Progress in Inflammation Research
Series Editor Prof. Michael J. Parnham PhD Senior Scientific Advisor GSK Research Centre Zagreb Ltd. Prilaz baruna Filipovic´a 29 HR-10000 Zagreb Croatia Advisory Board G. Z. Feuerstein (Wyeth Research, Collegeville, PA, USA) M. Pairet (Boehringer Ingelheim Pharma KG, Biberach a. d. Riss, Germany) W. van Eden (Universiteit Utrecht, Utrecht, The Netherlands)
Forthcoming titles: Chemokine Biology: Basic Research and Clinical Application, Volume II: Pathophysiology of Chemokines, K. Neote, G. L. Letts, B. Moser (Editors), 2006 The Resolution of Inflammation, A. G. Rossi, D. A. Sawatzky (Editors), 2006 (Already published titles see last page.)
In Vivo Models of Inflammation 2nd Edition, Volume I
Christopher S. Stevenson Lisa A. Marshall Douglas W. Morgan Editors
Birkhäuser Verlag Basel · Boston · Berlin
Editors Christopher S. Stevenson Novartis Institutes for Biomedical Research Respiratory Disease Area Novartis Horsham Research Centre Wimblehurst Road Horsham, West Sussex United Kingdom
Lisa A. Marshall Johnson and Johnson PRD Welsh & McKean Rd Spring House, PA 19477 USA
Douglas W. Morgan Portfolio, Program and Alliance Management BiogenIdec 14 Cambridge Center Cambridge, MA 02142 USA
A CIP catalogue record for this book is available from the Library of Congress, Washington D.C., USA
Bibliographic information published by Die Deutsche Bibliothek Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data is available in the internet at http://dnb.ddb.de
ISBN-10: 3-7643-7519-1 Birkhäuser Verlag, Basel – Boston – Berlin ISBN-13: 978-3-7643-7519-5 Birkhäuser Verlag, Basel – Boston – Berlin The publisher and editor can give no guarantee for the information on drug dosage and administration contained in this publication. The respective user must check its accuracy by consulting other sources of reference in each individual case. The use of registered names, trademarks etc. in this publication, even if not identified as such, does not imply that they are exempt from the relevant protective laws and regulations or free for general use. This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, re-use of illustrations, recitation, broadcasting, reproduction on microfilms or in other ways, and storage in data banks. For any kind of use, permission of the copyright owner must be obtained. © 2006 Birkhäuser Verlag, P.O. Box 133, CH-4010 Basel, Switzerland Part of Springer Science+Business Media Printed on acid-free paper produced from chlorine-free pulp. TCF ' Cover design: Markus Etterich, Basel Cover illustration: see p. 44; with friendly permission of Leo Joosten Printed in Germany ISBN-10: 3-7643-7519-1 ISBN-13: 978-3-7643-7519-5 987654321
e-ISBN-10: 3-7643-7520-5 e-ISBN-13: 978-3-7643-7520-1 www.birkhauser.ch
Contents Volume I (contents of volume II see last page)
List of contributors
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vii
Preface to the first edition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
xi
Preface to the second edition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Lisa R. Schopf, Karen Anderson and Bruce D. Jaffee Rat models of arthritis: Similarities, differences, advantages, and disadvantages in the identification of novel therapeutics . . . . . . . . . . . . . . . . . . . .
1
Leo A.B. Joosten and Wim B. van den Berg Murine collagen induced arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Stephen A. Stimpson, Virginia B. Kraus and Bajin Han Use of animal models of osteoarthritis in the evaluation of potential new therapeutic agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Roberto Benelli, Guido Frumento, Adriana Albini and Douglas M. Noonan Models of inflammatory processes in cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
83
Antonio Musarò and Nadia Rosenthal Advances in stem cell research: use of stem cells in animal models of muscular dystrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Karen F. Kozarsky Gene transfer technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 Matthias Müller and Nicole Avitahl-Curtis Transgenics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 Sreekant Murthy, Elisabeth Papazoglou, Nandhakumar Kanagarajan and Narasim S. Murthy Nanotechnology: Towards the detection and treatment of inflammatory diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Contents
Susan Brain UK legislation of in vivo aspects in inflammation research
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177
Naoko Kagiyama, Takuya Ikeda and Tatsuji Nomura Japanese guidelines and regulations for scientific and ethical animal experimentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187 Joanne B. Morris, Jeffrey Everitt and Margaret S. Landi United States guidelines and regulations in animal experimentation . . . . . . . . . . . . . 193 Index
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203
List of contributors
Adriana Albini, Dept of Translational Oncology, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy; e-mail:
[email protected] Karen Anderson, Novartis Institutes for Biomedical Research Inc, 250 Massachusetts Ave, Cambridge, MA 02139, USA; e-mail:
[email protected] Nicole Avitahl-Curtis, Novartis Institute for Biomedical Research, 100 Technology Square, Cambridge, MA, USA; e-mail:
[email protected] Roberto Benelli, Dept of Translational Oncology, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy; e-mail:
[email protected] Susan Brain, King’s College London, Cardiovascular Division, Guy’s Campus, London SE1 1UL, UK; e-mail:
[email protected] Jeffrey Everitt, GlaxoSmithKline Pharmaceuticals, LAS, 709 Swedeland Rd, King of Prussia, PA 19406, USA; e-mail:
[email protected] Guido Frumento, Dept of Translational Oncology, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy; e-mail:
[email protected] Bajin Han, GlaxoSmithKline, Research Triangle Park, NC 27709, USA; e-mail:
[email protected] Takuya Ikeda, GlaxoSmithKline, Tsukuba Research Laboratories, 43 Wadai, Tsukuba 300-4243, Japan; e-mail:
[email protected] Bruce D. Jaffee, Novartis Institutes for Biomedical Research Inc, 250 Massachusetts Ave, Cambridge, MA 02139, USA; e-mail:
[email protected]
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List of contributors
Leo A.B. Joosten, Rheumatology Research and Advanced Therapeutics, Department of Rheumatology, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands; e-mail:
[email protected] Naoko Kagiyama, Central Institute for Experimental Animals, 1430 Nogawa, Miyamae, Kawasaki 216-0001, Japan; e-mail:
[email protected] Nandhakumar Kanagarajan, Division of Gastroenterology and Hepatology, Drexel University College of Medicine, Philadelphia, USA; e-mail:
[email protected] Karen F. Kozarsky, Biopharmaceuticals, Center of Excellence for Drug Discovery, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA; e-mail:
[email protected] Virginia B. Kraus, Duke University Medical Center, Durham, NC 27710, USA; e-mail:
[email protected] Margaret S. Landi, GlaxoSmithKline Pharmaceuticals, LAS, 709 Swedeland Rd, King of Prussia, PA 19406, USA; e-mail:
[email protected] Joanne B. Morris, GlaxoSmithKline Pharmaceuticals, LAS, 709 Swedeland Rd, King of Prussia, PA 19406, USA; e-mail:
[email protected] Matthias Müller, Novartis Pharma AG, WSJ-386.409, 4002 Basel, Switzerland; e-mail:
[email protected] Narasim S. Murthy, Associated Radiologists, PA, 322 E. Antietam Street, Suite 106, Hagerstown, MD 21740, USA; e-mail:
[email protected] Sreekant Murthy, Division of Gastroenterology and Hepatology and Office of Research, Drexel University College of Medicine, Philadelphia, USA; e-mail:
[email protected] Antonio Musarò, Department of Histology and Medical Embryology, CE-BEMM and Interuniversity Institute of Myology, University of Rome “La Sapienza”, Via A. Scarpa 14, 00161 Rome, Italy; e-mail:
[email protected] Tatsuji Nomura, Central Institute for Experimental Animals, 1430 Nogawa, Miyamae, Kawasaki 216-0001, Japan; e-mail:
[email protected]
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List of contributors
Douglas M. Noonan, Dept of Clinical and Biological Sciences, University of Insubria, Varese, Italy; e-mail:
[email protected] Elisabeth Papazoglou, School of Biomedical Engineering, Drexel University, Philadelphia, USA Nadia Rosenthal, European Molecular Biology Laboratory, Mouse Biology Unit, Monterotondo, 00016 Rome, Italy; e-mail:
[email protected] Lisa R. Schopf, Abbott Bioresearch Center, 100 Research Drive, Worcester, MA 01605, USA; e-mail:
[email protected] Stephen A. Stimpson, GlaxoSmithKline, Research Triangle Park, NC 27709, USA; e-mail:
[email protected] Wim B. van den Berg, Rheumatology Research and Advanced Therapeutics, Department of Rheumatology, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands; e-mail:
[email protected]
ix
Preface to the first edition
The purpose of this volume in the series Progress in Inflammation Research is to provide the biomedical researcher with a description of the state of the art of the development and use of animal models of diseases with components of inflammation. Particularly highlighted are those models which can serve as in vivo correlates of diseases most commonly targeted for therapeutic intervention. The format is designed with the laboratory in mind; thus it provides detailed descriptions of the methodologies and uses of the most significant models. Also, new approaches to the development of future models in selected therapeutic areas have been highlighted. While emphasis is on the newest models, new information broadening our understanding of several well-known models of proven clinical utility is included. In addition, we have provided coverage of transgenic and gene transfer technologies which will undoubtedly serve as tools for many future approaches. Provocative comments on the cutting edge and future directions are meant to stimulate new thinking. Of course, it is important to recognize that the experimental use of animals for human benefit carries with it a solemn responsibility for the welfare of these animals. The reader is referred to the section on current regulations governing animal use which addresses this concern. To fulfill our purpose, the content is organized according to therapeutic areas with the associated models arranged in subcategories of each therapeutic area. Concepts presented are discussed in the context of their current practice, including intended purpose, methodology, data and limitations. In this way, emphasis is placed on the usefulness of the models and how they work. Data on activities of key reference compounds and/or standards using graphs, tables and figures to illustrate the function of the model are included. The discussions include ideas on a given model’s clinical correlate. For example, we asked our contributors to answer this question: How does the model mimic what is found in human clinical practice? They have answered this question in many interesting ways. We hope the reader will find the information presented here useful for his or her own endeavours investigating processes of inflammation and developing therapeutics to treat inflammatory diseases. October, 1998
Douglas W. Morgan Lisa A. Marshall
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Preface to the second edition
Since our first edition of “In Vivo Models of Inflammation” published in 1999, there has been amazing progress, and an abundance of exciting new information in inflammation research: new technologies, new therapeutics, new understanding of inflammatory processes, … and on and on, have emerged in the past 6 years. Supporting all of this are the fundamentals of inflammation research, i.e., the animal models, known mechanisms, and therapeutic standards, that have continued to provide the basis for generating these advances. Given the great progress, we have chosen to provide a second edition to our original text. The second edition of “In Vivo Models of Inflammation” comes to you in two volumes and provides an update of the models included in first edition with expanded coverage and more models. Again, these volumes emphasize the standard models regarded as the most relevant for their disease area. The intent is to provide the scientist with an up-to-date reference manual for selecting the best animal model for their specific question. Updates on previously described models are specifically focused on references to any additional pharmacology that has been conducted using these systems. The sections on arthritis models have been expanded and now include models relating to osteoarthritis. New areas described herein include models of neurogenic, cancer, and vascular inflammation. Additionally, coverage of in vivo technologies includes updates on transgenic and gene transfer technologies, and has also been expanded to include chapters on stem cells and nanotechnologies. The second edition continues to emphasize that conducting in vivo research carries with it a great responsibility for animal respect and welfare. The coverage of this concern has been extended to include chapters describing current regulations in the United States, the United Kingdom, and Japan. The ultimate aim of the second edition is to provide current best practices for obtaining the maximum information from in vivo experimentation, while preserving the dignity and comfort of the animal. We hope the information provided here helps in advancing the reader’s endeavors in investigating processes of inflammation and in developing therapeutics to treat inflammatory diseases. May, 2006
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Christopher S. Stevenson Lisa A. Marshall Douglas W. Morgan
Rat models of arthritis: Similarities, differences, advantages, and disadvantages in the identification of novel therapeutics Lisa R. Schopf, Karen Anderson and Bruce D. Jaffee Millennium Pharmaceuticals, Cambridge, MA, USA
Introduction The aim of this chapter is to update and expand the information reviewed by Carlson and Jacobson on the topic of rat models of arthritis [1]. Animal models of rheumatoid arthritis (RA) have been extensively used for many years in the evaluation of anti-arthritic agents [2–4]. The most widely used model, adjuvant-induced arthritis (AA) in rats, is discussed in detail here [4–6]. Another common model, which was not included in the first edition and is perhaps more relevant to human RA in terms of cartilage damage, is collagen-induced arthritis (CIA) in the rat [3, 7, 8], and is also outlined here. These two models are compared with a relatively new model, monoarticular streptococcal cell wall–induced arthritis (SCW) in the rat [1, 3, 9, 10]. All of these models share key features related to human RA that make them critical tools in drug development. They have provided information regarding genetic predisposition, prominent cell types, protein and molecular mediators involved in the immunological and inflammatory processes that leads to arthritic pathology.
Historical background The first reported observation that complete Freund’s adjuvant (CFA) could induce polyarthritis in rats was demonstrated by Stoerk and colleagues in 1954 [11] using spleen extracts emulsified in CFA. Shortly thereafter, Pearson showed that CFA alone could induce arthritis in rats [12]. Over the next decade, the AA rat model was used to test a variety of anti-arthritic therapies such as steroids and nonsteroidal anti-inflammatory drugs (NSAIDs) [1, 4]. More recently, this model has been used to assess immunomodulatory drugs such as methotrexate and cyclosporine A as well as therapies designed to block COX-2, TNF-_ or IL-1 [13–21]. Overall, the AA model has been the most extensively used arthritic rat model by the pharmaceutical industry, and has an excellent track record for predicting both activity and toxicity [19]. In Vivo Models of Inflammation, Vol. I, edited by Christopher S. Stevenson, Lisa A. Marshall and Douglas W. Morgan © 2006 Birkhäuser Verlag Basel/Switzerland
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The CIA model was first described in 1977 by Trentham and colleagues, and has since gained favor by providing clues into the pathogenesis of arthritis and related disorders as well as its predictive value for testing anti-rheumatic therapies [7, 8]. This model has been used to evaluate NSAIDs, methotrexate and cyclosporine A as well as newer therapies, which block TNF-_ and/or IL-1 [19, 22–27]. Although there are more data using the AA model, the rat CIA model has also proven to have predictive value for many current therapies and tends to be favored when examining protection against cartilage destruction because the lesion is more comparable to human RA than in the AA model [19]. Although we have known since the 1950s that injections of streptococcal cell wall components or more specifically covalent complexes of peptidoglycan and polysaccharide (PG-PS) from group A streptococci can induce rheumatic-like lesions, the monoarticular SCW model which is described in this chapter, was not developed until the mid-1980s, and has not been routinely used for pharmacological screening [1, 28–31]. However, in this review we provide details on methods and disease parameters such as joint swelling, histopathology, gene expression, serum acute-phase proteins and cartilage and bone markers. We also report efficacy determinations using this model to evaluate some of today’s current therapeutics.
Drug therapies There has been and continues to be extensive research in the area of drug development to treat human RA, but it is not the intention of this review to fully recount or explore all of these efforts [1–3, 32–36]. However, we have provided information on some of the more commonly used RA therapies and their efficacy in the three rat arthritic models (Tab. 1). This table focuses solely on therapeutic dosing regimes, although there is a vast amount of literature that explores prophylactic treatments as well [1, 2, 13, 14, 22, 23, 30, 37–44]. When patients first present with symptoms, the primary care physician will typically suggest the use of NSAIDs to provide some relief from pain and stiffness. Two examples of commonly used NSAIDs are ibuprofen and naproxen in Table 1, where we provide efficacy data in the rat models of arthritis. The main disadvantage to these treatments is that they only provide partial relief from pain and stiffness, but do not radically change the course of disease progression, as also predicted using the animal models (AA and CIA) [40]. They are typically only tolerated for short periods of time, after which patients can experience any number of gastrointestinal toxicity problems. Sometimes, corticosteroids are prescribed at this early stage, but, despite their potent anti-inflammatory action, they too have many dose-dependent side effects. Again, both the AA and CIA rat models predicted this outcome observed in patients [2]. Alternatively, corticosteroids such as prednisone are used
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Rat models of arthritis: Similarities, differences, advantages, and disadvantages…
Table 1 - Efficacy of standard RA drugs using therapeutic dosing regimes in rat models of arthritis: AA, CIA and monoarticular SCW Drug class
AA ED50 (mg/kg)
Ref.
CIA ED50 (mg/kg)
75a; 65a
[123, 124]
Naproxen Corticosteroids Prednisolone Dexamethasone DMARDS Methotrexate
7a
[125, 126]
49% @ [42] 25 mg/kge ND
ND
0.3a 0.005a; 0.01b
[124] [1]; MPI
ND 0.01f
ND 0.01i
Inactivea,b
[1]; MPI
0.1g
Cyclosporine A Leflunomide
2.4c 53% @ 32 mg/kgd
[127] [128]
Enhancede,f MPI; [42, 43] ND
56% @ 0.1–0.5i 18i ND
11h
<5j
NSAIDs Ibuprofen
Biologics Etanercept
ND
Ref.
MPI
MPI
SCW Ref. ED50 (mg/kg)
ND
MPI MPI MPI
MPI
ND, limited or no data available MPI, unpublished data from Millennium Pharmaceuticals, Inc. a Once a day oral dosing from days 12 to 29 b Once a day oral dosing from days 10 to 19 c MWF oral dosing (6 doses) d Once a day oral dosing from days 15 to 24 e Once a day oral dosing from days 14 to 27 f Once a day oral dosing from days 12 to 20 g Once a day oral dosing from days 6 to 21 h s.c. dosing days 12, 15, 18 i Once a day oral dosing from days 21 to 24 j s.c. dosing day 21
at low doses (< 10 mg/day) during acute flares [34]. Therefore, the primary care physician, or at this point a rheumatologist, will plan to prescribe a disease-modifying antirheumatic drug (DMARD), such as the examples given in Table 1 of methotrexate, cyclosporine A or the newer drug leflunomide. One major drawback of DMARDs is that they only begin to demonstrate efficacy after several weeks of
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therapy, and remission is rare, only 20–25% of patients [33]. Out of these examples, methotrexate is clearly the gold standard of care, and has been used since the 1950s. Patients on methotrexate often take a folic acid supplement to decrease toxic side effects. The most significant disadvantage to methotrexate therapy is that up to two thirds of RA patients on methotrexate will have an inadequate response to monotherapy. In the animal models, methotrexate is most efficacious when administered prophylactically and during the developing disease. This may be due to the need for prolonged dosing in vivo, analogous to what is observed clinically [2]. Methotrexate is given weekly to patients, which is difficult to mimic in the rat models due to the accelerated nature of disease progression in animals. Cyclosporine A is used as a common comparator drug in pharmaceutical drug evaluation because of its directed effects on T cells, but its relative toxicity has precluded its widespread use in RA patients. In fact, it has been demonstrated that in the rat CIA model, when given therapeutically, cyclosporine A can actually enhance disease, perhaps due to an alteration in the sensitive balance of helper versus suppressor T cells [43]. Leflunomide has only shown relatively modest efficacy in patients when used alone, and it also suffers from safety and tolerability issues, mostly related to elevations in liver enzymes and gastrointestinal issues. In the animal models, leflunomide administration has been most effective when given prophylactically [38, 45]. The most widely used biologics on the market inhibit the action of TNF-_; there are three products available: infliximab, adalimumab, and etanercept. We have provided animal data using etanercept in Table 1, and it is important to keep in mind that this product has been specifically designed to inhibit the action of human TNF_ and its differential potency to rat TNF-_ is not known. The major disadvantages of this therapy are the high cost (a third of patients will not respond), and the risk of opportunistic infection. Although we have been quick to point out the disadvantages of these selected therapies, they are currently the best the field has to offer, and they do provide relief and in some cases disease modifying activity to many patients. More recently, a combination therapy approach has provided added benefit to patients [14, 16, 19, 24, 25, 32, 36, 45–47]. Overall, the treatment of RA has dramatically improved over the last decade, with early diagnosis, and with new therapies on the horizon that will offer continued progress in caring for these patients.
Disease initiation and pathogenesis Most of the RA therapeutics currently in use have been evaluated using rat arthritis models, which is a testament to the similarity in mechanisms that drive disease development. Although many pathological features are the same, the rat models progress much more rapidly, with acute, severe inflammation and dramatic changes in bone that include both formation and resorption [2, 48, 49].
