, 181, 182, 183, 185, 194 mutations, xi, 12, 36, 41, 42, 45, 56, 73, 122, 171, 172, 173, 175, 181, 183, 197, 203 mycobacteria, 99 mycobacterium, 73, 74 myeloid, 19, 54, 59, 99 myeloid cells, 19, 59, 99 myeloma, 62 myeloperoxidase, 82, 112 myocardial infarction, 115, 131 myocardium, 131 myocyte, 203 myofibroblasts, 75 myosin, 60

N Na+, 176, 202 N-acety, x, 143, 147 N-acetylserotonin, x, 143, 147 natural, 2, 3, 20, 26, 43, 44, 105, 140, 149, 209, 215 natural killer, 2, 3, 20, 43, 209 natural killer cell, 43, 209 Nd, 140 neck, 96, 105 necrotic cell death, 176, 193 neonatal, 26 neonates, 23 neoplasia, 187 neoplasm, 92, 172 neoplasms, xi, 68, 149, 172 neoplastic, ix, 70, 80, 84, 90, 93, 99, 145, 163, 178, 180, 182, 185, 190, 191, 194 neoplastic cells, 190, 191, 194 neovascularization, 109, 114, 115, 117 nephrectomy, 196 nerve, ix, 10, 95, 105, 107 nerve growth factor, 10 Netherlands, 112, 119, 121 network, 19, 22 neuroendocrine, 147, 149, 200 neurohormone, 149 neuronal death, 201 neurons, 202

237

neutralization, viii, 30, 49, 59, 64, 69, 70, 208, 209 neutrophil, 51, 154, 156, 172, 180, 190, 191, 192, 193, 195 neutrophils, 12, 20, 54, 99, 152, 178, 180, 185, 189, 190, 192, 197, 198 New Orleans, 102 New York, 50, 62, 96, 105 NFkB, 116 NF-κB, 2, 15, 19, 21, 80, 84, 85, 89 Nielsen, 201, 202 nitrate, 119, 121 nitric oxide (NO), 82, 128, 132 nitrogen, ix, 96, 102, 107, 148 nitrosative stress, 82 NK, 2, 3, 15, 19, 20, 29, 132, 146, 147, 213, 214 NK cells, 19, 29, 146, 213 NK-cell, 147 Nobel Prize, 62 nodes, 185 nodules, ix, 96, 104, 107 non-Hodgkin lymphoma, 141 non-infectious, 55 non-small cell lung cancer, 203 normal, ix, xi, 2, 12, 22, 29, 46, 53, 56, 74, 81, 90, 92, 95, 98, 107, 108, 111, 114, 116, 117, 118, 121, 138, 147, 148, 171, 175, 190, 196, 218 normal conditions, 53 North America, 57, 64, 126 NOS, 118, 119, 120, 121 N-terminal, 2, 7, 11, 13, 17, 22, 26, 116 nuclear, xi, xii, 2, 17, 21, 31, 33, 37, 68, 70, 80, 82, 84, 90, 93, 108, 149, 171, 172, 179, 180, 181, 185, 187, 191, 192, 193, 197, 205, 206, 217 nuclear factor, 45, 206, 212 nuclear factor kappa, 206 nucleation, 202 nuclei, 85, 175, 179, 187 nucleic acid, 92 nucleocapsids, xii, 205, 209 nucleoli, 186 nucleolus, 200 nucleotides, 23, 56 nucleus, 16, 85, 145, 174, 175, 179, 180, 186, 192, 193 nutrient, 188, 191, 194 nutrients, 178, 188

O obesity, 37, 153

Index

238

observations, ix, 51, 56, 57, 85, 96, 97, 98, 108, 109, 136, 191 obstruction, 61, 181 occlusion, 115, 181 OCT, 38 oedema, 109 oligodendrocytes, 100 oligomeric, 13 oligomerization, 56 oligomers, 12 omission, 194 oncogene, 46, 130, 163, 174, 195, 196, 213 oncogenesis, 16 oncological, 104 oncology, 34, 118, 126, 163, 168, 213 oncoproteins, 182 oncosis, 172, 176, 200 onycholysis, 134 optimism, 97 oral, x, 51, 71, 151, 152, 153, 154, 155, 158, 162, 202 oral cavity, x, 151, 152, 155 oral health, x, 151, 153, 155, 158 organ, 15, 19, 20, 24, 47, 62, 81, 101, 102 organelles, 172, 176, 188, 192 osmium, 191 osmotic, 175 osteoclastogenesis, 40, 152 osteoporosis, vii, 1 osteosarcoma, 196 ovarian cancer, 93, 187 ovaries, 199 ovary, 144, 184, 200 oxidants, 20 oxidative, 40, 54, 82, 83, 92 oxidative stress, 40, 82, 83, 92 oxide, 35, 128, 132, 215 oxygen, 148, 176, 178, 182