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Rat models of arthritis: Similarities, differences, advantages, and disadvantages…
Susceptibility of rats to the development of arthritis is dependent on a variety of factors including strain, environment, age, and gender. Also, as with many disease states, susceptibility may not be attributed to any one factor alone. For an excellent review of individual strain susceptibility, refer to the chapter by Carlson and Jacobson in the first edition of this book [1]. Simply stated the specific genetic basis of differing susceptibility is not known, but apparently it is not just a matter of MHC expression. For example two different strains, which share the same MHC, can exhibit different susceptibilities. As related to environmental influence, the role of endogenous flora is illustrated by the differing susceptibility of F344 rats to AA and SCW. More specifically, animals maintained in a conventional facility are resistant to the models, whereas animals in germ-free housing are susceptible [50, 51]. We and others have observed that very young and much older animals are relatively resistant to arthritis development [4, 52]. The short life span of rodents compared to humans thereby makes age dependency a difficult parameter to correlate. However, as in human RA, females are more prone to arthritis development in the rat models of CIA and SCW, although there is equal gender susceptibility to AA. Lewis rats are an example of a strain that is susceptible to all the models discussed here. Susceptibility in this strain is perhaps related, at least in part, to a defect in their hypothalamo-pituitary-adrenal axis and resultant reduced ability to suppress inflammation [53]. We routinely use female Lewis rats approximately 2 months of age (or 150–170 g) in all of our studies, as this allows for comparison of the disease process between models, and for better assessment of efficacy of known and novel therapeutics without additional variables associated with gender and strain differences [54]. All three models discussed here are initiated by the introduction of foreign antigen, either of bacterial origin as in AA (mycobacterial) and SCW (PG-PS 100P from Streptococcus) or using xenogeneic type II collagen (in this case bovine) in CIA. In general the antigens are rapidly redistributed from the site of introduction, and there is subsequently a T cell response. T cells have been shown to play an essential role in the development of all three models [55]. The supporting evidence includes lack of disease development in athymic rats, transfer of disease to naïve animals using lymph node cells or thoracic duct cells from arthritic rats, and efficacy of anti-T cell therapeutics including antibodies or cyclosporine A [56–61]. In human RA, evidence such as the predisposition of certain MHC class II alleles and the large synovial T cell infiltrate suggests that the pathogenesis of the human disease involves antigen recognition by T cells, particularly in the initial phase of disease development, and probably in disease flare-ups as well [36, 62, 63]. In addition, the efficacy of immunosuppressants, and more recently of targeted anti-T cell therapies such as the anti-costimulatory CTLA4-Ig, supports this concept [64]. The appearance of lesions in rat joints following the introduction of bacterially derived antigens implies that there is a cross-reactive, joint-localized tissue antigen. It has been recently determined the pathogenic immunizing antigen of CFA is the
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mycobacterial heat shock protein mHsp65 [65], suggesting a possible case of antigenic mimicry. Hsps are molecular chaperone proteins that are highly conserved among species. Hsp60 is the closest related mammalian hsp to mHsp65, and is present in normal and RA synovium [6, 66]. Interestingly, Hsp60 (or any Hsp) have not been shown to be the direct antigen(s) implicated in AA pathogenesis. However, careful characterization of the mHsp65 protein has identified the existence of a pathogenic epitope, cross-reactive with endogenous cartilage link protein of a cartilage proteoglycan [67]. In addition, there is a distinct regulatory epitope that can actually provide protection from disease and is cross-reactive with endogenous Hsp60. Furthermore, it is possible to induce a protective immune response with mHsp65 protein that is effective in not only against AA, but also SCW and pristaneinduced arthritis (the latter model not involving bacterial products) [68–71]. The synovium of animals immunized with mHsp65 contains T cells reactive against Mhsp65/Hsp60 that include both effector and regulatory subtypes [6, 66, 72]. In the monoarticular SCW 100P model, the antigen is introduced directly into the joint, and actually remains at the site for a prolonged period afterwards [73]. After an initial acute episode, swelling subsides virtually completely. However, subsequent systemic administration of SCW 100P fraction, LPS, or superantigen can reactivate the arthritis [3]. T cells isolated from SCW 100P arthritic rats are cross-reactive, recognizing not only SCW, but also mHsp65 [61]. The ability of a variety of bacterial products, including LPS and the products of intestinal overgrowth, to reactivate the arthritic process suggests a possible link to the mechanism of RA disease flare activity [3, 29, 74, 75]. In CIA, the specific targeting of the joint in the subsequent inflammatory response is relatively straightforward. T cells capable of recognizing the collagen molecule, coupled with an appropriately susceptible genetic background, result in the production of both reactive cells and antibodies against autologous type II collagen in articular cartilage [3, 7, 8, 55]. Of the rat models, a significant humoral component has been attributed only to CIA; high titers of anti-collagen type II IgG antibodies are detected, and disease can be transferred to naïve animals using serum from arthritic rats [76, 77]. This autoantibody induction is one of the reasons this model may be favored over the others. In human RA, autoantibodies are a prominent feature of the disease; however, anti-collagen type II antibodies are only present in about 30% of patients, whereas the characteristic rheumatoid factor (RF), an anti-Fca autoantibody, is present in 70–80% of patients [78]. RA patients may also have autoantibodies to cyclic citrullinated peptides (CCP) and to cartilage antigens derived from collagen and aggrecan [79]. A pathogenic role for the humoral immune response in RA is further supported by the efficacy of the B cell-depleting anti-CD20 antibody [80, 81]. Rodent models are not characterized by either RF or anti-CCP antibodies [82]. Additionally, despite high circulating anti-collagen antibody levels in CIA, there is no evidence for local (synovial) antibody production as in RA [83, 84]. We and others have observed that, although foci of B cells can be
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Rat models of arthritis: Similarities, differences, advantages, and disadvantages…
seen in arthritic rat synovium, true lymphoid follicles such as those found in RA synovium are not present [2, 48]. Despite different means of induction, the developmental sequence of lesions in the joints of the rat arthritis models is quite similar. As early as 72 h after antigen introduction, T cells appear in the perivascular space in the synovium [8, 31, 48, 85]. Subsequently there is fibrin deposition in joint spaces, synoviocyte proliferation, and the appearance of increasing, and eventually very large and predominating, numbers of myeloid lineage cells, particularly neutrophils. Neutrophils are a prominent feature of rat arthritis models, and are the most numerous inflammatory cell in both tissue and synovial fluid [2, 86, 87]. These cells represent a significant source of cytokines, oxygen metabolites, and proteases with the potential to perpetuate joint destruction. Depletion of neutrophils has a significant therapeutic effect on established rat arthritis [59, 87]. Although numerous in human RA synovial fluid, neutrophils are not a conspicuous component of the RA synovium. Other myeloid cells, such as activated macrophages, are the major source of TNF_ and IL-1`. Increased levels of these cytokines has been documented in both RA and the rat models [3, 33, 36, 88, 89]. Besides their pro-inflammatory activities, these cytokines can potentiate both cartilage and bone damage. TNF-_ can drive the maturation of osteoclast precursors, and both TNF-_ and IL-1` can increase the resorptive ability of mature osteoclasts [90, 91]. In addition, IL-1` particularly induces chondrocytes and synoviocytes to produce matrix-degrading metalloproteases. Both cytokines decrease synthesis of normal cartilage components [92–96]. Anti-TNF and anti-IL-1`-directed therapies have a positive effect in the rat models [2, 40], and are currently used successfully in the clinic for RA [33–35]. In both human RA and rat arthritis models, initial synovial cell hyperplasia transitions into the development of invasive pannus tissue. Pannus formation and the progression of significant cartilage and bone lesions are later features of the arthritic disease process. The relatively late appearance of lesions in the hard tissues has contributed to the idea that bone and cartilage destruction were sequelae of the joint inflammatory process. Some recent evidence from RA clinical trials suggests that this may not be entirely correct. In patients, although anti-inflammatory therapy was shown to reduce clinical signs referable to inflammation, the progression of joint destruction was unchecked [97–99]. Furthermore, in trials involving anti-TNF therapies, synovitis and/or clinical response was unimproved; however, bone erosion was reduced [100, 101]. Rats and humans treated with IL-1R antagonist demonstrated greater protection against bone erosion than joint inflammation [18, 102]. These data suggest that the processes underlying joint inflammation and bone destruction may be mechanistically distinct, at least to some degree. In general, the bone alterations seen in the rat models develop rapidly and are severe compared to human RA. There is a notable periosteal bone formation component as well as bone resorption by osteoclasts. Increased osteoclast precursors are found in both the synovium and synovial fluid in both RA and the rat models [49, 103, 104].
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Table 2 - Comparing rat models of arthritis (AA, CIA and monoarticular SCW) with human RA Disease characteristic Antigen Sex predisposition Acute phase proteins Polyarthritis Synovitis Bursitis/tendonitis Cartilage involvement Bone involvement Rheumatoid factor Neutrophil influx CD4+ lymphocyte influx Macrophage in soft tissue B cells/Plasma cells
Elevated synovial cytokines: TNF-_ IL-1` IL-6 Edema Disease course Disease flares
Rat models
Human RA
Bacterial (AA, SCW) Bovine collagen type II (CIA) None (AA) Female (CIA, SCW) ++ + (AA, CIA) ++ ++ ++ (targeted in CIA) Early and aggressive (+++ AA; ++ CIA; +/– SCW) –
?
++ (tissue, synovial fluid) ++ ++ Random lymphocytic infiltrates (CIA); Role of B cells unclear (AA, SCW) ++ + + + ++ ~3 weeks subsides into fibrosis/ ankylosis eventually – (AA, CIA) + (SCW)
Female ++ + ++ ++ ++ ++ +** **70–80% of patients ++ (synovial fluid) ++ ++ Synovial lymphoid aggregrates; Local antibody production ++ + + + ++ Frequently decades +
In summary, many of the cell types involved and the molecular mechanisms implicated in human RA and the rat models of arthritis are identical. Most obviously, the prolonged time frame, and the recurrent nature of disease exacerbations of human RA are different (although recurrence is reported to occur in SCW), as are some other features of disease, including presence of RF and synovial lymphoid follicles. The preventative and disease-modifying therapeutic effects of a variety of nonsteroidal anti-inflammatory drugs suggests a greater dependence on prostaglandins for disease development and maintenance of the rat models [1, 4, 105]. No such activity has been seen in RA patients; however, it is likely that none are treated as near to the onset of their disease. Table 2 highlights the key features of the rat model
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pathology as compared with RA. Between the three models, which we run in female Lewis rats, we find that differences are primarily quantitative, and the general features of the disease are very similar overall. These are reviewed in the next section.
Disease parameters The onset of ankle swelling is monitored to indicate the development of arthritic disease following administration of an inducing agent. After the injection of CFA, there is a significant increase in the ankle volume of the injected group compared to controls. Paw swelling in AA is robust, with increases up to 3.5-fold times control volume. In general, maximal swelling occurs by day 19 and then plateaus. Our studies are generally completed at this point. If the study is continued out to 40–50 days, the paw volume may come down somewhat, but remains significantly elevated compared to non-arthritic controls. Paw swelling in CIA is less severe, generally reaching only about a 2-fold increase over the control volume. In onset, swelling generally occurs slightly later than in AA, beginning around day 14 after the first injection of bovine type II collagen. The greatest paw volume is seen at about day 21, and also plateaus in severity. The least severe paw swelling occurs in the SCW model, with affected paws only increasing in volume by 1.5-fold. In this model, there is a small initial paw swelling observed immediately following the intra-articular (i.a.) injection of SCW 100P, but this rapidly subsides by days 3–4. After the second exposure to antigen by intravenous (i.v.) challenge 2–3 weeks later, there is a rapid and predictable reappearance of paw swelling of the magnitude described. This swelling is maximal at 2–3 days after i.v. SCW and also plateaus. Due to the synchronization and predictability of the response, this model is favored as a model of arthritic flare, and is being used more commonly to evaluate novel therapies. A detailed description of each of these models is provided in a later section, including examples of paw swelling.
Clinical pathology The systemic manifestation of the inflammatory response to the inciting agents used in animal models of arthritis is reflected in several peripheral blood-based parameters. Normal female Lewis rats used in our studies typically have total leukocyte counts around 9 × 109– 10 ×109 cells/L, 80% of which are lymphocytes. Neutrophil counts are usually low, not exceeding 2 ×109 cells/L (Tab. 3). In the AA model, within the 1st week after CFA administration prior to the development of contralateral ankle swelling, neutrophil counts increase dramatically, about 5–7-fold normal. These high counts persist throughout the typical study period (19 days). Concomitantly, there is an increase in serum fibrinogen levels, from normal levels (roughly
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Table 3 - Comparison of various blood parameters in rat models of arthritis Parameter
Additional parameters for assessing inflammation and tissue destruction in rat arthritis models Control value, Day 19 AA, Day 22 CIA, Day 24 SCW mean ± SD mean ± SD mean ± SD (100P), mean ± SD
Clinical pathology 9.1 ± 1.4 Total leukocyte count, ×109/L 9 1.2 ± 0.6 Total neutrophils, ×10 /L 7.4 ± 0.7 Total lymphocytes, ×109/L Blood fibrinogen, mg/dL 213 ± 15 Acute phase proteins _1 acid glycoprotein, µg/mL 175 ± 90 Haptoglobin, mg/mL 0.5 ± 0.1 Soluble bone & cartilage markers Collagen type I telopeptides 35 ± 9 (RatLaps) ng/mL Collagen oligomeric matrix 1.4 ± 0.2 protein (COMP), µg/mL Quantifiable µCT-derived bone parameters Bone volume (arbitrary units) 35 ± 1 Bone roughness 1640 ± 250 (arbitrary units)
17.5 8.8 7.2 806
± ± ± ±
2.2 1.3 0.9 40
11.5 4.8 6.0 436
± ± ± ±
1.5 1.4 0.8 25
9.5 1.2 7.8 438
± ± ± ±
1845 ± 200 3.9 ± 0.9
490 ± 150 1.4 ± 0.3
82 ± 20
57 ± 15
ND
4.7 ± 0.5
3.3 ± 0.5
ND
21 ± 8 ND
29 ± 2 10950 ± 3840
ND ND
1.3 0.3 1.0 150
440 ± 100 1.5 ± 0.3
ND, not done.
200 mg/dL) to 4-fold increase (800 mg/dL). An elevated erythrocyte sedimentation rate (ESR) has also been documented in AA, from day 4 and peaking at day 12 but remaining high up to day 50 [89]. In CIA, the maximal neutrophil increase is less (3–4-fold), as is the fibrinogen increase (400 mg/dL). No increase in neutrophils is detected in monoarticular SCW model. However, fibrinogen levels increase to approximately the same levels as those seen in CIA (400 mg/dL). Typical values for each model are shown in Table 3.
Acute-phase proteins The acute inflammatory nature of all three models is reflected in the increases in serum levels of acute-phase proteins (APPs). We have typically used alpha-1-acid gly-
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coprotein and haptoglobin as disease biomarkers in our rat models. CRP, the APP most commonly followed in human RA patients, is not a major induced APP in the rat, and is therefore not as useful as it is in humans. Similar to the other parameters we have examined, the trend for the greatest magnitude of increase in AA is repeated. We have observed roughly 10-fold increased levels of alpha-1-acid glycoprotein and 7–8-fold increase in haptoglobin levels. CIA and monoarticular SCW produce similar elevations in both alpha-1-acid glycoprotein and haptoglobin, roughly 3-fold increases in both parameters. Representative values are shown in Table 3.
Gene expression We and others [30, 106] have profiled the expression of a number of genes in the joint during the development and establishment of rat arthritis. By semi-quantitative PCR, Schmidt-Weber et al. [106] detected increased mRNA expression for IFN-a, IL-1`, IL-5, IL-6, TNF-_ and IL-10 in the draining (popliteal) lymph node in AA. Most of these genes peaked in expression on day 6, before the onset of clinical arthritis. In the affected synovial membrane, peak IL-6 was found on day 16 and peak IL-1` occurred from days 13 to 20. Interestingly, these researchers reported that no TNF-_ mRNA was detected in the dissected synovial membrane. Another approach, including our own, has been to use the entire affected ankle for RNA preparation. The mRNAs we have examined in the paws reflect multiple parameters associated with arthritis, including markers of cellular infiltration [CD4, B29 (B cell), CD11b], NF-gB-induced inflammatory cytokines and related molecules (IL1`, TNF-_, IL-6, iNOS, COX-2), matrix metalloproteases (MMP3, MMP13), antiinflammatory cytokines (IL-10, TGF-`), and bone-associated markers (TRAP, RANKL, cathepsin K). In our quantitative PCR studies, the earliest mRNAs to achieve earliest statistically significant elevation in AA joints at day 12 were CD11b, MMP3, IL-10. In our hands we were also able to consistently detect TNF-_ in these whole joint preparations. The majority of genes examined achieved peak mRNA expression at day 16 and remained significantly elevated at day 19. This contrasts with paw swelling, which often is still increasing on day 16 and peaks at day 19. Interestingly, a very similar pattern of gene expression has been seen in CIA, although the increases generally occur 2–3 days later than in AA. Of the genes examined in all three models, we have primarily observed a difference in the expression level. The magnitude of mRNA increase was less in CIA than AA for a number of genes, including CD11b, TNF-_, IL-1`, iNOS, MMP13, and TRAP. The magnitude of increase is least in monoarticular SCW, although there is a marked induction of IL-6 at 24 days (3 days after reactivation); greater than CIA or AA at their respective final study day. Table 4 summarizes the relative magnitude of mRNA increase compared to housekeeping gene GAPDH on the final day of each model. Not all of the genes we have analyzed demonstrated regulation: for example, IL-4,
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Table 4 – Comparison of joint gene expression in rat models of arthritis
Gene Cell marker CD11b (myeloid) CD4 (T helper) Pro-inflammatory TNF-_ IL-1` IL-6* COX-2 iNOS Anti-inflammatory IL-10 TGF-` Metalloprotease MMP3 MMP13 Osteoclast-associated RANKL TRAP Cathepsin K
Joint mRNA expression: fold increase over baseline AA CIA SCW (100P)
10–25× 2–4×
5–20× 2–3×
6–10× 2×
5–15× 10–100× 250–3000×* 5–25× 30–150×
3–10× 10–60× 500–6000× 2–6× 15–70×
4–5× 15× > 6000× 6× 7×
2–3× 2–5×
2–3× 2–3×
ND ND
15–50× 15–40×
15–40× 3–20×
40× 15×
50× 5–10× 20×
20× 2–5× 10×
6× 5× ND
*Higher baseline associated with mineral oil injected control group (AA baseline) compared to saline or IFA injected controls (SCW, CIA baseline)
COX-1, and B29 (Ig beta) failed to demonstrate significant change in expression or differential expression between the experimental groups in all models.
Cytokine levels Plasma/serum and tissue cytokine levels have been studied by several investigators, using a variety of bioassay and ELISA methods. In AA, Philippe et al. [5] noted a spike in serum levels of TNF-_ and IL-6 (not IL-1`, which remained unchanged) between 6 and 12 h after CFA injection. These levels returned to baseline and then gradually increased up to day 20, with a greater magnitude of increase observed for IL-6 than TNF-_. Szekanecz et al. [89] reported concomitant increases in serum and joint cytokine levels between days 11 and 25 for TNF-_ and IL-6, and increased
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joint IL-1` as well. Smith-Oliver et al. [107] were able to detect TNF-_ in AA joints at day 20. In CIA joints (days 14–28), Magari et al. [108] found elevated levels of IL-1` and IL-6 protein, but not TNF-_. Rioja et al. [30] have examined joint cytokine levels in monoarticular SCW and found a good correlation of mRNA and protein increases for IL-6 and IL-1`, both immediately following the i.a. injection and 3 days after the i.v. challenge. Increased TNF-_ mRNA, but not protein, was also detected. The consistent detection of elevated joint TNF-_ mRNA (Tab. 4), as well as the anti-arthritic efficacy of anti-TNF-_ therapies, in all of the models supports the concept that perhaps the current methods are not sufficiently sensitive to detect small but significant increases of TNF-_ protein in these joints.
Cartilage pathology The articular cartilage can be assessed using histological methods, although routine hematoxylin and eosin (H&E) staining is generally not sufficient for visualizing subtle changes. Special stains for proteoglycan such as toluidine blue or SafraninO can be used to demonstrate a compromise of the cartilage layer. However, histology-based methods are not quantitative. Methods of measuring cartilage synthesis using uptake of radiolabeled sulfate or glycosaminoglycan quantitation has been used to some extent and show reduced cartilage formation in arthritis [5, 109]. However, these methods require the sacrifice of the animal. Recently, assays have been developed that utilize non-invasive, blood-based parameters to assess cartilage status. Useful analytes include, cartilage-associated oligomeric matrix protein (COMP), which can be measured in the blood as an indicator of cartilage turnover, and an assay for C terminal collagen type II fragments (CartilapsTM), which can be measured in serum or urine. The latter is generated by the proteolytic cleavage (destruction) of cartilage. Increased levels of COMP have been detected in CIA by us (Tab. 3) and by others [110, 111]; these levels correlated reasonably well with the clinical score at the end of the study. Regardless of method of assessment, cartilage destruction is a prominent feature of both AA and CIA. In AA, however, cartilage destruction often pales in comparison to the striking destruction of bone. Therefore, it is possible that loss of cartilage may in fact be due at least in part to the loss of the underlying bone. In CIA, there is a strong humoral immune response to type II collagen, with high titers of anti-collagen IgG antibodies; thus the articular cartilage is a primary target. In monoarticular SCW, at the peak of ankle swelling, i.e., 3 days following i.v. challenge with the bacterial component, there are minimal changes in the cartilage or bone of the joints. This is likely due to the short course (3 days) of the response. It has been observed that cartilage damage does occur if a greater post-inoculation interval (10–20 days) and/or additional reactivation of the disease is used [29, 31]. Figure 1 illustrates the H&Estained histopathology of all three models.
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Figure 1 The hind paws were decalcified in Immunocal (American MasterTech Scientific Inc., Lodi, CA) for 4 days. After decalcification paws were embedded in paraffin, sectioned, and stained with H&E. Histological evaluation of the ankle joints (peak disease) was based on five parameters, each scored on a scale of 0–4: composite inflammation (average of cellular infiltrate, edema and joint/tendon effusion), composite bony change (average of periosteal new bone formation and osteolysis), synovial alteration, pannus (fibrinocellular debris/granulation tissue within joint space), and cartilage degeneration. Higher scores indicated more severe disease as defined by each histopathological parameter. H&E-stained sagittal sections through the tibiotalar joint (upper panels) and midfoot region (lower panels) of a normal rat and diseased rats from three different models of arthritis. In the normal rat, the bone (B) and cartilage (C) are intact, and the synovium (S) is of low cellularity, consisting of mainly adipose cells with a synovial cell lining that is only a few cells thick. In the monoarticular SCW 100P joint, taken 3 days after i.v. reactivation, the synovium is hypercellular due to infiltrating inflammatory cells and synoviocyte proliferation. Bone and cartilage are intact, however. In the CIA joint, harvested on day 21 after collagen injection, synovial inflammation is evident, and there are foci in the distal tibia, talus, and midfoot where pannus tissue (black arrows) is invading the bone and cartilage. In AA, at day 19 after CFA injection, synovial inflammation is marked, and the invasion by pannus and resulting bone destruction is dramatic. Total magnification of images is 50×; black bar represents 150 µm.
Bone pathology Changes in bone are prominent features of AA and CIA. By histopathology, there is an extensive effacement of bone by the invasive inflammatory pannus tissue, and there is marked periosteal new bone formation and osteoclastic bone resorption.
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Figure 2 Comparing bone destruction by micro-computed tomography in rat models of arthritis. µCT imaging was performed on a Scanco µCT-40 (Scanco Medical AG, Zurich, Switzerland). The excised hind paws were secured in 36-mm imaging tubes and bathed in 10% buffered formalin. Approximately 200–300 slices, with 37.6-µm slice thickness, were acquired on a 1024 × 1024 image matrix with digital resolution of 36 µm × 36 µm × 37.6 µm. Other imaging parameters included a 150-ms exposure time, 55 peak kilovolts (kVp) 145 milliamp (mA) and 1000 projections over 360°. A normal rat ankle is shown for reference. In AA (day 19), severe bone loss is evident in the distal tibia and midfoot such that normal structures are difficult to recognize. This loss of bone volume can be quantitated and is significant. In contrast, bone volume loss is less in CIA (day 21). An alternative parameter, bone roughness, has been developed to quantitate the more subtle bony changes that occur in CIA (see Table 3). In monoarticular SCW (day 56), there is minimal bone damage and it is too little for quantitation. On day 24, after the first reactivation flare, in SCW (not shown) there is essentially no bone damage.
These features are seen in both models but are more dramatic and extensive in AA. Chronologically, investigators have noted the presence of large numbers of osteoclasts and their precursors, and bone destruction as early as day 5 in AA, and day 10 onward in CIA [49]. Bone pathology can also be assessed by newer, imagingbased methods. Micro-computed tomography (µCT) has been used by ourselves and others [112–114]. The µCT appearance of a representative arthritic joint from each model is shown in Figure 2, illustrating the differing degrees of bony involvement. The information captured by µCT can be used to quantitate the bone volume loss in AA (% decrease of 40 ± 20), and prove that it is indeed greater than CIA (% decrease, 16 ± 6) (Tab. 3) [114]. Typically, the bone volume loss in CIA is too small
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to afford a sufficient window for evaluating therapeutics. To address this issue, an alternative µCT-generated parameter, bone roughness, has been developed [114] to quantitate the more subtle bone changes seen in CIA. It is now possible to assess these bony changes in live, anesthetized rats as well as in post-mortem specimens. Magnetic resonance imaging (MRI) has also been used by some investigators, although effective quantitative methods are still under development [1, 115]. Other direct, quantitative methods that have been used include measurement of bone mineral density, and bone mineral content [113, 116, 117]. Indirect methods include blood-based assays for bone metabolites that offer a means of assessing bone destruction in the living rat. Useful analytes include collagen type I fragments (RatLapsTM) [112] (Tab. 3), osteocalcin [115, 118–120], and bone sialoprotein (BSP) [111].
Methods Adjuvant-induced arthritis As previously stated, there are multiple rat strains that can be used in the AA model and there appears to be no differences in susceptibility due to gender, however, in our laboratory we have exclusively used female Lewis rats with a starting weight of 150–170 g. Rats (n = 12) are randomly divided into experimental groups and weighed to determine the average body weight of each group. We also measure each rat’s ankle and paw (maximal lateral) individually with a plethysmometer (Ugo Basile Biological research Apparatus, Italy) to determine the baseline ankle measurement. Calipers can be used to measure ankle thickness as well but using a volume displacement method tends to be less biased by the researcher taking the measurement and encompasses the entire ankle instead a of single location. Rats receive an 0.1-mL injection using a 30 gauge needle of CFA (containing Mycobacterium tuberculosis H37 Ra ATCC 25177 heat killed from Difco Laboratories, Lee Labs, Grayson, GA) at a concentration of 3 mg/mL in mineral oil intradermally (i.d.) into the right hind footpad. Control rats are injected with an equal volume of mineral oil alone. The rats are lightly anesthetized during this injection process. Disease can also be induced if the injection is given i.d. at the base of the tail; however, we have found the robustness of disease between rats is much more consistent when the footpad injection site is used. Additionally, we have reduced the typical dose of 1.0–0.5 mg CFA to 0.3 mg. This does not compromise the therapeutic window, but has reduced the primary lesion on the injected footpad, thereby reducing pain and distress. The onset of arthritis, as indicated by contralateral paw swelling, appears on approximately day 10 post injection (Fig. 3). Both paw volume and body weights are measured throughout the study every 2–3 days. We have found for typical compound screening experiments that 19 days is sufficient to evaluate novel compounds
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Figure 3 Rat adjuvant-induced arthritis model (AA)
by providing a large enough therapeutic window to determine accurate half maximal effective dose ED50. The longer the disease progresses, the more bone damage occurs and the lesion on the injected paw worsens and even begins to develop on the contralateral paw. There can actually be a reduction in paw swelling over time due to reduced edema and inflammation. In our hands, we have not found the need to cull animals due to lack of disease development or too severe lesion development. However, if this is an issue, refer to an excellent review of this practice in the previous edition of this chapter [1]. Compound testing can be either prophylactic (beginning just prior to CFA injection and continuing throughout the experiment, typically 19 days) or therapeutic (beginning on day 10 and continuing to day 19). To get a sense of drug coverage over the course of one day, we serially bleed animals for pharmacokinetic (PK) analysis. Usually, for PK analyses, we add four additional animals per group and encompass the time points of 0, 1, 3, 6, 8 and 24 h. Therefore, these animals are bled serially five times by restraining the animal using a rat holder and warming the tail with a heat lamp or immersing it in warm water to dilate the vessels. The last time point (24 h) should be done after euthanasia via cardiac puncture. If the day chosen for the full PK determinations is late in disease (day 18 or later) it can be dif-
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ficult to obtain blood from the tail vein because arthritis in evident in the tail due to the cartilage destruction within the tail. We continue measuring each rat's ankle individually with a plethysmometer (paw volume meter) every 2–3 days to determine the response. We also weigh each rat in each group and determine the average body weight of each group every 2–3 days. A local nonspecific inflammatory reaction occurs at the site of the CFA injection. The initial acute inflammation is observed around day 3 and can progress over time into a lesion. The contralateral hind paw volume increase 65–70% over time (plateau occurs around day 19). Arthritic rats also experience about a 20% reduction in body weight when compared to nonarthritic controls by days 16–60. Body weight loss should be monitored carefully, because if rats experience more than a 20% reduction in their initial body weight, they should be humanely euthanized. As disease progresses, both hind limbs become immobile, and animals drag themselves with their front paws, but they are perfectly capable of reaching their food and water sources. However, to allow for easier access and to encourage them to eat, food pellets or gel packs can be placed within their cages. Swelling can still be observed up to 60 days. No response should be observed in the negative (mineral oil) control group of rats. The typical duration of each novel compound study is 19 days, but on occasion it may be necessary to extend the time period, e.g., to provide a longer treatment period to test for improvement in efficacy, or for removal of a compound after successful efficacy is achieved, to see if disease reduction is maintained or if the disease returns. Other physical changes observed as the disease progresses are decreased food intake, roughening of the hair, and lethargy [4]. Another common sign of distress is chromodacryorrhea [121]. This condition can be mistaken for a bloody discharge from the eyes and nose, but is actually caused by an ocular secretion of a porphyrin-containing pigment deep within the orbits and has been correlated with a stress response [1].
Collagen-induced arthritis The basic principles and procedures, as well as the clinical signs of disease for the CIA model are very similar to those just described for AA in the rat [8]. The biggest difference, as the name implies, is the material used to induce the disease and how it is introduced. On days 0 and 7, shaved female Lewis rats are administered three i.d. injections at the base of the tail using a 25–27 gauge needle (100 µg/0.1 mL/site) of incomplete Freund’s adjuvant (IFA; Difco, Franklin Lakes, NJ) only or IFA plus type II collagen (CII). We use nasal bovine type II collagen (CN276) obtained from Elastin Products Company, Inc. (Owensville, MO) at an initial concentration of 2 mg/mL in 0.01 M acetic acid. This solution is stirred overnight, and then emulsified with equal volume of IFA. A total volume of 0.3 mL is injected at multiple locations (0.1 mL/site); other sites such as intrascapular and flank regions
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Figure 4 Rat collagen-induced arthritis model (CIA)
can be used too. The rats are anesthetized with isoflurane for the injections. The rats get a booster injection again on day 7. As in the AA model, both paw volume and body weights are measured throughout the study approximately every 2–3 days. Paw volumes are measured using a water displacement plethysmometer and the onset of arthritis, as indicated by increased paw volume, appears on approximately day 14 following the initial injection of collagen. Disease onset and plateau are slightly delayed compared to the AA model and less robust (Fig. 4). No response should be observed in the negative (IFA only) control group of rats. The typical duration of a study for each novel compound is 21 days, but once again it may be necessary to extend the time period. A local nonspecific inflammatory reaction occurs at the site of the collagen injections, which also progresses into a lesion over time. The hind paw volumes increase 45% over time and plateaus around day 21. Arthritic rats may experience up to a 20% reduction in body weight compared to non-arthritic control animals at peak disease (day 21). Arthritic rats then start to gain weight again more rapidly and return to a body weight near that of their non-arthritic controls by day 45. One advantage of this model is that both hind paws can be assessed as part of the disease measurements, whereas in the AA model only the contralateral is appropriate. Although clearly showing the signs and symp-
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toms of distress as described for the AA model, in all cases it appears to be milder disease. The preparation of the collagen emulsion is one of the most critical steps in this model. The collagen mixture should be added dropwise to the IFA while gently sonicating the resulting mixture. After all the collagen is added, sonicate the solution more robustly for about 30 s. Care needs to be taken not to overheat the collagen, and to maintain a uniform, cool temperature, this entire procedure should be done on ice. The collagen emulsion thickens and should not spill out of beaker if turned upside-down. If the emulsion does not thicken, it should not be used. The collagen model requires more time and skill in the preparation when compared to AA, but there are also advantages. As previously mentioned, both hind limbs can be used for analysis and the milder disease causes less distress to the animals. Other advantages, as discussed in the previous sections, include the antibody component and the directed nature of the cartilage destruction.
Streptococcal cell-wall-induced arthritis Once again many of the comments regarding procedure and general principles of rat arthritis models have already been discussed and apply to SCW as well. There are two different versions of SCW, a polyarthritic or systemic 10S model and the monoarticular 100P model. In the polyarthritic model, a single injection of the 10S fraction of a SCW preparation is given intraperitoneally (i.p.) [31, 70, 122]. An initial acute response occurs and consists of a sharp increase in ankle measurements, which are typically 25% above baseline measurements. This rise reaches a peak in 3–5 days and is followed by a decline in the measurements on subsequent days. A chronic response should manifest around 12–14 days post injection and be seen as a slow increase in the ankle measurements. This is typically seen as 35% over baseline measurements (Fig. 5). The chronic response is remittent and erosive, and should persist for the remainder of the experimental period. Not all animals (~25%) exhibit this initial swelling and are considered non-responders and must be removed, this is a distinct disadvantage of this model. Additionally, the SCW material is much more costly than either adjuvant or collagen preparation. Lastly, we hypothesize that because the antigen is given i.p., animals have organ damage particularly the liver, spleen and kidney. The model we prefer, although it has not been used extensively in the pharmaceutical industry to date, is the monoarticular 100P model [1, 9, 29, 31]. Technically, this model is a bit more difficult, but it provides some advantages over the traditional AA and CIA models. First, in regard to the preparation of the SCW material, PG-PS 100P (Lee Laboratories) is sonicated and diluted in sterile, pyrogen-free saline to a concentration of 500 µg/mL rhamnose units. To induce disease, 10 µL (5 µg rhamnose units per joint) is injected i.a. into the tibiotalar joint space using a 27-
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Figure 5 Rat streptococcal cell wall-induced systemic arthritis model (SCW)
gauge needle on day 0 and slowly i.v. (400 µL, 250 µg/mL rhamnose) on day 21 via tail vein. Rats are lightly anesthesized during the i.a. injection to relieve the pain associated with the injection and to prevent movement, aiding in accuracy. The first administration of PG-PS 100P causes an acute response with minor paw swelling that peaks after 2–3 days (typically 20% above baseline) and then declines returning to baseline. The i.v. boost causes paw swelling to increase typically 35% above baseline (Fig. 6). [9, 10]. The monoarticular SCW model most closely resembles an arthritic flare seen in patients and has the additional advantage of fewer systemic complications. Overall, this model is less severe than the previously discussed models; as a comparison the AA model has an increase of 65–70% in paw swelling and CIA has a 45% increase over a 10-day period. In the SCW model, the rats continue to gain weight between days 0 and 21 and then experience a 5–10% drop in weight after i.v. reactivation of disease. No response should be observed in the normal (saline) control group of rats. Additionally, to ensure that the model represents a reactivation of disease, it is important to establish that the i.v. injection of PG-PS 1000P alone is nonarthropathic [31].
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Figure 6 Rat streptococcal cell wall-induced monoarticular arthritis model (SCW)
Conclusions This chapter has provided information on three distinct rat arthritic models, and has demonstrated that they are perhaps not that dissimilar. A summary of disease-related parameters for each of these models is given in Table 5. These models have few qualitative differences; the major departures are quantitative in nature. The AA model clearly exhibits the greatest magnitude of disease as measured by edema, cellular influx, cytokine levels, and bone destruction within the joint. Systemic markers of disease, such as APPs and markers of cartilage and bone destruction, are also the most elevated in this model of arthritis. The CIA model in rats is very similar to the AA model in terms of disease outcome as characterized by the parameters given in Table 5; however, the magnitude of such responses is approximately one third to one half less than what is observed in AA. One important difference that should be highlighted is that cartilage is the primary target in the CIA model, whereas cartilage destruction is a secondary consequence of bone damage in the AA model. Also, the monoarticular SCW model exhibits very little cartilage damage after its first reactive flare (day 24). Another distinction of the CIA model is that autoantibodies to type II collagen have been identified and are known to participate in the disease process, and to date no such autoantibodies have been clearly identified as disease
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Table 5 - Summary of rat models of arthritis, AA, CIA and monoarticular SCW CIA
SCW (monoarticular SCW; not SCW polyarthritis)
Initiation
Complete Freund’s adjuvant (contains Mycobacteria); ID; in footpad Single injection, day 0
PGPS 100 intra-articularly in ankle on day 0 followed by 3 week sensitization period; Inflammation reactivated by PGPS 100 IV on day 21
Paws affected
Polyarthritis; only contralateral ankle is relevant
Type II collagen in incomplete Freund’s Adjuvant; ID; in base of tail day 0 plus boost required at day 7 Polyarthritis; Both ankles and knees can be taken
Paw volume
GREATEST in magnitude 4.0 mL volume 2.5mL volume Earliest onset day 10; day 14; max/plateaus around day 19 max/plateaus around day 21 Equivalent in magnitude of Onset score to CIA (day 13) Earlier onset in AA (day 9) Equivalent CD4 & CD14 mRNA Equivalent CD4 & CD14 mRNA CD11b 2X greater in AA than CIA Cartilage destruction is secondary Cartilage is primary target; to bone & soft tissue damage Greater loss of proteoglycans Higher serum COMP levels
Pathology score
Cellular influx Cartilage destruction/ turnover (cartilage oligomeric matrix protein =COMP) Bone destruction/ turnover (RatLaps measures type I collagen)
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Greatest (RATLAPS scores higher) (µCT shows significant bone volume loss)
Inflammation can be induced in both ankles or one side can be used as internal control Small window in paw swelling; 2.0 mL volume Maximum swelling 2–3 days after IV Lower pathology scores (No/minimal bone or cartilage destruction; soft tissue changes primarily) Lower CD4, CD14, CD11b mRNA than both AA, CIA Essentially none
Bone damage; µCT detects Essentially none; significant bone roughness µCT supports histopathology but usually not significant bone volume loss
Rat models of arthritis: Similarities, differences, advantages, and disadvantages…
AA
AA
CIA
Other pro-inflammatory molecules: IL-1` TNF-_ IL-6 COX-2 INOS MMP13
2× higher level of joint mRNA in AA than CIA (Joint cytokine levels also higher in AA) Serum cytokine levels in all three models are generally not detectably increased Acute phase proteins Markedly increased levels of haptoglobin, _1-acid glycoprotein (8–10× over normal) Other systemic inflammation Neutrophils increased to 5–7× indicators: total neutrophils, normal numbers; Fibrinogen 4× fibrinogen levels increased
SCW (monoarticular SCW; not SCW polyarthritis)
Lower mRNA levels than AA or CIA of all of these except COX-2, IL-6 which are comparable to AA
Levels 1/2 to 1/3 of AA levels (3× over normal)
Levels 1/2 to 1/3 of AA levels (3× over normal)
Neutrophils increased to 3–4× normal numbers; Fibrinogen 2× increased
Neutrophils not significantly increased; Fibrinogen 2× increased
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Table 5 (continued)
Rat models of arthritis: Similarities, differences, advantages, and disadvantages…
contributors in the AA or the SCW models. The monoarticular SCW model most closely resembles an arthritic flare seen in human patients, and although this model shares many of the described features, it is much milder in terms of joint damage and systemic markers when compared to AA and in most cases CIA as well. As related to human disease, these models progress much more rapidly and exhibit marked bone resorption and formation, which is perhaps more similar to ankylosing spondylitis than RA. However, as highlighted in this chapter, these animal models have proven effective in predicting the outcome of many of the current drugs used in the treatment of RA and will continue to be useful tools for discovery of new therapies.
Acknowledgements The authors would like to sincerely thank Anneli Savinainen, Julie Kujawa, Michelle DuPont, Kristina Perry, Matt Silva, Elizabeth Siebert, and Sudeep Chandra from Millennium Pharmaceuticals for their technical support and dedication without which the quality data presented here would not have been possible.
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107 Smith-Oliver T, Noel LS, Stimpson SS, Yarnall DP, Connolly KM (1993) Elevated levels of TNF in the joints of adjuvant arthritic rats. Cytokine 5(4): 298–304 108 Magari K, Miyata S, Ohkubo Y, Mutoh S (2004) Inflammatory cytokine levels in paw tissues during development of rat collagen-induced arthritis: effect of FK506, an inhibitor of T cell activation. Inflamm Res 53(9): 469–474 109 Seed MP, Gardner CR (2005) The modulation of intra-articular inflammation, cartilage matrix and bone loss in mono-articular arthritis induced by heat-killed Myobacterium tuberculosis. Inflammopharmacology 12(5): 551–567 110 Larsson E, Erlandsson Harris H, Larsson A, Mansson B, Saxne T, Klareskog L (2004) Corticosteroid treatment of experimental arthritis retards cartilage destruction as determined by histology and serum COMP. Rheumatology (Oxford) 43(4): 428–434 111 Larsson E, Mussener A, Heinegard D, Klareskog L, Saxne T (1997) Increased serum levels of cartilage oligomeric matrix protein and bone sialoprotein in rats with collagen arthritis. Br J Rheumatol 36(12): 1258–1261 112 Sims NA, Green JR, Glatt M, Schlict S, Martin TJ, Gillespie MT, Romas E (2004) Targeting osteoclasts with zoledronic acid prevents bone destruction in collagen-induced arthritis. Arthritis Rheum 50(7): 2338–2346 113 Badger AM, Griswold DE, Kapadia R, Blake S, Swift BA, Hoffman SJ, Stroup GB, Webb E, Rieman DJ, Gowen M et al (2000) Disease-modifying activity of SB 242235, a selective inhibitor of p38 mitogen-activated protein kinase, in rat adjuvant-induced arthritis. Arthritis Rheum 43(1): 175–183 114 Silva MD, Savinainen A, Kapadia R, Ruan J, Siebert E, Avitahl N, Mosher R, Anderson K, Jaffee B, Schopf L et al (2004) Quantitative analysis of micro-CT imaging and histopathological signatures of experimental arthritis in rats. Mol Imaging 3(4): 312–318 115 Jacobson PB, Morgan SJ, Wilcox DM, Nguyen P, Ratajczak CA, Carlson RP, Harris RR, Nuss M (1999) A new spin on an old model: in vivo evaluation of disease progression by magnetic resonance imaging with respect to standard inflammatory parameters and histopathology in the adjuvant arthritic rat. Arthritis Rheum 42(10): 2060–2073 116 Yamane I, Hagino H, Okano T, Enokida M, Yamasaki D, Teshima R (2003) Effect of minodronic acid (ONO-5920) on bone mineral density and arthritis in adult rats with collagen-induced arthritis. Arthritis Rheum 48(6): 1732–1741 117 Okazaki Y, Tsurukami H, Nishida S, Okimoto N, Aota S, Takeda S, Nakamura T (1998) Prednisolone prevents decreases in trabecular bone mass and strength by reducing bone resorption and bone formation defect in adjuvant-induced arthritic rats. Bone 23(4): 353–360 118 Okamoto A, Yamamura M, Iwahashi M, Aita T, Ueno A, Kawashima M, Yamana J, Kagawa H, Makino H (2003) Pathophysiological functions of CD30+ CD4+ T cells in rheumatoid arthritis. Acta Med Okayama 57(6): 267–277 119 Osterman T, Virtamo T, Lauren L, Kippo K, Pasanen I, Hannuniemi R, Vaananen K, Sellman R (1997) Slow-release clodronate in prevention of inflammation and bone loss associated with adjuvant arthritis. J Pharmacol Exp Ther 280(2): 1001–1007
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120 Osterman T, Kippo K, Lauren L, Hannuniemi R, Sellman R (1995) Effect of clodronate on established collagen-induced arthritis in rats. Inflamm Res 44(6): 258–263 121 Harkness JE, Ridgway MD (1980) Chromodacryorrhea in laboratory rats (Rattus norvegicus): etiologic considerations. Lab Anim Sci 30(5): 841–844 122 Wilder RL (1988) Streptococcal cell-wall-induced arthritis in rats: an overview. Int J Tissue React 10(1): 1–5 123 Neuman RG, Wilson BD, Barkley M, Kimball ES, Weichman BM, Wood DD (1987) Inhibition of prostaglandin biosynthesis by etodolac. I. Selective activities in arthritis. Agents Actions 21(1–2): 160–166 124 Tanaka K, Shimotori T, Makino S, Aikawa Y, Inaba T, Yoshida C, Takano S (1992) Pharmacological studies of the new antiinflammatory agent 3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-o ne. 1st communication: antiinflammatory, analgesic and other related properties. Arzneimittelforschung 42(7): 935–944 125 Calhoun W, Gilman SC, Datko LJ, Copenhaver TW, Carlson RP (1992) Interaction studies of tilomisole, aspirin, and naproxen in acute and chronic inflammation with assessment of gastrointestinal irritancy in the rat. Agents Actions 36(1–2): 99–106 126 Ackerman NR, Rooks WH, 2nd, Shott L, Genant H, Maloney P, West E (1979) Effects of naproxen on connective tissue changes in the adjuvant arthritic rat. Arthritis Rheum 22(12): 1365–1374 127 Carlson RP, Hartman DA, Tomchek LA, Walter TL, Lugay JR, Calhoun W, Sehgal SN, Chang JY (1993) Rapamycin, a potential disease-modifying antiarthritic drug. J Pharmacol Exp Ther 266(2): 1125–1138 128 Magari K, Miyata S, Nishigaki F, Ohkubo Y, Mutoh S (2004) Comparison of antiarthritic properties of leflunomide with methotrexate and FK506: effect on T cell activation-induced inflammatory cytokine production in vitro and rat adjuvant-induced arthritis. Inflamm Res 53(10): 544–550
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Murine collagen induced arthritis Leo A. B. Joosten and Wim B. van den Berg Rheumatology Research and Advanced Therapeutics, Department of Rheumatology, Radboud University Nijmegen Medical Center, PO Box 9101, 6500 HB Nijmegen, The Netherlands
Introduction A major research goal in the field of arthritis is to unravel the pathogenesis of chronic arthritis and the concomitant joint destruction. A second, more practical goal is to define targeted therapies, selectively inhibiting the progression of destructive arthritis, yet leaving host defense mechanisms virtually intact. Although animal models are not ideal in terms of precise mimicry of human arthritic disease, they do reflect key aspects of their human counterparts and offer a useful approach to understand arthritic processes and to improve therapeutic treatment. Rheumatoid arthritis (RA) is characterized by chronic inflammation in the joints and progressive destruction of cartilage and bone. Histopathological features include immune complexes in the articular cartilage layers and variable amounts of macrophages and T cells in the synovium, accompanied by fibrosis and synovial hyperplasia. The disease is often considered as an autoimmune process, the articular cartilage being an intriguing component, since it is the victim but also a likely trigger of the disease. Arguments for this are based on the observation that destructive forms of RA tend to decline when the cartilage is fully destroyed. Moreover, total joint replacement often results in a complete remission of arthritis in that particular joint, without the need of concomitant synovectomy. This is compatible with cartilage components being joint specific autoantigens or cartilage tissue functioning as an avascular reservoir, retaining yet unidentified arthritogenic triggers. Models have been developed that have proved the arthritogenic potential of cartilage autoantigens, such as collagen type II (CII) and proteoglycan [1–4]. More recently [5–7], arthritogenic potential has been demonstrated for novel cartilage components such as collagen types IX and XI, cartilagederived oligomeric protein (COMP) and hyaline cartilage glycoprotein 39 (HC gp-39). These models all elude to the same principle: arthritis due to the loss of tolerance against a cartilage-specific autoantigen. Based on the hypothesis that RA is initiated by cross-reactivity of T cells to bacterial fragments and cartilage components, models of experimental arthritis were generated using bacteria as antiIn Vivo Models of Inflammation, Vol. I, edited by Christopher S. Stevenson, Lisa A. Marshall and Douglas W. Morgan © 2006 Birkhäuser Verlag Basel/Switzerland
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gens. Adjuvant arthritis and streptococcal cell wall-induced arthritis are the most common models of arthritis, using Mycobacterium tuberculosis and group A streptococci, respectively [8–11]. The present chapter is confined to detailed discussion of key events in arthritis induced with CII. Collagen-induced arthritis (CIA) is a widely accepted arthritis model, based on T cell and antibody-mediated autoimmune reactivity against cartilage CII. The model is characterized by severe cartilage and bone erosions. Induction has been demonstrated in various strains of rats and mice, susceptibility showing tight genetic restriction. More recently, CIA has been induced in non-human primates as well. The model of CIA is highly suited to analyze principles of autoimmune disease expression and antigen-specific immunosuppression. Moreover, it can be used to study mechanism and mediators involved in autoimmune cartilage and bone destruction. The following sections mainly deal with features of CIA in the mouse.