P p38, 17, 38, 46, 89, 90, 214 p53, xi, 83, 171, 172, 173, 174, 181, 182, 183, 185, 187, 191, 194, 197, 198, 199, 201, 203, 204 pain, 20, 24, 50 palmoplantar pustulosis, 135, 138, 139, 141 pancreas, 24, 84, 145, 184, 190 pancreatic, 197, 203 pancreatitis, 24, 31, 35, 39, 43 paracrine, 55, 81, 149

paradox, 139 paradoxical, ix, 135, 136, 137 parasite, 166 parasites, 99, 165 parenchymal, 19 parenchymal cell, 19 parenteral, 51 Paris, 162, 169 pars tuberalis, 145 particles, 175 passive, 148, 176 pathogenesis, vii, viii, xi, 1, 30, 50, 53, 60, 70, 71, 79, 80, 81, 82, 85, 90, 135, 137, 140, 141, 153, 162, 165, 211, 218 pathogenic, x, 19, 45, 53, 135, 144, 207, 215, 216 pathogens, 22, 56, 82, 152, 153, 155, 157, 165 pathologists, 172, 183, 187, 194 pathology, xi, 159, 162, 165, 168, 171, 203 pathophysiological, 110, 112, 119, 157 pathophysiology, viii, ix, 49, 58, 79, 80, 90, 135, 136, 159 pathways, 16, 17, 19, 22, 27, 31, 43, 47, 58, 77, 80, 101, 120, 131, 145, 147, 173, 175, 176, 189, 195, 198, 212 pattern recognition, 56 PBC, viii, 79, 80, 81, 82, 83, 90 PBMC, 206, 208 PCR, 2, 23, 73, 88 PDC, 81 pediatric, 103 peers, 57, 96 Pennsylvania, 95, 109, 114, 117, 119 peptidase, 2 peptide, 2, 3, 6, 19, 88 peptides, 53, 88 perforin, 82 perfusion, ix, 24, 25, 34, 35, 43, 47, 95, 102, 103, 111, 116, 117, 126, 127, 128, 129, 130, 132 periodic, 12, 139, 178 periodontal, x, 151, 152, 153, 155, 157, 158 periodontal disease, x, 151, 153, 157 periodontitis, 152, 153, 154, 155, 156, 157, 158 peripheral blood, 40, 121, 123, 155, 208 peripheral blood mononuclear cell, 208 peripheral nerve, ix, 95, 105, 107 peritoneal, 130 permeability, viii, 49, 60, 112, 115, 116, 117, 118, 131, 132, 188 permeabilization, 198 permeation, 119

Index perseverance, 97 perturbations, 53 PG, 195 pH, 180 phage, 121 phagocytic, 189, 193 phagocytosis, 20, 54, 177, 190, 192, 195 pharmaceutical, 69 pharmaceutical industry, 69 pharmacodynamics, 39 pharmacokinetic, 30 pharmacokinetics, 39, 127 pharmacological, 50, 145, 146 phenotype, 39, 51, 54, 55, 58, 61, 73, 74, 108, 175, 177, 179, 183, 197, 213 phenotypes, 35, 54, 61, 75, 84 Philadelphia, 95 phorbol, 12, 47 phosphate, 191 phospholipase C, 145 phosphorylates, 15, 32 phosphorylation, vii, 1, 8, 14, 15, 20, 43, 90, 118, 132, 146 photographs, 106 photoperiod, 144 physicians, 96, 112 physiological, ix, xi, 3, 21, 31, 34, 53, 92, 95, 118, 144, 145, 148, 171, 175 physiology, 3, 39, 131, 144 PI3K, 2, 17 pig, 131 pigs, 98 pilot study, 218 pineal, ix, 143, 144, 149 pineal gland, ix, 143, 144 pinealectomy, 146 PKC, 12 placebo, 38, 62, 64, 65, 66, 67, 68, 69, 75, 77, 217, 218, 219 placenta, 144 placental, 118, 132 plants, 144 plaque, 134, 135, 139, 157 plaques, 51, 100, 135 plasma, 6, 12, 15, 26, 35, 64, 100, 119, 121, 154, 172, 175, 176, 186, 199, 208, 209, 210 plasma levels, 35, 64, 210 plasma membrane, 6, 12, 15, 100, 172, 175, 176, 186, 199 plasminogen, 153