Induction of CIA The model of CIA was first described in 1977 by Trentham and colleagues [1], as a coincidental finding in protocols to induce autoantibodies to purified collagen preparations. The initial observations indicated that arthritis was confined to sensitization with native CII; denatured CII or native CI not showing arthritogenicity. The crucial element in this arthritis is the induction of immunity to foreign CII, subsequently cross-reacting with homologous CII. Plain immunization with homologous instead of heterologous CII can also be used, but then much stronger immunization regimens are needed to override natural tolerance. In Lewis rats, a single immunization with CII in complete Freund's adjuvant (CFA) at the base of the tail is sufficient to get full-blown expression of a polyarthritis within 14 days. In mice, the disease expression is more gradual, starting after 3–4 weeks in some animals, whereas a 10% incidence commonly takes 8–10 weeks. Both chicken and bovine CII preparations are proper heterologous antigens for inducing CIA in mice. Susceptible mouse strains include DBA/1j and B10RIII mice, which have the H-2q and H-2r haplotype, respectively. The dominant epitopes of the CII molecule differ between the DBA/1j and B10RIII mice, consistent with the different haplotype [7, 8]. Male mice show higher susceptibility as compared to female mice. In general, we use DBA/1j male mice and bovine CII to induce this model. Bulk quantities of bovine CII can be isolated from articular cartilage slices, taken from a knee joint of 1–2-year-old cows, according to Miller and Rhodes [9]. In brief, proteoglycans are extracted from the cartilage with 4 M guanidinium chloride in a neutral 0.05 M Tris buffer, for 24 h at room temperature. After washing, the cartilage is digested with pepsin (1 mg/ml in 0.1 M acetic acid) for 48 h at room tem-
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perature. The suspension is then centrifuged at 1000 g and the supernatant is adjusted to pH 7.4 with 2 M NaOH. Bulk protein is precipitated with addition of solid NaCl, reaching a final concentration of 20% NaCl, and equilibration for 2 days at 4°C. After centrifugation at 27 000 g for 30 min, the pellet is resolved in 0.5 M acetic acid and dialyzed against this solution overnight at room temperature. The collagen is then redissolved, and remaining material is removed by centrifugation (10 min at 2000 g). The collagen is then selectively precipitated at a final concentration of 5% NaCl, overnight at 4°C. The precipitate is spun down, redissolved and reprecipitated with 5% NaCl twice more to obtain a purer collagen preparation. Final dialyzation is done against 0.05 M acetic acid and the preparation can be stored as such at –20°C, or lyophilized. Pure, native CII preparations are poorly soluble in water, which provides a first, simple check on quality. Purity can be analyzed by gel electrophoresis. In addition, newly prepared CII batches are first screened in an ELISA, and compared with former arthritogenic CII batches and the use of a standard set of anti-CII antibodies, obtained from a pool of arthritic mice. To obtain a defined solution for immunization, CII is slowly dissolved in 0.05 M acetic acid overnight at 4°C (concentration 2 mg/ml) and then emulsified with an equal volume of CFA, containing 2 mg/ml Mycobacterium tuberculosis, strain H37Ra. Mice are then immunized intradermally at the base of the tail with 100 µl emulsion (100 µg CII). On day 21 a booster injection is given with 100 µg CII in 100 µl PBS, administered intraperitoneally. Onset of arthritis starts around days 25–28, often first affecting some digits of hind and fore paws, then spreading to multiple sites in the paw, including the ankle compartments (Fig. 1). When not heavily boosted the onset may be rather gradual, not even reaching a 100% incidence at 8 weeks and with limited numbers of joints affected (Fig. 2A). The model is a mixture of an immune complex disease and a delayed-type hypersensitivity reaction in the joint. Although anti-CII antibodies alone are able to induce arthritis after passive transfer, high concentrations are needed, in particular of complement-activating subclasses, recognizing multiple epitopes, and even then at best a transient arthritis occurs [10]. Passive transfer with bulk T cells or T cell clones has also been shown to yield poor disease expression [11]. Antibodies are probably needed to bind to the cartilage surface, promoting there the further release of collagen epitopes upon complement fixation and the attraction of leukocytes. Attachment of granulocytes to the cartilage surface is a crucial element of CIA. Subsequent influx of anti-CII-specific Th1 cells further drives the arthritic process. Of interest, the cytokine pattern in the lymphoid organs shows a dominant Th1 pattern after immunization with CII in CFA [12]. Susceptibility to CIA is enhanced when the amount of M. tuberculosis in the CFA preparation is enhanced. Moreover, severe arthritis can also be induced upon immunization with CII in incomplete Freund's adjuvant (IFA), provided that the mice are then treated with recombinant IL-12 dur-
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Figure 1 Macroscopic appearance of murine CIA, ranging from normal (A) to one affected toe (B) and full expression in the whole paw (C).
ing the immunization period, which strongly promotes a Th1 response. Bacterial preparations are potent inducers of IL-12. Remarkably, high doses of IL-12 given during standard immunization with CII in CFA were shown to suppress the CIA, associated with a marked reduction in CII-specific antibodies [13]. Although there is no doubt that Th1 reactivity is needed, the critical importance of high levels of anti-CII antibodies is further underlined in studies in susceptible and resistant mice strains. Additional IL-12 treatment during immunization with CII in CFA was shown to enhance CII-specific Th1 responses in C57BL/6 and B10.Q mice, but this protocol still failed to induce arthritis in these mice. Analysis of anti-CII antibody titers revealed that the levels remained markedly lower in these strains as compared to those found in susceptible DBA mice [14].
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Figure 2 (A) Incidence of CIA, as scored in one selected paw or in all paws. Note that the incidence in the right hind paw is still less than 50% after 50 days. Reflects a group of 20 mice. (B) Accelerated expression of CIA and more severe arthritis after i.p. injection of Zymosan at day 26. Reflects groups of 10 mice.
Expression of arthritis Apart from the generation of adequate levels of complement fixing anti-CII antibodies and the presence of anti-CII-specific Th1 cells, it is clear that expression of
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autoimmune arthritis depends on local conditions in joint tissues. CIA shows a higher incidence in male as compared to female mice, whereas other models such as antigen- or Zymosan-induced arthritis show the opposite sex preponderance. Apart from the autoimmune character, the elicitation of the joint inflammation by direct injection into the knee joint is a major difference between these models and CIA. In CIA no local insults are given and arthritis develops “spontaneously”. The male preponderance of CIA might be linked to the impact of hormones on this arthritic process, but it is tempting to suggest that the consistent fighting of male mice also makes a major contribution, causing microtrauma in joint tissues, which are crucial in triggering of onset of arthritis. In line with this, it is also our experience that disease incidence is generally higher when the mice are housed in large groups instead of small groups. Threatening autoimmune reactivity is generated by the immunization protocol, but precipitation of the autoimmune process in the joint is facilitated by nonspecific inflammation at such sites or systemic generation of proinflammatory mediators. It has long been recognized that systemic administration of IL-1, shortly before onset of the disease, markedly accelerates CIA expression [15]. This seems related to activation of endothelium, facilitating influx of inflammatory cells. Moreover, IL1 is a potent cartilage destruction mediator, causing loss of cartilage proteoglycans and thereby denuding the autoimmune target in CIA, the CII in the articular cartilage. In addition, it has been shown that local injection of TNF-_ or TGF-` potentiates CIA expression in the injected joint [16, 17]. TNF-_ is a pivotal proinflammatory cytokine in arthritis and an inducer of IL-1. TGF-`, although having immunosuppressive potential, is a potent chemoattractant. All of this fits with unmasking of dormant autoimmune reactivity by nonspecific attraction of inflammatory cells to the joints, including CII-reactive T cells, and amplification of the process by inflammation-mediated exposure of autoimmune epitopes. A single injection of LPS provides an elegant alternative for the acceleration of CIA by systemic administration of recombinant IL-1 [18]. In our standard protocol we give a booster immunization with 100 µg CII at day 21 and a single i.p. injection of 10–40 µg LPS around day 28. This not only greatly enhances the severity of the arthritis, but also synchronizes the expression in a group of mice and enlarges the number of affected joints in one animal. This is useful since spontaneous expression of CIA after plain immunization often affects only a limited number of joints, such as one or two toes, complicating grading and histological analysis. A critical prerequisite for the accelerated expression still remains the proper immunization with CII. In animals showing poor immunity to CII, the CIA can not be accelerated, either with a single or with repeated LPS challenge. The mechanism behind CIA acceleration with LPS can be linked to the generation of TNF-_ and IL-1, as well as marked IL-12 production. LPS-induced acceleration can be blocked with antibodies against IL-12, and acceleration can be induced with systemic administration of recombinant IL-12 [19].
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Table 1 - Factors influencing expression of murine CIA -
Isotype of the anti-CII antibodies Degree of Th1 anti-CII reactivity Acceleration with the cytokines IL-1, TNF-_ and TGF-` Control by the suppressive cytokines IL-4 and IL-10 (TGF-`) Nonspecific trauma in joint tissues promotes expression
Apart from LPS-induced cytokine generation, it is conceivable that the LPSinduced elicitation of IL-12 promotes Th1 generation. When applied shortly after the booster injection with CII, it will influence the critical process of generation of cross-reactive T cell activity from heterologous CII to the homologous CII of the mouse, including the process of epitope spreading. Although CIA can be induced by immunization with a small CII fragment, containing the dominant epitope, the arthritis is less severe as compared to the one induced with the whole CII molecule, suggesting multiple epitope involvement in classic CIA [9]. In line with the notion that any inflammatory stimulus, generating proinflammatory mediators such as TNF-_ or IL-1, will accelerate CIA expression (Tab. 1), we have demonstrated that a systemic injection with Zymosan (yeast particles) highly accelerates CIA in DBA/1j mice [20]. Consistent enhancement of CIA incidence and severity was seen with a dose of 3 mg Zymosan, injected i.p. This injection induces a marked peritonitis and onset of accelerated CIA expression was just noted after a few days. With increasing dosages of Zymosan, the arthritis expression could not be further enhanced (Fig. 2B). We even observed a delay in day of onset, apparently linked to a more prolonged, distracting inflammation in the peritoneal cavity.
Unilateral CIA We have developed a variant of the polyarthritic Zymosan-induced CIA, by local injection of Zymosan in one knee joint [20]. This injection is given around day 25 after the first immunization with CII at day 0 and boosting at day 21. Upon local injection in nonimmunized DBA mice, Zymosan induces a transient arthritis, with reversible cartilage proteoglycan depletion. When injected in CII-immunized DBA mice, a dose of 60 µg Zymosan is sufficient to accelerate expression of CIA in that knee joint, as reflected by the characteristic, aggressive cartilage destruction and prolonged joint inflammation. When a higher dose is injected, 180 µg Zymosan, the expression of CIA is not restricted to the injected joint, but also extends to the ipsilateral ankle joint, whereas the expression in the other paws was not enhanced. With the 180 µg dose of Zymosan the accelerated and synchronized expression in
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the ankle reached an incidence of 90%, whereas the incidence dropped to 10–50% with lower Zymosan dosages. Anti-TNF-_ treatment markedly reduced this spreading to the ipsilateral paw, whereas anti-IL-1 treatment fully prevented the expression in that ankle joint, making it likely that the Zymosan-induced expression is related to TNF-_ and IL-1, produced locally in the knee joint and diffusing to the ankle. To obtain optimal advantage of a fully synchronized and localized expression model of CIA, it is essential that the initial immunization and boosting with CII is not too optimal, creating already a considerable number of mice with affected ankles around day 25. In daily practice, when aiming for this unilateral model, we apply normal amounts of M. tuberculosis (1 mg) in the initial immunization, give no boosting with LPS and perform the CII boosting at day 21 in saline and not in Freund's adjuvant. Finally, we do a prescreening of the mice at day 25, discarding all animals having any sign of arthritis from the experiment and performing the local Zymosan acceleration in this negative-selection group. This approach highly reduces the variation normally encountered in the spontaneous polyarthritic CIA.
CIA in C57BL/6 mice It was shown previously that susceptibility of CIA was linked to the H-2q haplotype (DBA-1) and that H-2b (C57BL/6) mice were less sensitive for developing CIA. Recently, several studies have demonstrated that it is possible to induce CIA in C57BL/6 mice, although the disease incidence is lower than in the DBA-1 mice [21, 22]. C57BL/6 mice are immunized with 100 µg chick CII in enriched CFA (5 mg/ml M. tuberculosis) at the base of the tail at days 0 and 21. The first signs of CIA can be seen at day 30, and about 60–70% of the animals will get CIA at 40–50 days after the first immunization. The clinical and histological appearance of CIA in the C57BL/6 mice resembles the CIA in the DBA-1 mice. One of the great benefit of CIA in C57BL/6 mice is the applicability of this arthritis model in gene-deficient mice, since the most gene-deficient mice are on the C57BL/6 background.
Passive transfer model of CIA There is a growing interest in the use of passive immune complex models of RA, along with the availability of transgenic animals to unravel crucial pathways of inflammation and tissue destruction. The advantage of passive immune complex models is the applicability in several mouse strains. The first study was performed by transfer of whole serum or immunoglobulins concentrates isolated from arthritic DBA-1 mice to naïve DBA-1 mice [23]. Histopathologically, this passively transferred arthritis resembled the early disease of immunized DBA-1 mice. Several years later Terato et al. [24] described a passive transfer model using monoclonal anti-
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bodies directed against CII. The monoclonal antibodies against CII were developed from DBA/1 mice immunized with chick CII. Several antibodies were reactive with CB11, previously identified as containing a major immunogenic and arthritogenic epitope, which cross-reacted strongly with mouse CII. Individual antibodies were able to induce mild lesions consisting of minimal synovial proliferation but not overt arthritis. However, a combination of four antibodies induced severe arthritis with marked destruction of articular cartilage. Arthritis developed within 48–72 h after injection of the antibodies and persisted for the duration of the observation period of 3 weeks. Sets of antibody pairs are commercially available at the moment. In combination with LPS, the amount of antibodies needed for the passive transfer model can be reduced. Although this arthritis model can be induced in all mouse strains, DBA-1 is the most sensitive strain.
Arthritis score and histopathology The course of CIA is routinely scored by macroscopic analysis of arthritic signs in peripheral joints. In general, mice are examined every other day, from day 25, to get an impression of the course of the disease. Clinical severity of arthritis is graded on a scale of 0–2 for each paw, according to changes in redness and swelling. At late stages of the disease ankylosis of the ankles can be included. The macroscopic score is expressed as a selective value in one paw or as a cumulative value for all paws, with a maximum of 8. Histologically, the inflammation is characterized by a florid exudate in the joint space, containing numerous amounts of granulocytes, and a progressive destruction of the articular cartilage. Erosion of bone is pronounced, but periosteal new bone formation is also seen. Bone marrow is affected, but markedly less as compared to the polyarthritic adjuvant disease. Apart from the high number of granulocytes in the joint cavity, the synovial tissue contains large numbers of macrophages and lymphocytes. However, in this compartment granulocytes are also prominent in the first 2 weeks after onset. The most characteristic feature of CIA is the aggressive attack of the inflammatory process at the articular cartilage (Fig. 3A–F). In this model, heavy adherence of granulocytes at the cartilage surface is a common finding. Moreover, cartilage damage is not limited to loss of proteoglycans from the matrix, but in a short period of time roughening and deep erosions of the surface are consistently observed, further facilitating attachment of granulocytes at these sites. In that sense, granulocytes probably play an active role in the cartilage destruction, linked to sticking to anti-CII immune complexes in the surface layers. It has long been recognized that granulocytes contain high amounts of elastase and cathepsin G, which are potent mediators of cartilage proteoglycan depletion in co-cultures of cartilage and activated granulocytes in vitro. When such cultures are done in the presence of full serum or synovial fluid, the destruction is limited, due to large
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Figure 3 Characteristic histopathology of murine CIA in the area of the patella and opposite femur of the knee. Homogeneous Saffranin O staining of proteoglycans in cartilage surface layers of a naïve mouse (A). Depletion of proteoglycan in the superficial cartilage but still intact surface in arthritic knee joint (B) and marked depletion as well as severe erosion in late stage CIA (C). Focal bone destruction in the femur between the growth plate and the cartilage layer. Note the invasive synovium that erodes the bone from the outside (D, E). Severe cartilage erosion but also bone erosion underneath by ingrowth of granulation tissue in CIA (F).
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amounts of high molecular weight enzyme inhibitors in these fluids. However, when the granulocytes are pelleted on the cartilage surface, heavy destruction is again noted, implying direct extrusion of enzymes in the matrix and escape from natural inhibitors once the cells are in full contact with the cartilage surface [25]. This process of pronounced sticking is seen in vivo in the model of CIA, provided that the anti-CII antibody levels are high. In addition, we also only noted such sticking in murine mBSA-induced arthritis when the joints are immobilized [26]. Apparently, the immune complex formation between antibodies and cationic mBSA, planted in the cartilage surface, is on its own insufficient to generate heavy sticking under normal movement conditions, but when the cells are allowed to settle under immobilized conditions, the interaction remains intact. Like the situation in CIA, this condition results in erosion of the surface and further settlement and digging in into the roughened surface by the attached granulocytes. With respect to granulocyte involvement in cartilage destruction in human RA, it is clear that immune complexes can be found in the articular cartilage surface of a large number of patients. Whether the concentration is high enough to allow for consistent granulocyte attachment is yet unclear, and considerable variation between different RA patients seems obvious. Apart from this process, cartilage erosion at the cartilage margins, linked to pannus overgrowth, is considered to make a significant contribution in RA patients. At later stages of CIA, cartilage erosion at the margins and pannus formation is a prominent feature as well (Fig. 3E, F). After a few weeks, the model often progresses to complete loss of the whole cartilage, ending up in bone to bone contact and variable degree of ankylosis. This dramatic destruction of the cartilage reflects the directed autoimmune attack at the cartilage, and the arthritis in the synovial tissue burns out in a particular joint, when the cartilage is fully destroyed. The synovium then displays a mixture of an immune infiltrate, macrophages and a pronounced fibrotic reaction. The highly destructive character of CIA is also reflected in the massive occurrence of the proteoglycan breakdown neoepitope VDIPEN throughout the cartilage. This epitope is indicative for the involvement of metalloproteases, in particular stromelysin. In contrast to the lack of such epitopes in reversible cartilage proteoglycan depletion in Zymosan arthritis, and the variable degree of these neoepitopes in murine antigen induced arthritis, only showing expression at particular sites of the cartilage displaying irreversible lesions, the expression is fast and much more pronounced in CIA (Fig. 4). Detailed studies in antigen-induced arthritis made it clear that VDIPEN expression is linked to IL-1-driven processes [27] and colocalizes with collagen breakdown neoepitopes. All of this is compatible with a role of stromelysin in activation of collagenase, and a dominant role of this process in cartilage erosion in CIA. Stromelysin is produced in a latent form after activation of synovial cells or cartilage with IL-1, suggesting that further activation by granulocyte enzymes may contribute as well, elastase being a likely candidate in this process. Recent studies with elastase inhibitors revealed efficacy in murine CIA [28].
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Figure 4 Expression of cartilage proteoglycan breakdown neoepitope VDIPEN in the tibia-femur region. Note the absence in normal cartilage (A); local expression in murine CIA (B); fully affected cartilage in CIA (C).
Given the rapid development of the arthritic changes in this model, it is clear that consistent histological scoring of the severity of the arthritis is seriously complicated by variable days of onset of arthritis in individual mice, variation in onset between different paws or even between digits in one paw. The latter variability furthermore asks for highly standardized semi-serial sectioning of complicated joint structures of the whole paw. All of this flaws the design of proper drug studies. Attempts, discussed above, to synchronize expression in groups of mice, or perhaps even better, to precipitate the arthritis in a given joint by a local inflammatory insult, provide valuable improvements of applicability of the CIA model.