239

plasmodium falciparum, 166, 167, 168, 169 plasmodium malariae, 167 plasmodium ovale, 167 plasmodium vivax, 166, 168, 169 plastic, 153 plasticity, 35 platelet, 12, 111, 112, 128, 154, 156 platelet activating factor, 12 platelet aggregation, 112, 128 platelet count, 154, 156 platelets, 162, 166, 168 play, viii, x, 19, 30, 58, 79, 81, 82, 83, 143, 145, 146, 147, 148, 211 pleural, 203 pleuritis, 32 pneumonia, 23, 47, 155 polarity, 184 polarization, 54, 185 polycystic kidney disease, 22 polyethylene, 26, 67 polymerase, 2 polymerase chain reaction, 2 polymorphism, 4, 23, 31, 33, 34, 37, 38, 39, 42, 44, 47, 61, 175, 210, 211, 213, 214, 216 polymorphisms, xii, 2, 23, 30, 31, 32, 33, 35, 38, 40, 47, 56, 75, 205, 211, 212, 213, 214, 215, 217, 218, 219 polymorphonuclear, 99, 110, 112, 128, 195 polymorphonuclear cells, 112 polypeptide, 26, 99, 100, 112, 128, 129 polysaccharide, 97 pools, 196 poor, xi, 25, 106, 161, 165, 172, 174, 183, 189, 190, 196, 197 population, 31, 56, 59, 69, 72, 73, 96, 135, 140, 153, 157, 178, 214 pore, 29 positive correlation, 101 potassium, 145 potassium channels, 145 power, 58 preclinical, 30, 43, 44, 101, 102, 108, 109, 112, 113, 116, 117, 119, 121, 125 pre-clinical, 64 prediction, 22, 196 predictors, 43 predisposing factors, 134 prednisone, 51 premenopausal, 149 premenopausal women, 149

240

Index

pressure, 153, 180 prevention, xi, 148, 205 preventive, 146, 147 PRI, 5 primary biliary cirrhosis, viii, 79, 80, 81, 91, 92 primary prophylaxis, 214 primary tumor, 180, 199 priming, 147 pro-apoptotic protein, 21, 59, 173, 198 probe, 153 probiotic, 58 procoagulant, 112, 128, 216, 217 production, vii, viii, x, xii, 1, 6, 19, 20, 23, 24, 25, 27, 29, 30, 32, 33, 36, 38, 40, 42, 54, 55, 56, 62, 70, 72, 74, 80, 82, 87, 88, 89, 94, 98, 99, 112, 114, 119, 122, 124, 135, 141, 143, 146, 147, 166, 175, 176, 181, 184, 199, 205, 208, 209, 210, 217 pro-fibrotic, 61 prognosis, xi, 25, 86, 92, 104, 105, 172, 174, 182, 183, 184, 190, 196, 197 prognostic marker, 24, 30, 167, 172, 203 program, 2, 34, 188 proinflammatory, vii, xi, 19, 22, 29, 40, 48, 54, 71, 83, 99, 112, 128, 147, 152, 157, 189, 205, 206, 207, 208, 215 proliferation, vii, x, 1, 16, 17, 21, 29, 30, 36, 54, 55, 61, 75, 83, 87, 100, 109, 129, 144, 145, 146, 148, 149, 181, 182, 183, 194, 203, 208, 209, 214, 218 promote, 187, 189, 208, 214 promoter, xii, 7, 23, 32, 34, 37, 38, 39, 41, 42, 44, 113, 167, 168, 169, 205, 210, 211, 212, 213, 214, 215, 216, 219 promoter region, 23, 38, 41, 210, 216 propagation, 191 property, ix, 95, 159, 190 prophase, 187 prophylaxis, 70, 214 prostaglandin, 20, 42, 44, 87, 94 prostaglandins, 6, 87 prostanoids, 6 prostate, x, 12, 25, 41, 42, 143, 145, 147, 148, 149, 184, 188 prostate cancer, 25, 41, 42, 145, 147, 148, 149, 188 proteases, x, xi, 2, 27, 144, 171, 173, 176 protection, 21, 162, 167, 168, 169, 188, 191, 207, 209 protective role, 207, 209, 211 protein, vii, 1, 2, 3, 6, 7, 12, 14, 15, 16, 17, 19, 20, 22, 25, 26, 28, 34, 35, 37, 39, 41, 42, 56, 60, 61, 72, 75, 80, 84, 89, 92, 93, 98, 99, 100, 101, 108,