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Table 2 - Histology of CIA: comparison of affected knee and ankle joints
Infiltrate Cartilage damage Proteoglycan depletion
Day 37* Knee joint
Ankle joint
Day 42 Knee joint
Ankle joint
1.5 ± 0.6 1.0 ± 0.6 1.9 ± 0.8
1.5 ± 0.3 0.9 ± 0.4 1.6 ± 0.4
1.3 ± 0.4 1.2 ± 0.3 2.2 ± 0.5
1.7 ± 0.4 1.1 ± 0.2 1.9 ± 0.5
*Days after the first immunization with CII. The values represent the mean ± SD of at least 20 knee or ankle joints. Histology was scored on a scale ranging from 0 to 3. Infiltrate is scored as the amount inflammatory cells in synovial tissue and joint cavity. Cartilage damage reflects surface erosions, proteoglycan depletion indicates loss of Saffranin O staining.
Although the classic macroscopic scoring of CIA is always done in paws, with additional analysis of histology of the ankle joints, it is our experience that there is a high correlation between occurrence of arthritis in the knee and the ankle. Since the standardized joint sectioning is much easier in the knee compared to the ankle, histological analysis should preferably be done in the knee. We have carefully compared the characteristic histopathology in ankles and knees and the patterns of synovitis and cartilage destruction are very similar (Tab. 2). It should be noted that this is unlike the situation in adjuvant arthritis in the rat. The latter model of arthritis shows predominant expression in the ankles, whereas knee joints are rarely affected.
Cytokine involvement In line with a major role of the cytokine TNF-_ in human RA, the onset of CIA is TNF-_ dependent. Studies with neutralizing anti-TNF-_ antibodies or soluble TNF_ receptors revealed a major suppressive effect, when treatment was started shortly before onset of CIA [29, 30]. When the arthritis is fully expressed, subsequent blocking of TNF-_ appeared only marginally effective, implying that TNF-_ is crucial in the onset but less so in propagation of arthritis. In clear contrast, blocking of IL-1 with neutralizing antibodies or IL-1 receptor antagonist (IL-1Ra) markedly reduced severity of the arthritis [31], also when the arthritis was fully established (Fig. 5A, B). Moreover, anti-IL-1 treatment markedly reduced cartilage damage. Elegant studies in IL-1`-deficient mice showed full resistance to CIA induction and the critical importance of IL-1` was also emphasized by greatly reduced CIA in IL-1converting enzyme (ICE)-deficient mice and the efficacy of ICE inhibitors in CIA in
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Leo A. B. Joosten and Wim B. van den Berg
Figure 5 CIA is treated with systemic administration of neutralizing antibodies against TNF-_ (A) or IL-1_,` (B). Treatment was started in the various groups at either day 28 or 32. Anti-TNF-_ is still effective shortly after onset, but not in established disease. In contrast, anti-IL-1 is highly effective, even in late arthritis.
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Murine collagen induced arthritis
normal mice. Studies in TNF-_ receptor-knockout mice revealed a lower incidence and a milder form of CIA in the absence of proper TNF-_-receptor interaction. However, once a joint was afflicted, the progression of arthritis in that joint was indistinguishable from that in wild-type mice [32], again underlining the limited role of TNF-_ in propagation and cartilage destruction. It emphasizes that TNF-_ is helpful in acceleration of arthritis expression, but that TNF-_-independent onset can occur as well. Although it is claimed in human RA that TNF-_ is driving most of the IL-1 production and that TNF-_ blocking would be sufficient to block the whole arthritic process, this is not found in CIA. Recent studies in murine streptococcal cell wall-induced arthritis also revealed major TNF-_ dependence of initial joint swelling, but IL-1` dependence of cartilage destruction [33]. This was found using neutralizing antibodies and confirmed in TNF-_- and IL-1`-knockout mice. Again, TNF-_ blocking did not sufficiently prevent IL-1 production. These findings demonstrated that anti-TNF-_ treatment in RA patients would be beneficial when the disease is in fact a chronic process, due to repeated flares, with each acute exacerbation showing strong TNF-_ dependency. Of interest, when expression of CIA is not highly stimulated by additional boosting or synchronizing injections with LPS or additional cytokines, onset of arthritis starts only in a small number of joints, with gradual involvement of additional joints with time. This creates a seemingly extended period of TNF-_ dependency of the model, which is lost upon synchronization and speedy propagation to established arthritis in most joints. An intriguing element in control of CIA expression is formed by the synovial lining cells. This layer consists of synovial fibroblasts and macrophages. When macrophages are selectively depleted from this layer by local injection of toxic liposomes and the subsequent process of engulfment of liposomes by these phagocytes and subsequent apoptotic cell death, such a joint becomes refractory to the onset of CIA [34]. Further analysis revealed that the lining macrophages are a major source of chemotactic factors, needed to direct the initiating leukocyte influx into the joint. TNF-_ and IL-1` are potent inducers of chemokine production in lining cells, and when these recombinant cytokines are injected in a lining-depleted, naive joint, they do not induce leukocyte influx. In contrast, C5a is still fully capable of attracting leukocytes in such a joint. This further establishes the TNF-_/IL-1` dependence, with an intermediate role of the lining cells, in CIA. Subsequent studies in other models revealed that immune complex arthritis was totally abolished in lining depleted joints, whereas a strong T cell driven arthritis was hardly affected. Moreover, immune complex arthritis showed strong IL-1 dependence, sharing this feature with CIA, and further emphasizing that onset of CIA is more an immune complex phenomenon than a T cell process. Apart from a pivotal role of TNF-_ and IL-1` in onset and propagation of CIA, regulation of the arthritis is exerted by the cytokines IL-4 and IL-10. These socalled modulatory cytokines have a critical impact on the arthritic process at vari-
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ous levels, including the control of the Th1/Th2 balance, inhibition of macrophage TNF-_/IL-1` production and stimulation of chondrocytes in the articular cartilage. Expression of CIA is under the control of endogenous IL-10 [35]. High levels are found in the synovial tissue and anti-IL-10 antibodies, given shortly before expected onset, enhance incidence and severity. Anti-IL-4 antibodies were without effect, in line with the difficulty to detect significant levels of IL-4 in the synovium, but combined anti-IL-4/IL-10 treatment promoted the strongest expression of CIA (Fig. 6A). The opposite approach, i.e., treatment of CIA with systemically injected recombinant IL-4 or IL-10 revealed that IL-10 reduced the joint swelling in CIA, but more marked suppression, including reduced cartilage destruction was noted with the combination treatment with IL-4 and IL-10 (Fig. 6B, [36]). Of interest, IL-10 is a potent reducer of macrophage TNF-_ production, but IL-1 production was only suppressed with the combination of IL-10 and IL-4. Moreover, this combination also up-regulated the IL-1Ra/IL-1 balance, both in the synovium and in the cartilage. Additional studies, using gene transfer technology to overexpress IL4 locally in the knee joints of collagen-immunized DBA-1 mice showed clearly that IL-4 protects against severe cartilage and bone destruction. Moreover, overexpression of IL-4 generation reduces the local IL-1`, IL-6, IL-17 and RANKL levels [37]. The members of the IL-12 family represent another group of cytokines deserving major attention. These cytokines, including IL-12 and IL-23 originate from macrophages and are produced after activation with bacterial components. IL-12 is a potent inducer of IFN-a and promotes Th1 generation and propagation. As stated above, IL-12 when given at the expected onset of CIA, greatly enhances incidence and severity and systemic anti-IL-12 treatment prevented LPS-accelerated CIA expression. This in line with the reduced incidence and severity of CIA in IL12-deficient mice [38]. DBA/1 mice primed with CII in IFA treated with IL-12 developed significantly higher incidence and more severe disease compared with controls. These were elevated further by combination treatment with IL-12 and IL18. The IL-12/IL-18 treatment led to markedly enhanced synovial hyperplasia, cellular infiltration, and cartilage erosion compared with controls [39]. In contrast, when anti-IL-12 was applied in the established phase of CIA, it appeared poorly suppressive and upon interruption of anti-IL-12 treatment we noted a marked exacerbation. Moreover, late treatment with recombinant IL-12 suppressed instead of enhanced the arthritis, prolonged IL-12 treatment markedly enhanced IL-10 levels and the suppressive effect of IL-12 could be abrogated with anti-IL-10 [19]. This suggests a dual role of IL-12 in early and late disease. The potent induction of IL10 reflects an intriguing feedback pathway to control for excessive and prolonged Th1 responses, but seriously hampers therapeutic targeting of IL-12 in autoimmune arthritis. It has been suggested that the inflammatory cytokine IL-15 plays an important role in the development of several autoimmune diseases, including RA. IL-15 is
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Murine collagen induced arthritis
Figure 6 Treatment of CIA with systemic neutralizing antibodies against IL-4 and/or IL-10 (A) or recombinant proteins IL-4 and/or IL-10 (B). Antibodies given at days 28, 32 and 36. Recombinant cytokines given daily, from day 34. For experiment in (A), mice were selected at day 28, having no signs of arthritis. In (B), groups of mice are depicted which were challenged with LPS at day 28, to obtain high arthritis expression in the control mice. Spontaneous CIA expression is under the control of endogenous IL-10. Suppression of established arthritis is still possible with additional IL-4/IL-10.
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derived from several cell types, including macrophages and fibroblasts. IL-15 induces T cell chemotaxis and activation, together with B cell maturation. Furthermore, it enhances NK cell cytotoxicity and cytokine production, activates neutrophils and modifies monokine secretion [40, 41]. IL-15 mediates several diverse effects at multiple stages of the immune response. Interestingly, administration of soluble IL-15 receptor a-chain prevents the onset of CIA, indicating a role of IL-15 in antigen-induced immunopathology [42]. In addition, it was elegantly shown that targeting of IL-15 receptor-bearing cells with an antagonist mutant IL-15/Fc protein prevents disease development and progression in murine CIA [43]. These data indicate that IL-15 may be an interesting target in autoimmune disease, like RA. The first clinical studies in RA that neutralized IL-15 revealed promising results. IL-17A is a pro-inflammatory cytokine, produced by activated CD4+ memory T cells, and it induces production of other pro-inflammatory cytokines by stromal cells [44]. It has been described in RA synovium and shows similar cellular responses to IL-1 [45]. IL-17A belongs to a family of proteins, including IL-17B, IL-17C, IL-17E and IL-17F. The trigger for IL-17 has not been fully identified; however, IL23 promotes the production of IL-17 and a strong correlation between IL-15 and IL-17 levels in synovial fluid has been observed. IL-17 induces NF-gB activation and IL-1, IL-6, IL-8, G-CSF and TNF-_ production in both fibroblasts and macrophages [46]. Interestingly, synergistic effects of IL-17 and IL-1 as well as TNF-_ were reported. In addition, IL-17 was associated with cartilage destruction and inhibition of chondrocyte proteoglycan synthesis due to increased catabolic enzymes and NO production [47]. Furthermore, IL-17 was shown to be a potent stimulator of osteoclastic bone resorption by enhanced prostaglandin E2 synthesis. Recently, it was nicely demonstrated that blocking of endogenous IL-17 in experimental arthritis models, such as CIA, using neutralizing antibodies against IL-17 prevented bone destruction [48]. In line with these observations, overexpression of IL-17 during onset of CIA clearly aggravated the joint destruction through loss of the receptor activator of NF-gB ligand/osteoprotegerin (OPG) balance [49]. It would be of high importance to perform blocking studies with anti-IL-17 in experimental arthritis models, such as CIA to unravel distinct IL-17 activity or its synergy with IL-1, IL15 or TNF-_. A novel factor for osteoclast differentiation, receptor activator of nuclear factor B ligand (RANKL), has been identified [50]. This TNF-related cytokine (also called OPG ligand, osteoclast differentiation factor, and TRANCE) is an essential factor for osteoclast differentiation and activation. RANKL-deficient mice have a complete absence of osteoclasts and exhibit osteopetrosis. In addition, RANKL is involved in the interaction of T cells and dendritic cells, and plays a role in immune cell differentiation [51]. OPG is a naturally occurring decoy receptor for RANKL. When bound to RANKL, OPG prevents the binding of RANKL to RANK, and thus inhibits the biological activity of RANKL. The relative local expression levels of RANKL and OPG (often represented as the RANKL/OPG ratio), is instrumental in
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Murine collagen induced arthritis
determining the degree of osteoclast-mediated bone resorption. RANKL binds to a cell-associated TNF receptor-related protein called receptor activator of nuclear factor B (RANK). This type I transmembrane protein is expressed on many cell types, including osteoclasts and osteoclast precursor cells, certain B and T cells, and dendritic cells. The interaction between RANKL and its receptor, RANK, has been shown to be critical in osteoclastogenesis and bone resorption. RANKL and OPG are important positive and negative regulators of osteoclastogenesis and bone resorption. Treatment of murine CIA or rat adjuvant arthritis with OPG reduces the number of osteoclasts and prevents bone erosion [52, 53]. IL-18 is a cytokine originally identified as IFN-a-inducing factor (IGIF), it is a member of the IL-1 family of proteins [54]. As IL-1`, IL-18 is produced as an inactive precursor and it is cleaved by ICE to the biologically active form. IL-18 acts synergistically with IL-12, IL-2 and antigens to induce the production of IFN-a. The crucial role of IL-18 in IFN-a synthesis was demonstrated in IL-18-deficient mice, where the IFN-a production was markedly reduced after injection of endotoxin, despite of normal IL-12 production in these animals [55]. IL-18 is produced by human articular chondrocytes, and it induces pro-inflammatory cytokines and catabolic factors like NO, cyclooxygenase and stromelysin. Studies in IL-18-deficient mice or blocking studies with anti-IL-18 demonstrated the proinflammatory role of IL-18 in arthritis [56, 57]. Recently, IL-18 binding protein (IL-18BP) was isolated and cloned. This protein blocks the endotoxin-induced IFN-a production in mice and belongs to a member of novel soluble receptors [58]. Since IL-18 is an early promotor of Th1 cells, IL-18BP probably plays a crucial role in the regulation of immune response. Studies in models of arthritis showed the potential therapeutic value of IL-18BP for treatment of RA [59]. IL-23 is a new member of the IL-12 family of regulatory cytokines produced by activated macrophages and dendritic cells. IL-23 is a heterodimeric cytokine composed of a p19 subunit and the p40 subunit of IL-12 [60]. IL-23 affects memory T cell and inflammatory macrophage function through engagement of a novel receptor (IL-23R) on these cells. Using gene-targeted mice lacking only IL-12 (p35–/–) or IL-23 (p19–/–), it was demonstrated that the specific absence of IL-23 is protective, whereas loss of IL-12 exacerbates CIA. IL-23 gene-targeted mice do not develop clinical signs of disease and are completely resistant to the development of joint and bone pathology. Resistance correlates with an absence of IL-17-producing CD4+ T cells despite normal induction of collagen-specific, IFN-a-producing T helper 1 cells. In contrast, IL-12-deficient p35–/– mice developed more IL-17-producing CD4+ T cells, as well as elevated mRNA expression of proinflammatory TNF-_, IL-1`, IL6, and IL-17 in affected tissues of diseased mice. These data indicate that IL-23 is an essential promoter of end-stage joint autoimmune inflammation, whereas IL-12 paradoxically mediates protection from autoimmune inflammation [61]. Figure 7 shows a schematic presentation of the cytokines involved in pathways of synovitis and concomitant cartilage and bone destruction.
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Figure 7 Schematic presentation of pathways of synovitis and concomitant cartilage and bone destruction. Note the amplifying elements through T cell activation and generation of autoantibodies. The latter will trigger macrophages after immune complex (IC) formation, through Fca receptors. T cell-derived IL-17 contributes to bone destruction via induction of RANKL in co-operation with IL-1 and TNF-_. Ag, antigen; APC, antigen-presenting cell; Ch, chondrocyte; Fibro, fibroblast; IFN, IFN-a.
Applicability of the model The model of CIA establishes that an autoimmune reaction to a cartilage component can lead to a chronic, destructive polyarthritis. Although it is far from accepted that CII is a crucial antigen in human RA, the findings in the model may exemplify common principles in arthritis directed against cartilage autoantigens. The model is highly suitable and widely used to try to understand the immunoregulation in autoimmune arthritis and to identify ways to induce tolerance using peptide fragments, or to selectively target the T cell receptors involved in collagen epitope recog-
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nition. Detailed discussion of these topics goes beyond the scope of the present chapter and a recent review of Myers et al. [9] as well as the chapter of Griffiths et al in this volume is advised for further reading. Apart from the immunoregulation, the model is highly suitable to try to understand the complex cytokine interplay in onset and propagation of arthritis, and to identify therapies aimed at prevention of cartilage destruction. Examples of cytokine involvement are already discussed above. As mentioned before, the onset of CIA is an immune complex phenomenon. This stage shows high sensitivity to NSAIDs and in fact, all therapies which will interfere with the initiating leukocyte influx will show efficacy. The more interesting part of the model is the established phase of the arthritis and the ongoing destruction of the articular cartilage. When the efficacy of drugs at onset of arthritis is investigated, it should be realized that this stage is rather stress sensitive. Daily treatment by i.p. or oral injection may have a large impact at that stage, and handling of mice should be done with great subtlety. We have often noted a significant suppression of arthritis onset with prophylactic daily vehicle treatment, whereas this effect was absent at later stages, when the arthritis is fully established. Treatment that is started after onset of arthritis has the further advantage that grouping of the mice can be done by weighted randomization, creating a similar mean index of arthritis at the start of the various control and treatment groups. When randomization has to be done before onset, higher variation between groups is unavoidable and in general, the use of at least 10 mice per experimental group in such protocols is warranted. To illustrate the efficacy of some drugs in murine CIA a few examples are discussed in more detail. The onset of CIA is sensitive to treatment with indomethacin. A dose of 1 mg/kg significantly suppressed the macroscopic signs of arthritis. Intriguingly, when low dosages of indomethacin are used, sensitivity is lost. However, when treatment with indomethacin is combined with a leukotriene synthesis inhibitor, marked synergistic suppression was observed (Fig. 8A). This clearly illustrates that both prostaglandins and leukotrienes are of importance at arthritis onset. The role of leukotrienes was also nicely illustrated in the poor induction of CIA in lipoxygenase-deficient mice [62]. Of interest, cartilage destruction was also markedly reduced with the combination treatment (Tab. 3). It suggests that NSAIDs with a combined profile would be the better anti-arthritic drug. More recent interest focused on the dominant COX-1 and/or COX-2 inhibitory pattern of NSAIDs, further pinpointing the profiling of suitable NSAIDs. A second example of therapeutic approaches in this model of arthritis is provided by the demonstration of synergy between steroids and IL-10. Steroids are potent suppressors of arthritis, but their clinical application is seriously hampered by side effects such as osteoporosis. It would be desirable to find ways to combine drugs at lower, nontoxic concentrations, yet retaining the beneficial effects. We found that prednisolone treatment suppressed CIA at a dose of 1–5 mg/kg per day, but dosages of 0.05 or 0.1 mg/kg were without effect. Interestingly, daily IL-10 treatment at a
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Figure 8 (A) CIA mice received daily oral treatment with indomethacin and/or a leukotriene synthesis inhibitor (Bay W 5676), from day 28 for 14 consecutive days. The data represent groups of 10 mice. Note the marked synergy. (B) CIA mice received daily i.p. treatment with prednisolone and/or murine IL-10. Note the synergy between low-dose steroid and IL-10.
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Table 3 - Histology of CIA after treatment with either COI, LSI or combination of COI/LSI Treatment
Dose
Infiltrate
Vehicle COI COI LSI COI/LSI
– 1 mg/kg 0.1 mg/kg 200 mg/kg 0.1/200
1.3 0.7 1.4 1.5 0.6
± ± ± ± ±
Cartilage damage
0.7 0.6 0.9 1.0 0.4
1.5 0.8 1.6 1.4 1.0
± ± ± ± ±
Proteoglycan depletion
0.7 0.5 0.6 0.8 0.3
2.0 1.5 2.2 2.1 1.2
± ± ± ± ±
0.7 0.8 0.9 0.5 0.8
Treatment of arthritic mice was started at day 28 after immunization with CII. Mice were injected i.p. twice a day with cyclooxygenase inhibitor (COI) indomethacin or leukotriene synthase inhibitor (LSI) Bay W 5676, or the combination for 14 consecutive days. The data represent the mean ± SD of at least ten mice per group. Histology was scored on a scale ranging from 0 to 3.
Table 4 - Histology of CIA after treatment with IL-10, prednisolone or IL-10/prednisolone Treatment
Dose
Infiltrate
Cartilage damage
Vehicle IL-10 Prednisolone IL-10/pred.
– 5 µg/day 0.05 mg/kg 5/0.05
1.7 1.3 1.9 1.0
1.5 1.2 1.7 0.8
± ± ± ±
0.9 0.7 1.0 0.8
± ± ± ±
1.0 0.6 0.9 0.7*
Proteoglycan depletion 2.3 2.0 2.1 1.5
± ± ± ±
1.3 1.0 1.0 1.0*
COMP (µg/ml) 8.2 8.0 10.2 4.8
± ± ± ±
0.8 0.7 1.8 0.6*
Treatment of arthritic mice was started at day 28 after immunization with CII. Mice were injected i.p. twice a day with murine IL-10, prednisolone or the combination for 14 consecutive days. The data represent the mean ± SD of at least ten mice per group. Histology was scored on a scale ranging from 0 to 3. Serum COMP levels were determined by ELISA, levels in normal sera amount 4.2 ± 0.6 µg/ml. *P<0.05 Student’s t-test compared to vehicle.
dose of 5 µg/mouse reduced macroscopic signs of swelling, but did not suppress other parameters of arthritis. When IL-10 was combined with the low-dose prednisolone treatment, this resulted in marked suppression of arthritis (Fig. 8B), including reduction in cartilage destruction (Tab. 4). The latter effect was also evident when COMP levels were measured in the serum. COMP is a marker of enhanced cartilage turnover [63], and its serum concentration raises from 4 µg/ml in control
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mice to 8 µg/ml in mice with active CIA. Moreover, we observed a straight correlation between COMP levels around day 40 and the degree of histological cartilage damage at that stage. After treatment with the combination of low-dose prednisolone and IL-10, the COMP values were fully normalized. A final application of therapeutic manipulation in CIA is illustrated with a gene therapy approach with IL-1Ra. Shortly before onset of CIA, we injected fibroblasts transfected in vitro with a retroviral gene construct, containing IL-1Ra, directly into the knee joint. Normal fibroblasts served as controls. The onset of arthritis was almost completely prevented in knee joints containing these IL-1Ra-producing cells. Intriguingly, this treatment also prevented the occurrence of arthritis in the ipsilateral paw, whereas it had no effect on arthritis in the other paws [64]. These findings underline the critical role of IL-1 in this model and furthermore support the applicability of gene transfer, allowing for treatment of a major joint, yet influencing arthritis expression in nearby, smaller joints.
Final remarks Murine CIA is highly IL-1 dependent and is, therefore, an excellent model to screen novel drugs with IL-1-related activity. However, it should be realized that the model is rather sensitive to other drugs as well, including a range of NSAIDs. The latter effect is less pronounced in established CIA, whereas IL-1 sensitivity still remains and it might be more appropriate to use the more chronic phase for screening of IL1 drugs. Another peculiar element of CIA is the dominant involvement of PMNs and the highly erosive character, with full destruction of the articular cartilage and major bone erosions. Apart from the discussion that CII might not be a crucial autoantigen in RA, the model reflects autoimmune arthritis driven by a cartilage autoantigen and as such represents patterns probably holding for a range of cartilage arthritogens. The high scientific interest in immunomodulation studies in this model will soon provide further insight in therapeutic applicability of tolerance induction [65], be it in an antigen-specific method or using the principle of bystander suppression. The latter stands for the suppression of inflammation by Th2 or Th3 cells, directed against a non-related antigen and causing suppression by local production of suppressive cytokines such as IL-4, IL-10 or TGF-` [66]. In that sense, defined tolerization against cartilage-specific “antigens” might prove of therapeutic value. CIA is only one of the many available arthritis models, and in the pharmaceutical industry the first screening of drugs is often done in adjuvant arthritis, mainly related to ease of handling and historical reasons. The choice for the one or the other model should be based on the desired drug profile and the characteristic arthritic aspects of the various models. Reviews on arthritis models [67–69] are suggested for further reading.
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established murine collagen-induced arthritis with anti-IL-1 treatment. Clin Exp Immunol 95: 237–243 Mori L, Iselin S, Delibero G, Lesslauer W (1996) Attenuation of collagen induced arthritis in 55kDa TNF receptor type 1 (TNFR1) IgG1 treated and TNFR1 deficient mice. J Immunol 157: 3178–3182 Kuiper S, Joosten LAB, Bendele AM, Edwards CK III, Arntz OJ, Helsen MMA, van de Loo FAJ, van den Berg WB (1998) Different roles of TNF_ and IL-1 in murine streptococcal cell wall arthritis. Cytokine 10: 690–702 Van Lent PLEM, Holthuysen AEM, van den Bersselaar LAM, van Rooijen N, Joosten LAB, van de Loo FAJ, van de Putte LBA, van den Berg WB (1996) Phagocytic lining cells determine local expression of inflammation in type II collagen-induced arthritis. Arthritis Rheum 39: 1545–1555 Kasama T, Strieter RM, Lukacs NW, Lincoln PM, Burdick MD, Kunkel SL (1995) IL10 expression and chemokine regulation during the evolution of murine type II collagen-induced arthritis. J Clin Invest 95: 2868–2876 Joosten LAB, Lubberts E, Durez P, Helsen MMA, Jacobs MJM, Goldman M, van den Berg WB (1997) Role of IL-4 and IL-10 in murine collagen-induced arthritis. Arthritis Rheum 40: 249–259 Lubberts E, Joosten LAB, Chabaud M, van Den Bersselaar L, Oppers B, Coenen-De Roo CJ, Richards CD, Miossec P, van Den Berg WB (2000) IL-4 gene therapy for collagen arthritis suppresses synovial IL-17 and osteoprotegerin ligand and prevents bone erosion. J Clin Invest 105: 1697–1710 McIntyre KW, Shuster DJ, Gillooly KM, Warrier RR, Connaughton SE, Hall LB, Arp LH, Gately MK, Magram J (1996) Reduced incidence and severity of collagen-induced arthritis in interleukin-12-deficient mice. Eur J Immunol 26: 2933–2938 Leung BP, McInnes IB, Esfandiari E, Wei XQ, Liew FY (2000) Combined effects of IL12 and IL-18 on the induction of collagen-induced arthritis. J Immunol 164: 6495–6502 Shah MH, Hackshaw KV, Caligiuri MA (1998) A role for IL-15 in rheumatoid arthritis? Nat Med 4: 643 McInnes IB, Liew FY (1998) Interleukin 15: a proinflammatory role in rheumatoid arthritis synovitis. Immunol Today 19: 75–79 Ruchatz H, Leung BP, Wei XQ, McInnes IB, Liew FY (1998) Soluble IL-15 receptor alpha-chain administration prevents murine collagen-induced arthritis: a role for IL-15 in development of antigen-induced immunopathology. J Immunol 160: 5654–5660 Ferrari-Lacraz S, Zanelli E, Neuberg M, Donskoy E, Kim YS, Zheng XX, Hancock WW, Maslinski W, Li XC, Strom TB, Moll T (2004) Targeting IL-15 receptor-bearing cells with an antagonist mutant IL-15/Fc protein prevents disease development and progression in murine collagen-induced arthritis. J Immunol 173: 5818–5826 Fossiez F, Djossou O, Chomarat P, Flores-Romo L, Ait-Yahia S, Maat C, Pin JJ, Garrone P, Garcia E, Saeland S et al (1996) T cell interleukin-17 induces stromal cells to produce proinflammatory and hematopoietic cytokines. J Exp Med 183: 2593–2603 Kotake S, Udagawa N, Takahashi N, Matsuzaki K, Itoh K, Ishiyama S, Saito S, Inoue
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K, Kamatani N, Gillespie MT et al (1999) IL-17 in synovial fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis. J Clin Invest 103: 1345–1352 Chabaud M, Durand JM, Buchs N, Fossiez F, Page G, Frappart L, Miossec P (1999) Human interleukin-17: A T cell-derived proinflammatory cytokine produced by the rheumatoid synovium. Arthritis Rheum 42: 963–970 Chabaud M, Lubberts E, Joosten L, van Den Berg W, Miossec P (2001) IL-17 derived from juxta-articular bone and synovium contributes to joint degradation in rheumatoid arthritis. Arthritis Res 3: 168–177 Lubberts E, Koenders MI, Oppers-Walgreen B, van den Bersselaar L, Coenen-de Roo CJ, Joosten LA, van den Berg WB (2004) Treatment with a neutralizing anti-murine interleukin-17 antibody after the onset of collagen-induced arthritis reduces joint inflammation, cartilage destruction, and bone erosion. Arthritis Rheum 50: 650–659 Lubberts E, van den Bersselaar L, Oppers-Walgreen B, Schwarzenberger P, Coenen-de Roo CJ, Kolls JK, Joosten LA, van den Berg WB (2003) IL-17 promotes bone erosion in murine collagen-induced arthritis through loss of the receptor activator of NF-kappa B ligand/osteoprotegerin balance. J Immunol 170: 2655–2662 Hsu H, Lacey DL, Dunstan CR, Solovyev I, Colombero A, Timms E, Tan HL, Elliott G, Kelley MJ, Sarosi I et al (1999) Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc Natl Acad Sci USA 96: 3540–3545 Kong YY, Feige U, Sarosi I, Bolon B, Tafuri A, Morony S, Capparelli C, Li J, Elliott R, McCabe S et al (1999) Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand. Nature 402: 304–309 Romas E, Sims NA, Hards DK, Lindsay M, Quinn JW, Ryan PF, Dunstan CR, Martin TJ, Gillespie MT (2002) Osteoprotegerin reduces osteoclast numbers and prevents bone erosion in collagen-induced arthritis. Am J Pathol 161: 1419–1427 Pettit AR, Ji H, von Stechow D, Muller R, Goldring SR, Choi Y, Benoist C, Gravallese EM (2001) TRANCE/RANKL knockout mice are protected from bone erosion in a serum transfer model of arthritis. Am J Pathol 159: 1689–1699 Okamura H, Tsutsi H, Komatsu T, Yutsudo M, Hakura A, Tanimoto T, Torigoe K, Okura T, Nukada Y, Hattori K (1995) Cloning of a new cytokine that induces IFNgamma production by T cells. Nature 378: 88–91 Dinarello CA, Novick D, Puren AJ, Fantuzzi G, Shapiro L, Muhl H, Yoon DY, Reznikov LL, Kim SH, Rubinstein M (1998) Overview of interleukin-18: more than an interferon-gamma inducing factor. J Leukoc Biol 63: 658–664 Plater-Zyberk C, Joosten LAB, Helsen MM, Sattonnet-Roche P, Siegfried C, Alouani S, van De Loo FA, Graber P, Aloni S, Cirillo R et al (2001) Therapeutic effect of neutralizing endogenous IL-18 activity in the collagen-induced model of arthritis. J Clin Invest 108: 1825–1832 Wei XQ, Leung BP, Arthur HM, McInnes IB, Liew FY (2001) Reduced incidence and severity of collagen-induced arthritis in mice lacking IL-18. J Immunol 166: 517–521
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Novick D, Kim SH, Fantuzzi G, Reznikov LL, Dinarello CA, Rubinstein M (1999) Interleukin-18 binding protein: a novel modulator of the Th1 cytokine response. Immunity 10: 127–136 Smeets RL, van de Loo FA, Arntz OJ, Bennink MB, Joosten LA, van den Berg WB (2003) Adenoviral delivery of IL-18 binding protein C ameliorates collagen-induced arthritis in mice. Gene Ther 10: 1004–1011 Oppmann B, Lesley R, Blom B, Timans JC, Xu Y, Hunte B, Vega F, Yu N, Wang J, Singh K (2000) Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 13: 715–725 Murphy CA, Langrish CL, Chen Y, Blumenschein W, McClanahan T, Kastelein RA, Sedgwick JD, Cua DJ (2003) Divergent pro- and antiinflammatory roles for IL-23 and IL-12 in joint autoimmune inflammation. J Exp Med 198: 1951–1957 Griffiths RJ, Smith MA, Roach ML, Stam EJ, Milici AJ, Scampoli DN, Eskra JD, Byrum RS, Koller BH, McNeish JD (1997) Collagen-induced arthritis is reduced in 5-lipoxygenase-activating CT: protein-deficient mice. J Exp Med 185: 1123–1129 Mansson B, Carey D, Alini M, Ionescu M, Rosenberg LC, Poole AR, Heinegard D, Saxne T (1995) Cartilage and bone metabolism in RA. Differences between rapid and slow progression of disease identified by serum markers of cartilage metabolism. J Clin Invest 95: 1071–1077 Bakker AC, Joosten LAB, Arntz OJ, Helsen MMA, Bendele A, van de Loo FAJ, van den Berg WB (1997) Prevention of murine collagen-induced arthritis in the knee and ipsilateral paw by local expression of human IL-1Ra protein in the knee. Arthritis Rheum 40: 893–900 Myers LK, Seyer JM, Stuart JM, Kang AH (1997) Suppression of murine collageninduced arthritis by nasal administration of collagen. Immunology 90: 161–164 Miossec P, van den Berg WB (1997). Th1/Th2 cytokine balance in arthritis. Arthritis Rheum 40: 2105–2115 Van den Berg WB (1998) Animal models of arthritis: Applicability. In: P Maddison, D Isenberg, P Woo, D Glass (eds): Oxford Textbook of Arthritis. Oxford University Press, Oxford, 559–573 Van den Berg WB, van den Broek MF, van de Putte LBA, van Bruggen MCJ, van Lent PLEM (1991) Experimental arthritis: Importance of T cells and antigen mimicry in chronicity and treatment. In: Kresina TF (ed): Monoclonal Antibodies, Cytokines, and Arthritis. Dekker, New York, 237–252 Van den Berg WB (1998) Role of T cells in arthritis: Lessons from animal models. In: P Miossec, WB van den Berg WB, GS Firestein (eds): T Cells in Arthritis (PIR). Birkhäuser Verlag, Basel, 75–92 Van den Berg WB (2004) Animals models. In: St Clair EW, Pisetsky DS, Haynes BF (eds): Textbook Rheumatoid Arthritis. Lippincott Williams & Wilkins, Philadelphia, 254–267
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Use of animal models of osteoarthritis in the evaluation of potential new therapeutic agents Stephen A. Stimpson1, Virginia B. Kraus2 and Bajin Han1 1GlaxoSmithKline,
Research Triangle Park, NC 27709, USA; 2Duke University Medical Center, Durham, NC 27710, USA
Introduction Osteoarthritis (OA) is the most common joint disease. It was once thought to be an inevitable consequence of mechanical wear and tear of the joint over time. It is now defined not simply as a wear and tear disease, but rather the result of a multifactorial set of predisposing factors superimposed on an individual’s quality and quantity of attempted repair processes leading to varying levels of pain, joint dysfunction, and structural damage [1, 2]. Predisposing factors can include genotype, increasing age, past trauma, malalignment and obesity, all of which can impact the biochemical and mechanical stability and function of joint components. While focal cartilage destruction is the structural feature most studied and identified with OA, the disease affects all components of the joint organ, leading to alterations in subchondral bone and supporting neuromusculature, marginal chondrophyte and subsequent osteophyte formation, capsular thickening, and variable degrees of mild synovitis, all of which can elicit pain and joint dysfunction. Different joints can be differentially impacted by these predisposing and mechanistic factors [3, 4]. This presents the biomedical researcher with a bewildering complexity when attempting to select the appropriate animal model system in which to evaluate potential targets for OA treatment and prevention. Not surprisingly then, many different animal models have emerged, each with its own particular relevance to disease predisposition, and joint damage, in humans. This chapter describes animal models of OA and focuses on their recent use in the evaluation of potential therapeutic modalities. It is hoped that this chapter will serve as a valuable reference to OA animal models and examples of their uses with various types of chemical and biological agents, and facilitate the design of studies to investigate new therapeutic agents.
In Vivo Models of Inflammation, Vol. I, edited by Christopher S. Stevenson, Lisa A. Marshall and Douglas W. Morgan © 2006 Birkhäuser Verlag Basel/Switzerland
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OA animal models OA animal models can be divided into two broad types: spontaneous and induced models. Within the spontaneous category, there are “naturally occurring” OA models, in which osteoarthritis develops at some time, without known purposeful induction, usually without knowledge of specific predisposing mutations, and progresses slowly as the animal ages. An extensively characterized example and commonly used model is spontaneous OA of the medial compartment of the knee in Hartley albino guinea pigs [5]. Spontaneous OA has also been studied in mice and Syrian hamsters [6]. Transgenic models, in which a mutation has been introduced into a key regulatory or structural protein inducing an OAlike phenotype, have also been developed and have recently been reviewed by Helminen et al. [7]. Within the induced group, there are surgically induced models in which joint instability has been introduced by transecting, damaging or removing a key joint structure, and models induced by the local injection of an exogenous substance (chemical or biological). Key features of induced models include known time and nature of induction and, relative to spontaneous models, accelerated progression. Therapies can be instituted either before or after OA induction to evaluate the ability of a therapeutic agent to prevent disease or slow progression. An extensively characterized and widely used surgical model involves transection of the anterior cruciate ligament (ACL) in dogs, often referred to as the Pond-Nuki dog model [8, 9]. Medial meniscal tear in the guinea pig and rat [10], and partial medial meniscectomy in the rabbit [11] are also commonly employed, and while joints are smaller and introduce some limitations on evaluation of cartilage defects, are an important and useful alternative to surgically induced models in dogs in that larger numbers of animals per group can often be used and different biological reagents are available. Small animals require smaller amounts of drug and therefore can also represent an advantage during early phases of drug development. The selection, practical use, and advantages and disadvantages of the various models have been extensively reviewed by several key investigators in recent years (Moskowitz [12]; Pritzker [13]; Billingham [14]; Doherty, Griffiths and Pettipher [15]; Oegema and Visco [16]; Bendele [6, 10]; Brandt [9]; Helminen [7]).
Use of OA animal models in the evaluation of potential therapeutic modalities The last 10 years have seen a vast increase in the number and diversity of therapeutic modalities under investigation for potential use in the treatment of human OA. Owing to the difficulty in obtaining sequential human joint tissue from developing OA, and the lack of validated surrogate and biomarkers for the evaluation of
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disease progression and efficacy, animal models of OA have remained an important practical way to assess new therapies. Table 1 is a list of agents tested in animal models of OA over the last decade. While an attempt was made to be thorough, Table 1 may not be inclusive of every study in the literature, and in the attempt to be inclusive, the table is not meant to indicate which studies are, in the authors’ opinion, most convincing. The intent is to reflect the vast variety of agents that have now been evaluated, the variety of different animal models employed and the variety of outcomes measured, to give the investigator a fairly comprehensive view of options and trends when considering the evaluation of a new agent. A remarkable variety of agents have been evaluated, including dietary supplements and vitamins, complex plant and animal tissue extracts, highly specific protein biologics, and chemical products of rational drug design strategies against specific molecular targets. The development of such a wide variety of agents is no doubt impelled by the high prevalence of OA, being the commonest joint disease, affecting a huge cross-section of the population, with a predicted vast future increase in patient numbers driven by the demographics of aging and increases in other predisposing factors such as obesity. Thus, while there is a pressing sociomedical need for major improvements in the treatment of both pain and structural joint changes, the common and chronic nature of the disease demand a low side-effect profile – hence interest in nutritional supplements with albeit modest efficacy but very high safety – along with the bold pioneering of new more efficacious agents. Several trends are evident in the use of OA animal models for evaluation of a new agent. Testing of an agent, for example, in both a spontaneous model and a surgically induced model, may give an indication of how broadly a new agent may address the heterogeneity of human OA initiation and progression. Examples include evaluations of hyaluronan and matrix metalloproteinase (MMP) inhibitors (see Tab. 1). The most frequently used spontaneous model is the guinea pig. The veterinary medicine literature provides a rich resource of options for evaluation of pain and joint function in spontaneous OA with several examples of veterinary clinical studies of spontaneous OA in dogs also included in Table 1. The most frequently used surgically induced model is the ACL transection in the dog, though use of rabbits is also common. Animals larger than the rat or mouse offer a greater surface area of cartilage and other joint structures in which to assess damage, and importantly, from which to measure release of biomarkers. Moreover, the larger the animal, the more readily synovial fluid can be harvested for analyses of drug and OArelated biomarker concentrations, and the more readily can intra-articular drugs be introduced into the animal joint. With practice, these procedures can be successfully performed in animals as smalls as rats and guinea pigs. However, these procedures are much more difficult in mice. In addition to assessing efficacy, OA animal models are important in developing and validating new biomarkers of disease activity and progression. Such studies also
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lead to the development of more acute, mechanistic models useful for the testing of specific potent agents with defined molecular targets, and can be an important option to the more variable, costly and long-term models of the full symptomatic, functional and structural features of OA. This biomarker/acute mechanistic model arena is beyond the scope of this chapter. For an excellent example of the evaluation of a new agent in conjunction with biomarker and model validation, see the work of Otterness et al. [17–20] in the evaluation of MMP-13 inhibitors and an in vivo hamster model for measurement of MMP-13-induced cartilage damage and the release of collagen type II epitopes.
Conclusion There is no one animal model of OA which can be considered a gold standard for predicting efficacy in human OA (for an assessment of human OA trial experience, see the excellent review by Brandt and Mazzuca of recent clinical studies, including trials of orally administered glucosamine sulfate, chondroitin sulfate, doxycycline, risedronate, and diacerein, and intra-articular injection of hyaluronan [79]). This is due in part to the paucity of agents evaluated to date, in both animal models and human clinical settings, that could serve to validate models for evaluation of new agents. Another complexity in model and outcome choice is the investigator’s intent to evaluate symptom (pain) relief versus the joint structural changes required to provide evidence for disease-modifying OA drug activity. It is also possible that different models reflect different forms of OA in humans – for example, the vast majority of studies are in surgically induced OA models that may be most representative of post-injury OA in humans. Therefore, the selection of models in which to evaluate a new potential therapeutic agent may well include an acute, mechanism-based model with a specific joint structural change outcome, and an evaluation of pain, function and structural changes in either a spontaneous model of naturally occurring OA or a surgically induced model, or both.
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Table 1 - Evaluation of potential therapeutic agents in animal models of osteoarthritis—experience over the last approximately ten years Osteoarthritis model
Reported effects include:
Ref.
Hyaluronic acid
Rabbit; ACL transection
[21]
Hyaluronic acid
Guinea pig; spontaneous OA
Hyaluronic acid Hyaluronan Hyaluronan
Rabbit; ACL transection Rabbit; ACL transection Rabbit; partial meniscectomy
Hyaluronic acid
Rabbit; fibronectin fragmentinduced cartilage proteoglycan loss Hamster; spontaneous OA
Reduction in chondrocyte apoptosis; reduction in cultured cartilage nitric oxide (NO) levels Protection against cartilage degeneration; decrease in subchondral bone density and thickness; trabecular change toward rod-like, compliant structure Decreased surface roughness on femoral cartilage Decreased NO production in meniscus and synovium Increased presence of glycosaminoglycans in the menisci; trend for less articular cartilage deterioration Decrease in proteoglycan loss
Exercise
Condrosulf (chondroitin 4,6-sulfate) Carboxymethylated chitin
Rabbit; chymopapain-induced OA Rabbit; ACL transection
Fursultiamine with glucosamine Rabbit; partial medial and chondroitin sulfate meniscectomy
Protects from increase articular cartilage fibrillation, and decreased proteoglycan content and synthesis seen with lack of exercise Increase in cartilage proteoglycan content
69
Less severe cartilage degradation judged by macroscopic score and Mankin’s grading score; decreased MMP-1 expression Reduction in macroscopic and histological lesions; reduction in MMP-1 staining in chondrocytes of cartilage superficial layer
[22]
[23] [24] [25] [26] [27]
[28] [29]
[30]
Use of animal models of osteoarthritis in the evaluation of potential new therapeutic agents
Agent
Agent
Osteoarthritis model
Reported effects include:
Ref.
Glucosamine
Rabbit; ACL transection
[31]
Mixture of glucosamine, chondroitin sulfate and manganese ascorbate Ascorbic acid
Rabbit; ACL transection
Detectable, site-specific disease modifying effect, but did not prevent fibrillation and/or erosions of the articular cartilage in all animals, and no effects in medial joint compartments. Reduced total linear involvement and total grade of histological sections of medial femoral condyles
Vitamin supplement and selenium
Guinea pig; spontaneous OA
Undenatured type II collagen
Mouse; STR/1N strain spontaneously developing OA at an early age Obese dog; spontaneous OA
Estradiol or the selective estrogen receptor modulator levormeloxifene Fibroblast growth factor-18
Rat; 6-month-old female rats with OA that develops over a 9-week period following ovariectomy Rat; meniscal tear
Connective tissue growth factor Soluble TFG-beta RII
Rat; monoiodoacetic acid-induced OA Mouse; papain intraarticular injection
Increased severity of histological knee OA; increase in size and number of marginal osteophytes Decrease in histological score for articular cartilage damage; increase in expression of antioxidative enzymes in the knee joint Decrease in overall pain, exercise-associated lameness, pain upon limb examination Inhibition of histological articular cartilage damage; inhibition of urinary excretion of collagen type II degradation products Increase in cartilage thickness and reduction in cartilage degeneration score; increase in chondrophyte size and remodeling of subchondral bone Repair of articular cartilage to histologically normal appearance Reduced osteophyte formation; increased articular cartilage proteoglycan loss; reduced cartilage thickness
[32]
[33] [34]
[35] [36]
[37]
[38] [39]
Stephen A. Stimpson et al.