111, 112, 113, 114, 115, 116, 117, 130, 131, 146, 148, 163, 175, 183, 189, 199, 201, 204, 206, 207, 209, 210, 213, 214, 215, 216, 217, 218, 219 protein binding, 15 protein kinase C, 2, 12, 20 protein kinases, vii, 1, 14, 146 protein sequence, 100 protein synthesis, 108 proteins, vii, xi, 5, 8, 14, 15, 16, 21, 22, 26, 29, 35, 45, 54, 56, 60, 80, 101, 108, 116, 118, 132, 144, 145, 147, 173, 174, 182, 183, 188, 198, 201, 202, 205, 210, 214 proteobacteria, 57 proteolysis, 6, 36 proteolytic enzyme, 12 protocol, 154 protons, 198 pseudo, 10 psoriasis, ix, 21, 22, 29, 133, 134, 135, 136, 137, 138, 139, 140, 141 psoriatic, 31, 133, 134, 135, 136, 137, 138, 139, 140, 141, 216 psoriatic arthritis, 31, 133, 134, 139, 140, 216 psychological stress, 134 psychological stressors, 134 public health, xi, 165, 205, 206 pulmonary edema, 101 purification, 62, 122, 124 P-value, 156 pyruvate, 81

Q quality of life, 136, 147 Quinones, 212

R radiation, 149, 187, 194, 198, 200 radio, 131 radioactive tracer, 104 radiolabeled, 116 radiotherapy, 105, 163 random, 96, 192 range, vii, ix, x, 7, 11, 13, 27, 62, 95, 106, 118, 144, 145, 152, 154, 156, 174, 177, 183, 193 RANKL, 2, 6, 157 RANTES, 2, 167, 169 rash, 134

Index rat, 46, 48, 84, 85, 91, 92, 93, 112, 114, 115, 119, 120, 127, 128, 130, 131, 211, 214 rats, viii, 79, 84, 85, 98, 109, 127, 129, 149, 209 reactive oxygen, 54, 82, 145, 148, 176 reactive oxygen species (ROS), 54, 82, 83, 145, 146, 147, 176 reactivity, 35, 185, 218 reading, 11 recall, 108 receptors, vii, viii, 1, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 25, 26, 30, 31, 33, 34, 35, 39, 40, 41, 43, 44, 45, 46, 47, 49, 80, 81, 82, 84, 91, 92, 93, 101, 108, 114, 116, 123, 124, 130, 135, 141, 145, 146, 148 recognition, ix, 33, 56, 136 recombinant human monoclonal antibody, 44 recombination, 114 recovery, 37, 114, 115, 211 rectum, 53 recurrence, 57, 65, 76, 104, 135, 174 red blood cell, 179 red blood cells, 179 redistribution, 60, 160 redness, 20 redox, 145, 148, 162 refractory, 21, 22, 41, 52, 64, 77 regeneration, 19, 21, 90, 109, 209, 212 regression, 30, 96, 98, 102, 111, 114, 115, 120 regular, 11, 116 regulation, 6, 12, 17, 22, 29, 30, 31, 33, 40, 43, 60, 73, 81, 84, 85, 88, 89, 90, 93, 94, 112, 116, 119, 124, 129, 130, 132, 140, 145, 148, 158, 175, 187, 191, 206, 208, 209 regulators, 6, 12, 16, 116, 195 rejection, 42, 43, 62, 75, 100 relapse, 64, 65, 69, 182 relationship, x, 29, 56, 84, 151, 153, 155, 158, 160, 162, 163, 172, 179, 180, 181, 184, 203, 215 relationships, 16, 157 relevance, 29, 37, 47, 73, 190, 218 remission, viii, 49, 52, 54, 59, 64, 66, 67, 69, 76, 98, 133, 141, 206 remodeling, viii, 49, 58 remodelling, 58, 61 renal, 24, 25, 39, 42, 45, 75, 120, 125, 196, 199, 202, 215 renal cell carcinoma, 25, 42, 45, 125, 196, 199, 202 renal failure, 215 renal function, 39, 120 renal replacement therapy, 24

241

repair, 61, 74, 174, 187 reperfusion, 24, 36, 37, 43, 45 replication, xii, 113, 187, 194, 205, 209, 213, 217 repression, 54 reproduction, 144 resection, 51, 52, 65, 76, 104, 105, 126 reservoir, 103 residues, 4, 7, 8, 10, 11, 13, 18, 21, 26, 35, 100, 101 resistance, vii, 12, 20, 32, 38, 42, 58, 59, 60, 68, 71, 74, 100, 108, 122, 123, 173, 181, 201 resistin, 158 resolution, 35, 107, 133, 136 resources, 115, 153 respiratory, 23, 47, 100, 125, 167, 169, 207, 213 respiratory failure, 23, 47 retardation, 50 retention, 62 reticulum, 176, 186, 191 retina, 144, 145 retinoids, 136 retinopathy, 114 retrovirus, 160 retroviruses, 162, 163 rheumatic, 34, 134, 137, 211, 212 rheumatic diseases, 34, 137 rheumatoid arthritis, vii, 1, 2, 21, 22, 29, 35, 36, 37, 38, 40, 46, 48, 62, 67, 75, 77, 100, 133, 134, 137, 138, 140, 217 rhythms, 144, 145 ribosomal, 91 ribosomal RNA, 91 rice, 38 risk, 24, 25, 32, 33, 37, 40, 42, 47, 56, 57, 70, 73, 104, 126, 153, 167, 169, 174, 189, 199, 206, 208, 214 risk factors, 153, 214 rituximab, 135, 141 RNA, 160, 207, 211 rodents, 64 rolling, 60 rural, 217

S safety, viii, 29, 30, 48, 49, 64, 68, 70, 76, 137 salivary glands, 178 sampling, 53 sanitation, 57 sarcomas, ix, 3, 25, 95, 96, 102, 104, 105, 106, 118, 126, 127, 129, 130

242

Index

satellite, 104 scalp, 139 scavenger, 144, 148 schema, 104 Schiff, 36, 137, 216 Schmid, 126 scores, 51, 215 SCP, 175 SDF-1, 86, 87, 89, 90, 93 search, 3 searching, 98 secrete, 98, 99, 189, 208 secretion, viii, 43, 54, 55, 59, 79, 90, 93, 152, 155, 158, 165, 184, 213 seizures, 31 selectivity, 117 self, vi, 171 self-destruction, 173 senescence, viii, 71, 79, 82, 83, 90, 92, 201, 203 sensitivity, 19, 22, 108, 112, 141, 173 separation, 53, 115, 123, 192 sepsis, vii, 1, 22, 23, 24, 25, 32, 33, 34, 40, 42, 43 septic shock, 19, 35, 101, 102, 118, 125 sequencing, 23 series, xi, 171 serine, 2, 8, 14, 15, 20, 34 serum, x, 3, 12, 31, 33, 67, 82, 99, 122, 143, 145, 151, 152, 153, 154, 155, 156, 157, 158, 160, 162, 163, 166, 167, 211, 212 severe acute respiratory syndrome, 213 severity, 23, 25, 31, 37, 50, 84, 153, 167, 169, 211 sex, 145 shape, 11, 177 shares, 17 shock, 19, 20, 23, 25, 35, 47, 101, 102, 118, 125, 132, 196, 200, 217 short-term, 76 sibling, 190 siblings, 57 side effects, 25, 30, 133, 138, 207, 216 sigmoid, 60 sigmoid colon, 60 signal peptide, 2, 6, 19 signal transduction, 14, 32, 47, 80, 101 signaling, vii, 1, 3, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 27, 31, 32, 33, 34, 35, 37, 38, 41, 44, 45, 46, 82, 83, 85, 89, 91, 92, 94, 121, 125, 145, 147, 148, 176, 202, 208, 209, 210, 213 signaling pathway, vii, 1, 16, 17, 18, 27, 38, 83, 85, 89, 92, 145, 148, 176, 210