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Table 1 (continued)
Dog; ACL transection
Calcium pentosan polysulfate
Dog; surgical treatment of ACL deficiency (effect of compound on recovery) Dog; spontaneous chronic osteoarthritis Dog; unilateral L4-S1 dorsal root ganglionectomy, followed 3 weeks later by ipsilateral ACL transection Dog; ACL transection
Pentosan polysulfate Diacerhein
PB-145 (inhibitor of serine protease C1s) and pentosan polysulfate Calcitonin
Dog; ACL transection Rabbit; partial meniscectomy Dog; spontaneous OA Dog; spontaneous arthritis
Dog; ACL transection
Reduction in macroscopic lesion severity on the tibial plateaus and femoral condyles; histological lesion severity decreased Dose-dependent protective effect on development of osteophytes and cartilage lesions Reduction in width of osteophytes and size of macroscopic cartilage lesions Improvement in gait, performance in daily life activities, and vitality No effect judged by peak vertical force and vertical impulse of affected limbs, using a force platform No difference in dogs’ owners assessment of function or radiographic grading; decrease in 5D4 keratan sulfate epitope Improvement in lameness, body condition, pain on joint manipulation and willingness to exercise Decrease in synovial fluid volume; trend to decrease cartilage fibrillation and ulceration
[40]
[41] [42] [43] [44] [45]
[46] [47]
71
Improvement in Mankin score; increased joint fluid levels of intact IGFBP-5 and IGF-1
[48]
Reduced severity of cartilage histological lesions; enhanced HA content and fast-sedimenting aggrecan aggregates in cartilage
[49]
Use of animal models of osteoarthritis in the evaluation of potential new therapeutic agents
Interleukin 1 (IL-1) receptor antagonist (in transduced synovial cells) IL-1 receptor antagonist (recombinant) IL-1 receptor antagonist (local gene transfer) Powder of quality elk velvet antler P54FP (a plant extract)
Agent
Osteoarthritis model
Alendronate (a bisphosphonate Rat; ACL transection bone antiresorptive)
Zoledronic acid (a bisphosphonate bone antiresorptive)
Rabbit; chymopapain-induced OA
NE-10035 (a bisphosphonate bone anti-resorptive)
Dog; ACL transection
Carprofen, deracoxib, etodolac
Dog; spontaneous, chronic, unilateral OA Dog; ACL transection
Carprofen
Meloxicam Licofelone (combined 5lipoxygenase and cyclo-oxygenase inhibitor)
Dog; spontaneous OA Dog; ACL transection
Licofelone
Dog; ACL transection
Reported effects include:
Ref.
Chondroprotection by histological and collagen degradation biomarker criteria; subchondral bone resorption suppression; inhibited vascular invasion into calcified cartilage; reduced local release of active TGF-` Decreased grossly and histologically detectable cartilage degeneration; decreased urinary levels of collagen cross-links Reduction of formation and resorption of cancellous subchondral bone; no effect on osteophyte formation or pathological changes in articular cartilage (mild OA lesions in controls and small sample size noted) Decrease PGE2 levels in synovial fluid
[50]
Decreased width of osteophytes, size of cartilage lesions and histological severity of cartilage lesions; decreased subchondral bone remodeling Improved general clinical score Reduction in OA bone morphological changes with reduction in MMP-13 levels in bone cells and reduction in number of cathepsin K and MMP-13 positive osteoclasts Reduced chondrocyte apoptosis; decreased levels of caspase-3, COX-2 and iNOS in cartilage
[51]
[52]
[53] [54]
[55] [56]
[57]
Stephen A. Stimpson et al.
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Table 1 (continued)
ML-3000 (a dual inhibitor of 5-lipoxygenase and cyclooxygenase) Tenidap
MMP-13 inhibitors Matrix metalloproteinase inhibitors Matrix metalloproteinase inhibitor Matrix metalloproteinase inhibitors Doxycycline Doxycycline and CMT-7 (matrix metalloproteinase inhibitor)
Decreased size and grade of cartilage lesions; decreased levels: PGE2 in synovial fluid; LTB4 production by synovium; collagenase 1 in cartilage; IL-1` in synovial membrane Dog; ACL transection Decreased size of osteophytes; decrease size and grade of cartilage lesions; no effect on histological evidence of synovial inflammation; decrease in collagenase-3 in cartilage and IL-1` in synovial fluid Hamster; IL-1 or MMP-13-induced Inhibition of release of glycosaminoglycans or collagen cartilage matrix degradation in type II fragments, respectively, induced by knee joint aggrecanse or MMP-13 Guinea pig; partial medial Inhibition of decrease in global histological score; meniscectomy inhibition of decrease in cartilage thickness Hamster; MMP-13-induced cartilage Inhibition of release of collagen type II fragments matrix degradation in knee joint Rabbit; ACL transection and Reduction in gross pathological cartilage changes meniscectomy Rat; medial collateral ligament and Inhibition of cartilage degradation and osteophyte meniscus transection formation Rat; iodoacetic acid-induced OA Inhibition of joint damage Dog; ACL transection Guinea pig; spontaneous OA
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Reduced NO levels in cartilage; no effect on stromolysin activity in cartilage Decrease by CMT-7 and not doxycycline in cartilage fibrillation and destruction, subchondral bone sclerosis and cyst formation in central compartment of medial condyle; elevation by CMT-7 in cartilage levels of collagen, hyaluronan and proteoglycan
[58]
[59]
[18]
[60] [17] [61] [62] [63] [64] [65]
Use of animal models of osteoarthritis in the evaluation of potential new therapeutic agents
Matrix metalloproteinase (MMP)-13 and aggrecanase inhibitors MMP inhibitor S-34219
Dog; ACL transection
Agent
Osteoarthritis model
Reported effects include:
Ref.
Doxycycline
Dog; ACL transection
[66]
Doxycycline
Dog; ACL transection 2 weeks after dorsal root ganglionectomy
Ro 32-3555 (collagenase inhibitor)
No significant effect on subchondral bone formation or resorption Decrease in cartilage lesions on distal aspect of the femoral condyle; no effect on osteophytosis or cartilage lesions in other regions Inhibition of joint space narrowing and osteophyte formation; histological evidence for protection from cartilage degradative changes Inhibition of degradation of articular cartilage
Mouse; STR/1N strain spontaneously developing OA at an early age Rat; cartilage breakdown induced by intra-articular injection of P. acnes Dog; ACL transection Lower cell scores for expression of iNOS, nitrotyrosine, COX-2, collagenase-1 and stromelysin-1 in cartilage; lower cell scores for IL-1`, COX-2, iNOS and nitrotyrosine in synovial lining and mononuclear cell infiltrate Dog; ACL transection Decreased size of cartilage lesions macroscopically, and decreased severity histologically Dog; ACL transection Reduction in incidence of osteophyte number and size; decreased size of cartilage lesions; decreased collagenase and general metalloproteinase activity in cartilage and decreased levels of IL-1`, PGE2 and nitrite/nitrate in synovial fluid Rabbit; partial meniscectomy Reduction in size of erosive area on tibial plateau
Ro 32-3555 N-iminoethyl-L-lysine [inhibitor of inducible NO synthase iNOS)] N-iminoethyl-L-lysine (inhibitor of iNOS) N-iminoethyl-L-lysine (inhibitor of iNOS)
CPA-926 [prodrug of esculetin (dihydroxycoumarin)]
[67]
[68]
[69] [70]
[71] [72]
[73]
Stephen A. Stimpson et al.
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Table 1 (continued)
Methotrexate
Mouse; collagenase-induced OA; spontaneous OA in STR/1N transgenic strain Rabbit; ACL transection
Guinea pig; partial medial meniscectomy
Slight reduction in histological cartilage damage; no effect on several other parameters tested
[74]
Reduced development of arthritic lesions (mainly lesion surface size reduction); reduction in MMP-13 expression Reduction in median histopathological OA scores in both models; reduction of urinary levels of collagen cross-links in the transgenic model Decrease in surface area of cartilage macroscopic lesions and osteophyte width on lateral chondyles; decrease in inflammation (villous hyperplasia) Decrease in surface area and grade of cartilage macroscopic lesions; decrease in MMP-13 and IL-1` in cartilage
[75]
[76]
[77]
[78]
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Use of animal models of osteoarthritis in the evaluation of potential new therapeutic agents
PD-0200347, an (alpha2delta ligand of voltage-activated Ca2+ channels) Pralnacasan (prodrug of RU 36384/VRT-18858 inhibitor of IL-1` converting enzyme) PD 198306 (an inhibitor of mitogen-activated protein kinase kinase 1/2) Pioglitazone (a PPAR gamma ligand)
Rabbit; partial meniscectomy and transection of medial collateral and cruciate ligaments Dog; ACL transection
Stephen A. Stimpson et al.
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4
5 6 7
8 9 10 11
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15
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Buckwalter J (2002) The many phases of osteoarthritis: Introduction. In: VC Hascall, KE Kuettner (eds): The Many Faces of Osteoarthritis. Birkhäuser, Basel, 3–4 Dieppe P (2002) Assessment of joint damage in osteoarthritis: Introduction. In: VC Hascall, KE Kuettner (eds): The Many Faces of Osteoarthritis. Birkhäuser, Basel, 323–328 Dieppe P, Cushnaghan J, McAlindon T (1992) Epidemiology, clinical course, and outcome of knee osteoarthritis. In: KE Keuttner, R Schleyerbach, JG Peyron, VC Hascall (eds): Articular Cartilage and Osteoarthritis. Raven Press, New York, 617–627 Cole A, Hauselmann H, Flechtenmacher J, Huch K, Koepp H, Eger W, Aurich ME, Rolauffs B, Margulis A, Muehleman C et al (2006) Metabolic differences between knee and ankle. In: VC Hascall, KE Kuettner (eds): The Many Faces of Osteoarthritis. Birkhäuser, Basel, 27–29 Bendele AM, Hulman JF (1988) Spontaneous cartilage degeneration in guinea pigs. Arthritis Rheum 31: 561–565 Bendele AM (2002) Animal models of osteoarthritis in an era of molecular biology. J Musculoskelet Neuronal Interact 2: 501–503 Helminen HJ, Saamanen AM, Salminen H, Hyttinen MM (2002) Transgenic mouse models for studying the role of cartilage macromolecules in osteoarthritis. Rheumatology 41: 848–856 Pond MJ, Nuki G (1973) Experimentally-induced osteoarthritis in the dog. Ann Rheum Dis 32: 387–388 Brandt KD (2002) Animal models of osteoarthritis. Biorheology 39: 221–235 Bendele AM (2001) Animal models of osteoarthritis. J Musculoskelet Neuronal Interact 1: 363–376 Moskowitz RW, Davis W, Sammarco J, Martens M, Baker J, Mayor M, Burstein AH, Frankel VH (1973) Experimentally induced degenerative joint lesions following partial meniscectomy in the rabbit. Arthritis Rheum 16: 397–405 Moskowitz R (1992) Experimental models in osteoarthritis. In: R Moskowitz, D Howell, V Goldberg (eds): Osteoarthritis: Diagnosis and Medical/Surgical Management. Saunders, Philadelphia, 213–232 Pritzker KP (1994) Animal models for osteoarthritis: processes, problems and prospects. Ann Rheum Dis 53: 406–420 Billingham MEJ (1998) Advantages afforded by the use of animal models for evaluation of potential disease-modifying osteoarthritis drugs (DMOADs). In: KD Brandt, M Doherty, LS Lohmander (eds): Osteoarthritis. Oxford Press, Oxford, 429–438 Doherty N, Griffiths RJ, Pettipher ER (1998) The role of animal models in the discovery of novel disease-modifying osteoarthritis drugs (DMOADs). In: KD Bradt, M Doherty, LS Lohmander (eds): Osteoarthritis. Oxford University Press, Oxford, 439–449 Oegema T, Visco D (1998) Animal models of osteoarthritis. In: Y An, R Friedman (eds): Animal Models in Orthopaedic Research. CRC Press, Boca Raton, 349–367
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Moreau M, Dupuis J, Bonneau NH, Lecuyer M (2004) Clinical evaluation of a powder of quality elk velvet antler for the treatment of osteoarthrosis in dogs. Can Vet J 45: 133–139 Innes JF, Fuller CJ, Grover ER, Kelly AL, Burn JF (2003) Randomised, double-blind, placebo-controlled parallel group study of P54FP for the treatment of dogs with osteoarthritis. Vet Rec 152: 457–460 Innes JF, Barr AR, Sharif M (2000) Efficacy of oral calcium pentosan polysulphate for the treatment of osteoarthritis of the canine stifle joint secondary to cranial cruciate ligament deficiency. Vet Rec 146: 433–437 Read RA, Cullis-Hill D, Jones MP (1996) Systemic use of pentosan polysulphate in the treatment of osteoarthritis. J Small Animal Pract 37: 108–114 Brandt KD, Smith G, Kang SY, Myers S, O’Connor B, Albrecht M (1997) Effects of diacerhein in an accelerated canine model of osteoarthritis. Osteoarthritis Cartilage 5: 438–449 Clemmons DR, Busby WH Jr, Garmong A, Schultz DR, Howell DS, Altman RD, Karr R (2002) Inhibition of insulin-like growth factor binding protein 5 proteolysis in articular cartilage and joint fluid results in enhanced concentrations of insulin-like growth factor 1 and is associated with improved osteoarthritis. Arthritis Rheum 46: 694–703 El Hajjaji H, Williams JM, Devogelaer JP, Lenz ME, Thonar EJ, Manicourt DH (2004) Treatment with calcitonin prevents the net loss of collagen, hyaluronan and proteoglycan aggregates from cartilage in the early stages of canine experimental osteoarthritis. Osteoarthritis Cartilage 12: 904–911 Hayami T, Pickarski M, Wesolowski GA, McLane J, Bone A, Destefano J, Rodan GA, Duong lT (2004) The role of subchondral bone remodeling in osteoarthritis: reduction of cartilage degeneration and prevention of osteophyte formation by alendronate in the rat anterior cruciate ligament transection model. Arthritis Rheum 50: 1193–1206 Muehleman C, Green J, Williams JM, Kuettner KE, Thonar EJ, Sumner DR (2002) The effect of bone remodeling inhibition by zoledronic acid in an animal model of cartilage matrix damage. Osteoarthritis Cartilage 10: 226–233 Myers SL, Brandt KD, Burr DB, O’Connor BL, Albrecht M (1999) Effects of a bisphosphonate on bone histomorphometry and dynamics in the canine cruciate deficiency model of osteoarthritis. J Rheumatol 26: 2645–2653 Sessions JK, Reynolds LR, Budsberg SC (2005) In vivo effects of carprofen, deracoxib, and etodolac on prostanoid production in blood, gastric mucosa, and synovial fluid in dogs with chronic osteoarthritis. Am J Vet Res 66: 812–817 Pelletier JP, Lajeunesse D, Jovanovic DV, Lascau-Coman V, Jolicoeur FC, Hilal G, Fernandes JC, Martel-Pelletier J (2000) Carprofen simultaneously reduces progression of morphological changes in cartilage and subchondral bone in experimental dog osteoarthritis. J Rheumatol 27: 2893–2902 Peterson KD, Keefe TJ (2004) Effects of meloxicam on severity of lameness and other clinical signs of osteoarthritis in dogs. J Am Vet Med Assoc 225: 1056–1060 Pelletier JP, Boileau C, Brunet J, Boily M, Lajeunesse D, Reboul P, Laufer S, Martel-Pel-
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Models of inflammatory processes in cancer Roberto Benelli1, Guido Frumento1, Adriana Albini1 and Douglas M. Noonan2 1Dept 2Dept
of Translational Oncology, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy; of Clinical and Biological Sciences, University of Insubria, Varese, Italy
Introduction: Inflammation and cancer In 1863 Rudolf Virchow noted leukocytes in tumor tissues and made the connection between inflammation and cancer; most intuitively, he suggested that the presence of leukocytes indicated an origin of cancer at sites of chronic inflammation [1]. These observations were largely ignored over the next century, where the presence of leukocytes in tumors was generally considered an indication of an aborted attempt to reject the tumor by the host. Not much later after Virchow, William B Coley, noting that some tumors regressed when accompanied by infection, proposed the use of bacterial extracts to treat cancer, in retrospect the first attempt in immunotherapy. Although the cure rates of Coley’s approach was similar to that of radiotherapy that was developing at the same time, the vastly superior potential of dosing radiation led to its regular use in cancer treatment and the Coley approach was again forgotten. Recently, the potentials of the observations of these pioneers and their importance for the cancer patient are being re-recognized [1–3]. It has been estimated that approximately 15% of the world’s tumor burden can be ascribed to infectious agents [3]. Contrary to the hypothesis that led to the USA’s “war on cancer”, in which oncogenic viruses were thought to directly transform normal cells, it now appears that often it is the host’s response itself that drives the tumorigenic process. If we add clinically recognized chronic inflammation to the list, the percentage of cancers associated with infection and chronic inflammation soars [3], the number continues to rise further if we then consider subclinical chronic inflammation [4]. We are forced to recognize that inflammation plays a key role in initiating and promoting cancer. We should not forget, however, that inflammatory responses can also eliminate tumors, contributing to both specific and innate tumor rejection [5–12]. We are now coming to grips with the concept that, as known for specific immunity, the innate cells themselves are often skewed in phenotype [4]; these can be either a tumor-promoting phenotype that is associated with tissue remodeling, angiogenesis and growth promotion characteristic of chronic inflam-
In Vivo Models of Inflammation, Vol. I, edited by Christopher S. Stevenson, Lisa A. Marshall and Douglas W. Morgan © 2006 Birkhäuser Verlag Basel/Switzerland
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mation, or an anti-tumor phenotype typical of the intense tissue damaging activation in acute inflammation. Here we largely discuss the former phenotype.
Inflammation as an initiator The local inflammatory milieu enriches the tissue in growth factors stimulating cellular replication, but also in oxygen, nitrogen and other free radicals that kill pathogens but also can directly damage DNA. DNA damaging agents, cytokines that repress the protective function of p53, and growth factors that drive the DNA replication that fixes mutations, combine to generate a pro-mutagenic environment [3]. In acute inflammation undergoing resolution, the brief risk of tissue damage is worth the benefit of pathogen elimination; when inflammation goes chronic the risk of transformation increases in proportion with time.
Inflammation as a promoter It is now well recognized that the inflammatory infiltrate can release a numerous cytokines, chemokines and growth factors that directly or indirectly stimulate tumor cell proliferation [13–15]. This appears to be derived form the regenerative capacity of the immune system to mediate repair of damaged tissues. Although traditional therapeutic approaches often view cancer as a group of indefinitely replicating cells, it is now clear that tumors must be considered complex tissues that undergo constant remodeling. In 1986, Divorak [16] put forward the concept of tumors as “wounds that never heal”, as in the final phases of acute inflammation there is tissue rebuilding, but a cancer does not ever resolve. Again, we see the parallels between chronic inflammation and cancer-associated inflammation. In addition to the direct stimulation of tumor cells by the growth factors provided by inflammation, we have the support of vessel formation, where inflammatory cells appear to play an important role [17–20], and of the stromal-tumor cell interplay, where the supporting stromal cells provide many components required, including even the enzymes for escape [21–23]. Finally, the expression of chemokine receptors by tumor cells appears to direct them to the tissues to colonize during the process of metastasis formation [24–26].
Inflammation as an immunosuppressor The immune system can confront transformed cells by either specific immunity, both humoral and cytotoxic, or by innate immunity via natural killer (NK) cells. However, even though cancer cells can express antigens that can be recognized by the
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specific immune system, or in an attempt to escape lose expression of MHC class I molecules, thus becoming a target for NK cells, the ability of these immune components is generally potently suppressed within the tumor microenvironment. This suppression appears to be due, in part, to inflammatory cells that promote tissue remodeling and suppresses cell killing [4]. Taken together, inflammatory cells within a tumor mass can be considered a double-edged sword: under specific stimulation they can produce anti-angiogenic factors and free radicals able to directly destroy tumor cells. However, most clinically successful tumors generate an environment of partial activation, converting these cells to promoters of tumor growth and dissemination by improving angiogenesis, tissue break-down and remodeling (mediated by proteolytic enzymes such as MMPs), growth factor and chemokine production, and immune system suppression [3, 27, 28]. While the Coley toxin approach was a primitive way of resetting the inflammatory immune components from a “chronic” back to an “acute” state, it proved unreliable in that systemic administration of generic inflammatory substances like LPS (similar to that sometime found after gastrointestinal surgery interventions) has been shown to favor growth and dissemination of metastases and increase angiogenesis [29]. It appears that the clinically successful tumors are those that can use the inflammatory infiltrate for their own benefit [3, 28]. It is clear that we need models to both further our budding knowledge in the role of inflammation and cancer, and to develop approaches for interrupting the inflammation-tumor cycle to isolate tumor cells from the host and increase their susceptibility to therapy.