signaling pathways, 17, 18, 27, 38, 89, 145, 148, 176 signalling, 17, 40, 47, 56, 59, 61, 91, 173, 189, 196 signals, vii, 1, 13, 14, 16, 37, 45, 74, 80, 114, 117, 174, 189, 215, 218 signs, 20, 165, 192 single nucleotide polymorphism, 2, 4, 42, 56, 212 siRNA, 88 sites, 7, 12, 21, 59, 64, 65, 105, 127, 135, 153, 166, 185 skeletal muscle, 36, 105 skin, ix, 95, 96, 103, 104, 107, 111, 117, 133, 134, 135, 136, 137, 138, 139, 141, 144 skin disorders, 136 small intestine, viii, 46, 49, 53, 72 smokers, 57 smoking, 57, 72, 154, 156 smoking cessation, 57 smooth muscle, 3, 72, 99, 105, 189 smooth muscle cells, 3, 72, 99, 189 SNP, 2, 31 SNPs, 23, 56, 210 soft tissue sarcomas, 25, 96, 102, 104, 118, 129 solid tumors, ix, 95, 102, 107, 108, 131, 172, 178, 188, 202 solubility, 26 solvent, 6 Spain, 56, 143 species, 39, 53, 54, 71, 82, 101, 115, 145, 148, 152, 165, 168, 176 specificity, 26, 33, 40, 41, 47, 75, 116, 118, 173 spectrum, vii, 1, 19, 52, 179 S-phase, 200 spindle, 181, 187, 196 spleen, 19, 62 spondyloarthropathy, 137, 219 sporadic, 183, 204 SPSS, 154 squamous cell carcinoma, 190 S-shaped, 11 stability, 6, 27 stabilization, 175 stabilize, 175 stages, 184, 188, 208 starvation, 188, 190 stasis, 181 STD, 162 stenosis, 83 steric, 26 steroid, 62, 65, 67, 77, 211 steroids, 65, 68, 218

Index stimuli, 177 stimulus, 22, 59 stoichiometry, 39 stomach, 84, 185 strain, 71, 213, 217 strains, 57, 97, 207 strategies, x, 25, 30, 56, 90, 144, 162 stress, 16, 40, 41, 82, 83, 92, 125, 146, 173, 174, 181, 203 stressors, 134 strictures, 61, 75 stroma, 90, 178, 180, 184, 190 stromal, 19, 41, 89, 90, 93, 182, 183, 185, 187, 190 stromal cells, 19, 41, 89 stromal fibroblasts, 19, 93 structural protein, 209 subcutaneous tissue, 104 subgroups, 14 submucosa, 58, 59 substances, 188 substitution, 23 subtilisin, 6 success rate, 97 suicide, xi, 34, 171, 204 supernatant, 112, 117 superoxide, 20 superoxide dismutase, 20 supply, 181, 182, 191, 194 suppression, xi, 27, 29, 34, 38, 114, 160, 171, 189, 209 suppressor, 54 suppressor cells, 54 suprachiasmatic, 145 suprachiasmatic nucleus, 145 surface area, 136 surgeons, 34, 102, 112 surgery, 24, 47, 50, 52, 92, 93, 96, 103, 105, 109, 112, 114, 117, 121, 126, 127 surgical, ix, 24, 33, 38, 39, 50, 51, 52, 66, 69, 95, 102, 103, 104, 105, 172, 191, 203 surgical intervention, 50 surgical resection, 51, 104 surveillance, 21 survival, vii, ix, x, 1, 3, 16, 17, 18, 21, 22, 23, 30, 36, 40, 41, 59, 80, 95, 104, 105, 108, 113, 131, 144, 147, 172, 174, 180, 182, 183, 188, 190, 194, 195, 203 survival rate, 105 survival signals, vii, 1, 80 survivin, 173, 174, 196, 201, 202

243

susceptibility, 23, 24, 25, 31, 37, 38, 40, 41, 44, 56, 72, 122, 209, 210, 211, 216 sustainability, 65 sweat, 135, 138 Sweden, 56 swelling, 20, 111, 175, 176, 177, 179, 180, 186, 192, 193, 195 symmetry, 13 symptoms, 20, 40, 50, 55, 96, 165 syndrome, 20, 25, 40, 50, 64, 67, 68, 69, 70, 90, 99, 101, 102, 110, 125, 138, 139, 175, 203, 213 synergistic, 60, 123, 132 synergistic effect, 60 synovial fluid, 12, 34, 44 synovial tissue, 105 synthesis, x, 20, 22, 32, 33, 108, 143, 144, 146, 147, 148, 203 systemic circulation, 24, 103 systemic lupus erythematosus, 22, 31 systomics, x, 159, 160

T T cell, x, 2, 7, 12, 19, 29, 38, 40, 71, 72, 74, 76, 77, 135, 141, 146, 159, 160, 161, 163, 165, 190, 208 T cells, 12, 19, 29, 38, 40, 71, 72, 74, 76, 135, 141, 146, 161, 190, 208 T lymphocyte, x, 59, 71, 74, 82, 121, 141, 159, 213 T lymphocytes, 71, 74, 82, 121, 141, 213 Taiwan, 213 target organs, 25, 102 targets, 200 T-cell, 3, 19, 54, 55, 71, 163, 218 T-cell receptor, 54, 55 T-cells, 19 technical assistance, 156 teeth, x, 151, 152, 153, 155 testes, 145 testicle, 144 tetracycline, 121, 132 tetroxide, 191 Texas, 133, 143 TGF, 54, 55, 58, 61, 93, 166, 168, 207 Thailand, 159, 165, 169 therapeutic, 207 therapeutic agents, vii, 1, 30, 167, 207 therapeutics, ix, 68, 70, 95 therapy, viii, ix, xi, xii, 3, 24, 29, 36, 39, 41, 42, 43, 49, 51, 52, 57, 58, 60, 62, 64, 65, 66, 67, 68, 69, 70, 71, 74, 75, 76, 95, 97, 106, 112, 117, 121,

244

Index

125, 127, 130, 131, 133, 134, 136, 137, 138, 139, 140, 141, 149, 160, 162, 163, 171, 172, 188, 202, 205, 211, 212, 213, 214, 216, 218, 219 Thomson, 3, 37 threat, 165 threatening, 25 three-dimensional, 196 threonine, 2, 14, 15, 34 threshold, 29 threshold level, 29 thrombocyte, 111 thrombocytopenia, 166, 168 thromboembolic, 180, 202 thromboembolism, 181 thrombosis, 181, 182, 201 thrombotic, 158, 181 thromboxane, 33 thrombus, 127 thymocytes, 12, 20, 201 thymus, 144, 146 thyroid, 2, 12, 172, 181, 198, 204 thyroid carcinoma, 181, 198, 204 thyroiditis, 163 timing, 42 TIMP-1, 61 tissue, ix, x, xi, 3, 19, 21, 24, 25, 54, 57, 59, 61, 74, 82, 84, 85, 86, 88, 89, 91, 95, 96, 100, 101, 102, 104, 105, 106, 107, 108, 111, 112, 114, 115, 116, 117, 119, 125, 126, 127, 128, 129, 130, 151, 153, 157, 166, 171, 172, 177, 178, 179, 183, 189, 191, 198, 210, 215 tissue homeostasis, xi, 171 title, 50 TLR, 2, 82 TLR4, viii, 32, 79, 82, 85, 90, 101 T-lymphocytes, 53, 54, 58, 59, 68, 99 TNF-alpha (-α,), ix, 3, 4, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 44, 45, 47, 51, 62, 63, 64, 74, 75, 79, 80, 81, 82, 84, 85, 86, 87, 88, 89, 90, 93, 95, 96, 98, 99, 100, 101, 108, 112, 113, 116, 118, 119, 120, 121, 125, 126, 127, 128, 130, 132, 137, 138, 139, 140, 141, 151, 152, 153, 154, 155, 156, 157, 163, 168, 212, 214, 215, 218 Tokyo, 99, 154, 205 tolerance, 53, 56, 71, 155, 158 toll-like, 2, 82, 91, 92, 93, 125, 140 topology, 6 tourniquet, 103, 113 toxic, 98, 101, 103, 108, 120, 175, 176 toxic effect, 101, 108

toxicity, ix, x, 6, 35, 39, 95, 102, 104, 121, 144, 146, 147, 148, 213, 218 toxicology, 201 toxin, 3 toxins, 19, 47, 96, 97, 99, 122, 195 TP53, 175, 203 tracers, 104 trachoma, 23, 34 trade, 63 traffic, 46 transcription, vii, viii, x, xi, 1, 4, 6, 7, 12, 14, 16, 17, 21, 22, 23, 39, 54, 79, 80, 84, 108, 143, 147, 203, 205, 208, 209 transcription factor, vii, viii, xi, 1, 7, 14, 16, 17, 21, 79, 80, 84, 205, 208 transcription factors, vii, 1, 7, 14, 16, 21 transcriptional, 23, 55, 61, 72, 175 transducer, 54 transduction, 16, 131, 145, 149 transfection, 88 transfer, 129, 191, 194, 198 transformation, x, 144, 189, 208 transforming growth factor, 54, 58, 215 transgenic, 14, 209, 213 transgenic mice, 209, 213 transition, 188 transitional cell carcinoma, 199 translation, 6, 12, 82, 121, 147 translational, 37, 69 translocation, 15, 57, 89, 90, 174 transmembrane, 2, 3, 5, 7, 10, 17, 37, 38, 39, 44, 74, 80, 100, 116, 145, 178 transmembrane glycoprotein, 7, 10, 178 transmission, 188, 217 transmission electron microscopy, 188 transplant, 43, 62, 90 transplant recipients, 62 transplantation, 42, 81, 100 transport, 60, 148, 180 trauma, 6, 25, 36, 40, 42, 100, 134 trial, 30, 34, 38, 63, 64, 65, 66, 67, 68, 69, 76, 77, 104, 125, 126, 137, 216, 217 triggers, 72, 100, 173, 201 trimer, 6, 13, 27 tuberculosis, 22, 51, 68, 69, 70, 97, 98 tumor cells, vii, ix, x, 3, 6, 18, 93, 96, 97, 98, 99, 107, 109, 121, 144, 145, 146, 159, 161, 163, 178, 179, 180, 181, 184, 185, 187, 188, 190, 191, 193, 194, 199, 208

Index tumor growth, 87, 97, 98, 99, 102, 114, 115, 117, 121, 130, 131, 145, 146, 180 tumor invasion, viii, 80, 93 tumor metastasis, 25 tumorigenesis, 25, 94, 146, 174, 200, 201, 208 tumours, 25, 175, 183, 191, 201, 203 turnover, 21, 188 two-way, x, 151 type 2 diabetes, x, 39, 151 tyrosine, 13, 17, 130, 132, 146

U UCB, 63 ulcer, 179 ulceration, 50, 55, 119 ulcerative colitis, 50, 52, 56, 59, 72 ultrasound, 119 ultrastructure, 188, 199 ultraviolet, 133, 136 underlying mechanisms, 25, 190 uniform, 193 United States, 25, 65 untranslated regions, 100 urinary, 61, 144, 199 urinary bladder, 61 urinary tract, 199 urine, 12, 35, 42 uterus, 184

V vaccine, 97, 207 vacuole, 192 validation, 126 valine, 100 values, 27, 156 variability, 211 variable, 173, 178 variables, 154, 156, 203 variance, 135, 154, 156 variation, 23, 41 vascular cell adhesion molecule, 2, 58, 60 vascular disease, 155 vascular endothelial growth factor (VEGF), 2, 17, 48, 130, 131, 134, 182, 199 vascular occlusion, 112, 181 vasculature, 108, 110, 111, 112, 114, 116, 117, 121, 129, 132, 135, 180

245

vasculitis, ix, 136 vasculogenesis, 114, 130 vasoconstriction, 145 VCAM, 2, 58, 60, 111 vector, 121, 132 VEGF, 2, 17, 114, 116, 130, 131, 134, 182, 199 VEGF protein, 116 vein, 58, 103, 111, 118, 146, 149, 154, 180 vertebrates, 144 vesicle, 188 vessels, 103, 104, 105, 109, 114, 115, 116, 118, 121, 177, 181, 182 vimentin, 183, 184, 200 viral, xi, 205, 206, 209, 211, 212, 213, 216, 217, 219 viral hepatitis, xi, 205, 206, 213, 216, 219 viral infection, 149 viral promoter, 113 virological, 212 virology, 215 virus, x, xi, 44, 121, 129, 146, 147, 159, 160, 162, 205, 206, 207, 208, 209, 212, 213, 214, 215, 216, 217, 218, 219 virus infection, 208, 209, 213, 214, 215, 216, 217, 218, 219 virus replication, 209, 213 viruses, 6, 99 visible, 104 visualization, 50 vitamin D, 136

Index

246

W warrants, 30, 122 waste disposal, 189 waste products, 178 water, 57, 176 weakness, 20 weight loss, 101 Western countries, 83 Western societies, 56 withdrawal, 65 women, 81, 149, 183 workers, 99, 144 World Health Organization, 126 wound healing, 99 wound infection, 33 writing, 65

X xeno-cannibalism, xi, 172, 190, 191, 194 xenograft, 114, 115, 127, 130 xenografts, 117, 121, 131, 146, 149 X-linked, 2, 21

Y yeast, 144 yield, 100

Z Zen, 90, 91, 92, 93, 94 zinc, 15, 86