Models of the initiator/promotor activities of inflammation Although in vitro studies suggest that inflammation can provide a milieu able to promote DNA damage and classic initiation events, this role has been more difficult to discern in vivo. Here gene-targeted mice are beginning to provide key indications of the role of inflammation and specific cytokines in cancer progression. One example is the effect of transforming growth factor-beta 1 (TGF-`1) on colon and cecal cancers. TGF-`1 belongs to a family of polypeptides those members are related through a 100-amino acid carboxy-terminal domain with varying degrees of sequence identity and a set of at least seven cysteine residues in common [30, 31]. TGF-`1 is involved in several biological processes including growth control, regulation of epithelial cell differentiation and cell matrix interactions, and it appears to provide protection from genetic damage caused by inflammatory cells [32]. Moreover, TGF-`1 is known to be at the apex of a signaling pathway that is one of the more commonly disrupted pathways in human colon cancer [33]. Homozygous TGF-`1–/– mice die within 2 or 3 weeks after birth due to autoimmune reactions, massive inflammatory cell responses and tissue necrosis [34]. In contrast, Rag2–/– mice spontaneously develop mucosal hyperplasia of the cecum and
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colon that rarely progresses, and that appears to be dependent on inflammation in response to the intestinal bacterial flora. If TGF-`1–/– mice are crossed with Rag2–/– and placed on a 129 strain background, the animals are rescued from the autoimmune phenotype survive as long as 8 months [32, 34]. In these TGF-`1–/– Rag2–/– mice, however, the Rag2–/– hyperplasia progresses and these animals develop multiple carcinomas in the cecum and colon within 5 months of age. Interestingly, no hyperplasia was observed in germ-free TGF-`1+ Rag2–/– mice and both hyperplasia and progression to carcinoma were absent in germ-free TGF-`1–/– Rag2–/– mice [34]. When these animals were exposed to Helicobacter hepaticus, carcinomas promptly reappeared. Taken together, the data suggest that flora-induced colitis was a necessary condition for hyperplasia, which, in mice lacking TGF-`1 in a 129 genetic context, could progress to carcinoma. An similar colon carcinogenesis effect with inability to suppress chronic inflammation has been observed in IL-10 gene-targeted mice. These animals show normal lymphocyte development and antibody responses, but grow slowly, are anemic, and have a chronic enterocolitis that progress to colorectal adenocarcinomas in adulthood. The lack of an IL-10 gene leads to uncontrolled immune response involving overproduction of T helper 1 cells due to an excessive production IL-12 and/or IFNa. The inflammation first appears in the cecum, then in the ascending colon and finally in the transverse colon, a phenomenon concurrent with bacterial gastrointestinal tract colonization [35]. These data clearly demonstrate that induction of inflammation is clearly a key factor in human and murine colon cancer. In the in vivo model using azoxymethane, tumors are produced when the animals are given oral administration of dextran sodium sulfate (DSS) as an inflammatory stimulus [36, 37]. Interestingly, after induction of colitis by continuous oral administration of DSS, a lack of signaling through the chemokine receptors CCR2 and CCR5 protects the mice from the consequent severe inflammation and mucosal damage [38, 39].
Models of angiogenesis Recent data now indicate that one of the key promoting activities of inflammation is participation in a program of angiogenesis induction. In the absence of sufficient vascularization, most tumors would not be able to exceed the dimension of a few mm3 and would rarely form metastases [40]. Angiogenesis is an early and critical event in tumorigenesis, where the “angiogenic switch” represents a critical conversion point to malignancy [41]. Inhibition of this angiogenic switch would be a potent mechanism for maintaining clinical tumor dormancy, prolonging survival and improving the quality of life. Inflammatory cells produce a wide variety of factors able to induce angiogenesis [42, 43], and angiogenesis is a known endpoint of chronic inflammatory conditions such as arthritis. Inhibition of tumor angiogenesis
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is an interesting pharmacological target for cancer; recently avastatin, an anti-VEGF antibody, became the first anti-angiogenesis drug approved by the FDA for use in combination with standard chemotherapy, others are certainly on the way [44]. Although angiogenesis is a physiological event occurring in response to different conditions, it is rare in adult humans, with the quiescent endothelial cells showing very low proliferative and turnover indices. Epidemiological studies suggest that blocking inflammation can also reduce cancer risk [45], perhaps in part by preventing the angiogenic switch. For both inflammation and angiogenesis, the target cells are not highly plastic tumor cells prone to develop drug resistance mechanisms, but rather normal, genetically stable cells whose behavior can be expected to be consistent across different tumor types. Anti-angiogenic therapies theoretically have few potential side effects in the adult host, supported by the relatively low toxicity of most angiogenesis inhibitors in pre-clinical and clinical trials. Interestingly, avastatin and anti-inflammatory COX-2-specific inhibitors show a similar risk factor: cardiovascular infarction. It has been hypothesized that this may be due to increased susceptibility of endothelial cells to apoptosis due to VEGF sequestration by avastatin, or reduction of COX-2-dependent VEGF production by the COX-2-specific inhibitors [46]. The advantages and disadvantages of basic models for studying angiogenesis in vitro [47] and in vivo [48, 49] have been previously discussed, here we rapidly review these with comments regarding aspects of these models. A myriad of models for angiogenesis in vivo have been developed, of these only three are the most widely used: the avian chorioallantoic membrane, subcutaneous Matrigel sponges and the corneal micropocket model.
The avian chorioallantoic membrane model This model, which consists of placing a disc or gel containing angiogenic (or antiangiogenic, for that matter) factors onto the chorioallantoic membrane of an open egg (usually a chicken egg) and observing the response in the vessels near the disc, has been used for decades as a model of angiogenesis [50, 51]. Since it involves a two-dimensional embryonic membrane rather than vertebrate tissues, but does measure angiogenesis, it provides a bridge between in vitro and in vivo models. It allows relatively high throughput, but can often show high variability that is compounded by the difficulties in quantifying the results. Further, it is not a mammalian system. This limits both the application of factors, which if of mammalian origin that may not be active, and the use of inhibitors such as monoclonal antibodies that may not recognize avian counterparts. Systemic administration of drugs or other factors is difficult in this model. However, the immune system is quite conserved with most components present, thus information as to the role of immune responses in regulating angiogenesis can be obtained.
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Matrix implant models In this method for studying angiogenesis, vascularization of an acellular matrix is induced, where new vessels are easily visualized and the user has control over the composition of the matrix [52, 53]. Matrices based on Matrigel are among the most widely used, particularly for the convenience of implantation: since Matrigel is liquid at 4°C and solidifies at body temperature, it can be simply injected rather than having to subject the animal to surgical (thus inflammatory per se) procedures. Angiogenic stimuli and eventual inhibitors are mixed with the Matrigel prior to injection, after some days the gels can be recovered and the angiogenesis quantified by either morphometric analyses or quantification of the hemoglobin present in the gel. Further, the intensity and types of cellular infiltrates can be assessed by histological and immunohistochemical procedures, providing clues as to potential interrelations between the cells recruited into the matrix. The test itself is easily done and relatively rapid, thus through-put is high but more dependent on the method of quantification rather than on the assay itself. Among the most important advantages of the Matrigel system is that it is murine, allowing use not only of well-characterized biomolecules in a context of a mammalian immune system, but also the full array of transgenic and gene-targeted animals that are invaluable in precisely defining mechanisms. We have successfully used Matrigel to examine angiogenesis induced by generic inflammatory stimuli such as LPS [54–56] as well as cytokines and chemokines. There are several disadvantages to the Matrigel system as well, including Matrigel itself. Matrigel does contain some endogenous growth factors that may compound some analyses; further, it is itself a murine matrix, mostly laminin1 but also collagen IV and proteoglycans, which can bind to or have independent activities when testing specific factors. The use of artificial matrices can circumvent this problem but creates that of the necessity for surgery for implantation. Heparin is often included in the Matrigel to avoid growth factor complexation in the matrix and enhance responses; this can also lead to hemorrhages within the gels. While the presence of vessels can be determined, it is not easy to visualize blood flow, thus vessels may be present but flow may be stagnant. Finally, as with any of these systems the local area used may not reflect endothelial behavior in other tissues.
Corneal models The mammalian cornea is a normally avascular, transparent and readily visible. It is possible to implant cells, tissue fragments or pellets made of hydron or Matrigel containing specific factors or extracts onto the cornea of, usually, rabbits or mice [57–59]. The angiogenic reaction can then be visualized over time in living animals
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as vessels grow from the sclera into the cornea in response to the stimulus. The efficacy of local or systemic inhibitors can be assessed. The drawbacks of this technique are numerous, beginning with the need for adequate surgical technique and surgery itself, largely limiting its use to those groups with substantial expertise. Diverse animal models are available, but while rabbits offer larger size for ease of both surgery and analysis, they do not have the flexibility in terms of genetics or syngenic materials that the much smaller mice provide. Quantification is not easy, as various parameters can be assessed and these may vary independently. Finally, and particularly important when considering inflammation, the eye is an immune privileged site, and this might drastically affect analysis of the role of inflammatory components in angiogenesis. For example, a notable difference in results using chemokine-induced angiogenesis has been observed between studies using corneal and Matrigel models [17], that imply significant differences in mechanisms of action.
Granuloma models Granuloma is a typical tissue reaction, with strong angiogenesis, elicited by chronic inflammation. A frequent model of granuloma is based on the direct infection of mice with Mycobacterium strains, although this model implies a true infection and cannot be finalized to the study of infection-unrelated events (see [60] as an example). As a consequence, more accessible models are based on immunological or mechanical stimulation of a target tissue. For example, lung granulomas can be induced by the injection of sensitizing agents, like NG-nitro-L-arginine-methyl ester, followed by challenge with purified mycobacterium protein derivative [61]. Other models have used, with great fantasy, any kind of stimuli to induce a granulomatous tissue, among these a simple injection of 0.2 ml of a 1:40 saturated crystal solution of potassium permanganate in mice that induces a granuloma in 1 week [62]. In a model of colon cancer progression, implantation of the non-tumorigenic adenoma cell line FPCK-1-1 attached to a small plastic plate induced first acute and then chronic inflammation, which allowed the system to progress to moderately differentiated adenocarcinoma [63]. Most of these methods can be applied without any need for extensive training, giving interesting indications on the temporal steps of granuloma formation and its inhibition. Unfortunately, these experiments are only partially able to give quantitative data, and most results are dependent upon histological examination of samples. In addition, the induction of granulomas by mechanical trauma involves a chronic, irreversible phenomenon of cell necrosis releasing important mediators not present in those granulomas where apoptosis is the natural way of cell death [64].
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Wound healing The wound healing assay was one of the first experimental in vivo protocols to be established and later adapted to specifically study angiogenesis. The model is initiated by wounding of a portion of skin, which can be performed by interventions such as surgical incision, cryoinjury or burning [65–67]. A variety of experimental settings can be applied to study different forms of angiogenesis: the presence of ischemia or hyperoxia, growth factors, inhibitors and so on. A particular modification of this test is the skin window model, where the wound is only applied to open a surface of the tissue used to implant active soluble mediators of the angiogenic process covered by a transparent screen [68]. This screen allows for direct observation of the angiogenic process, but the previous observations concerning the granuloma tissue model suggest it could be per-se active as stimulus. While the ease of application of the wound assay makes this test rapidly available, some concern comes from the possible influence of infections of the wounds by bacteria, yeasts and fungi. These infections would be particularly detrimental if latent, as the operator could observe opposite results due to pro- or anti-angiogenic molecules of infective origin.
Invertebrate models? All of the above models are based on vertebrates and, with the exception of the avian chorioallantoic membrane, on mammalian systems that arouse ethical and cost concerns. One interesting approach is that of invertebrate systems, in particular the leach Hirudo medicinalis. Surprisingly, these animals have been shown to mount an inflammatory angiogenic response during wound healing and to the injection of angiogenic factors, including VEGF, EGF and bFGF and the cytokine GMCSF [69–74], all of human origin, suggesting excellent conservation of key mechanisms. In addition to endothelial cells, the inflammation and angiogenic response appears to be in part guided by innate immune system equivalents to macrophages, granulocytes, and NK cells [73]. While this system lacks specific immunity and is clearly distant on an evolutionary scale, the possibility of high throughput at low cost while partially avoiding ethical considerations make it an interesting possibility as an initial screening system.
In vivo tumor models Tumor cell xenograft models clearly comprise one of the key and most widely used methods for addressing the role of inflammation and angiogenesis in promoting tumor growth, as well as for pre-clinical identification of pharmaceuticals for
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inhibiting these processes. This involves the introduction of tumor cells that have usually been cultured in vitro into a suitable host and following the growth of the tumor over time. There are also a number of animal models that have been genetically manipulated to increase the probability of cancers in one or more tissues. Numerous different tumor types have been used, and the advantages and disadvantages of these approaches have been previously discussed. Here we would like to emphasize the potential that use of genetically modified tumor cells or genetically modified host backgrounds can have on elucidation of molecular mechanisms, particularly regarding inflammation. As an example, we are taking chemokines and cancer.
Chemokines and cancer Chemokines are small proteins (8–12 kDa) divided into four subfamilies (CC, CXC, CX3C, and C) according to the sequence organization of the conserved cysteine residues at the N terminus. These proteins are recognized by a family of receptors that have a seven-transmembrane segment, G-protein coupled receptors. Although there are “monogamous” ligand-receptor couples, these proteins are often promiscuous, with ligands able to activate multiple receptors and receptors recognizing multiple ligands (see [75] for review). As their name implies, the chemokines are chemotactic cytokines that play a key role in both homeostatic and induced trafficking of leukocytes, with receptor expression often restricted to specific leukocyte types [76, 77]. Chemokines play a key role in cellular recruitment in inflammation and also appear to have a key role in cancer; however, what they are doing in cancer is not yet clear. Although chemokine overexpression in tumor cells can lead to reduced tumorigenicity and rejection in in vivo models, the chemokines themselves are often strongly expressed in human cancers (see [78] for review). One potential explanation may be derived from the observations on the effects of chemokine expression in melanoma cells by the group of Herlyn [79]. CCL2 (monocyte chemoattractant protein 1 or MCP-1) is a CC chemokine strongly acting on monocytes and macrophages whose major receptor is CCR2. When CCL2 was overexpressed in non-tumorigenic melanoma cells, low level of expression was permissive for tumor growth [79], possibly through induction of angiogenesis [80]. In contrast, high level expression in the same cells was not permissive for tumor growth, apparently via tumor rejection [79]. A similar situation was observed for IL-8 (CXCL8). CXCL8 is a chemokine highly active on neutrophils, inducing their recruitment, as well as being angiogenic [81] apparently through induction of a neutrophil-dependent cascade [54], leading to VEGF production [18] and angiogenesis that appears to be critical for tumorigenesis [19]. Introduction of CXCL8 into non-tumorigenic melanoma cells was again permissive for tumor formation at low levels of expression but repressed growth at high levels [82]. Interestingly, progressed melanoma
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cells seemed to be resistant to high level CXCL8 [82], which promoted growth and metastasis, suggesting that tumor progression involves tolerance toward intense inflammation. Studies in gene-targeted mice for chemokines and chemokine receptors will shed further light on the role of chemokines and inflammation in cancer development and metastasis. For example, mice gene targeted for CCR5 (CCR5–/–) showed slower tumor growth as compared to wild-type controls, as well as improved responses to cancer vaccines [83]. CCR5 binds CC chemokines like RANTES, MIP1_ and MIP1`, that recruit both macrophages and neutrophils [79]; CCR5 ligands can also stimulate endothelial cell chemotaxis and/or proliferation, two essential processes for angiogenesis. Accordingly, CCR5–/– mice showed a persistent inhibition of corneal neovascularization after denudation of corneal epithelium, suggesting CCR5 as an essential component in corneal neovascularization [84]. Expression of the CCR5 ligand CCL5 (RANTES) has been associated with enhanced melanoma formation and correlated with breast cancer stage. Finally, CCL5 antagonist have been observed to repress tumor growth in a breast cancer model [83].
Innate vs. specific immunity The group of Lisa Coussens demonstrated the angiogenic potential of mast cells [85] and later neutrophils [2] in an human papillomavirus oncogene-driven skin transgenic cancer model [86]. Using a combination of crosses between gene-targeted mice and bone marrow transplants, they demonstrated a key role of bone marrowderived MMP9, particularly from neutrophils, for driving carcinogenesis (see [2, 3] for review). Recently, the same group revealed a role for humoral immunity through local antibodies deposition in pre-malignant lesions that is required for tumorigenesis [87] that again appears to reflect chronic inflammation.
Inflammation-related immune regulatory mechanisms The presence of a large number of inflammatory cells in the reactive peri-tumoral stroma and, at a lesser extent, within the tumor parenchyma is a common finding in almost all human solid tumors. These cells are mostly macrophages, neutrophils and eosinophils, which are recruited at the tumor site by an ordinate sequence of chemotactic stimuli. In this process of extravasation and tumor tissue migration, inflammatory cells get activated, starting mechanisms that can lead to immune suppression and foster tumor immune escape. Tumor-associated macrophages (TAM) are a key component of inflammatory circuits that promote tumor growth and progression [27]. They produce growth factors, including IL-1 and platelet-derived growth factor, which may directly promote
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tumor growth [88]. Oxidative stress induced by TAM suppresses the expression of CD3 c chain of T cell receptor complex, impairing tumor-specific T cell responses [89]. TAM secrete proteases that degrade the surrounding tissue and could facilitate tumor cell expansion and invasion. Furthermore, as discussed above they secrete factors that promote angiogenesis, including vascular endothelial growth factor, a protein that further supports the growth and spread of tumors [90, 91]. Accordingly, TAM density significantly correlates with microvessel counts and disease-free survival in non-small cell lung cancer [92]. Recently, the concept that macrophages can polarize diverse phenotypes, nominated M1 and M2, has been proposed [4, 93]. TAM have the properties of a polarized M2 population, tuning adaptive Th1 immunity. TAM from mouse and human tumors showed defective production of IL-12 and increased production of the immunosuppressive cytokine IL-10 [94]. Like TAM, neutrophil granulocytes can prevent anti-tumor immune response by suppressing T cell function via reactive oxygen species, mainly hydrogen peroxide [95]. In humans, an immune suppressive enzyme, arginase I, is stored in granules of both neutrophils and eosinophils [96]. Arginase I is released within the tumor [97] where it represses T cell function by production of nitrotyrosines and repression of c chain expression of the T cell receptor [98, 99]. Arginase II has never been detected in cells of hematopoietic lineage. Eosinophils may also play a role in immune polarization and suppression in cancer, these cells can express COX-2 [100, 101], an enzyme associated with T regulatory type 1 responses [102]. However, eosinophils, as well as macrophages, are important sources of indoleamine 2,3-dioxygenase (IDO) expression. IDO is an enzyme endowed with a strong immune suppressive activity [103, 104] through mechanisms not as yet fully elucidated. All these mechanisms displayed by inflammatory cells, together with the regulatory ones exerted by tumor cells, result in a strong local immune suppression. As a matter of fact, in humans the lymphocytes infiltrating tumors are anergic and do not proliferate [105]. A subpopulation of T lymphocytes also takes part in tumor immune escape. Regulatory T cells (T regs) are CD4+ T cells that also express CTLA-4, the glucocorticoid-induced TNFR family-related protein (GITR), the forkhead/winged helix transcription factor Foxp3, and high levels of CD25. T regs effectively down-regulate immune response [106]. In ovarian carcinoma the amount of tumor-infiltrating CD25+ T cells correlates with reduced survival [107].
Considerations on methods and reagents Unfortunately mice are not the best model for studying immune suppression exerted by tumor-associated inflammatory cells: in general the inflammatory infiltrate is by far less massive in mice tumors than in human ones. Furthermore, the patterns
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of expression of arginase I and IDO differ in mice and men. In mice, mature granulocytes do not express arginase I, while circulating immature myeloid cells do [108, 109]; by contrast, arginase I-positive immature myeloid cells have not been found in humans [98], where the protein is expressed by mature granulocytes. Again in contrast to that observed in humans, murine eosinophils do not express IDO; instead, IDO expression has been found in neutrophils in Candida-infected mice [110]. Macrophages from both mice and humans can express IDO; however, induction of IDO activity can be detected in murine macrophages only when the synthesis of reactive nitrogen species is inhibited [111]. Murine macrophages, but not human macrophages, express arginase I when treated with IL-4 and IL-13 [112]. Since arginase is stored in granules, molecular biology techniques aimed at detecting arginase gene expression are not particularly useful in granulocytes. Instead, several antibodies against arginase I are available for immunohistochemistry and Western blot studies. Arginase activity can be detected by either a colorimetric assay detecting urea [98], or by HPLC detection of ornithine [113]. The latter method is recommended for samples with relatively low arginase activity. Great care should be taken in isolating arginase-positive cells in humans: isolation techniques can induce degranulation in both eosinophils and neutrophils. So far no commercial anti-IDO antibodies for immunohistochemistry on paraffin-embedded human samples are available, while analysis of IDO expression on cryostat sections has to face several problems: (i) both eosinophils and macrophages are endowed with strong endogenous peroxidase and phosphatase activity, so immunohistochemistry with these reagents cannot be done, (ii) eosinophils and macrophages also express Fc receptors and both immunohistochemistry and immunofluorescence must include an effective Fc receptor blocking step. IDO activity can be detected by either a colorimetric assay, or by reverse-phase HPLC [114]; however, the former technique should not be used for concentrations of kinurenine, the tryptophan catabolite produced by IDO activity, below 1 µM. T regs from tissues and from peripheral blood are usually detected by flow cytometry. However, activated T cells transiently express CD25, and thus can alter overall evaluation of CD4+/CD25+ cells, therefore a third marker should be added when the sample potentially contains recently activated T cells. A technique detecting CD4+/CD25+/FOXP3+ cells in tissues has been described [107].
Conclusions A variety of models reflecting a multitude of mechanisms are bringing into focus the role of inflammation as both a driving force in carcinogenesis as well as a potential weapon to combat tumors. Further, these models now provide the basis for screening for molecules able to break to the pro-cancer chronic inflammation cycle and turn the tide against tumors.
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Acknowledgements We are indebted to Patrizia Larghero and Giorgia Travaini of the Prevention Unit2, Centro di Biotecnologie Avanzate, Genova, Italy, for helpful discussion. These studies were supported by grants from the AIRC (Associazione Italiana per la Ricerca sul Cancro), the Ministero della Salute Progetto Finalizzato, the MIUR Progetto Strategico and Progetto FIRB, the Fondi di Ateneo of the University of Insubria, the PNR-Oncologia Citochine & Chemokine, the Compagnia di San Paolo and the Comitato Interministeriale per la Programmazione Economica (CIPE).
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Advances in stem cell research: use of stem cells in animal models of muscular dystrophy Antonio Musarò1 and Nadia Rosenthal2 1Department
of Histology and Medical Embryology, CE-BEMM and Interuniversity Institute of Myology, University of Rome “La Sapienza”, Via A. Scarpa 14, 00161 Rome, Italy; 2European Molecular Biology Laboratory, Mouse Biology Unit, Monterotondo 00016 (Rome), Italy
Introduction One of the most exciting aspirations of current medical science is the regeneration of damaged body parts. The capacity of adult tissues to regenerate in response to injury stimuli represents an important homeostatic process that until recently was thought to be limited in mammals to tissues with high turnover such as blood and skin. Functional regeneration of the injured central nervous system was considered impossible, as Santiago Ramón y Cajal described in the early 20th century, ‘‘once the development was ended, the fonts of growth and regeneration … dried up irrevocably’’. However, this central dogma of cell biology has been revised on the basis of recent experimental evidence that even the adult brain is able to undergo repair. It is now generally accepted that each tissue type, even those such as nerves or muscle that are considered post-mitotic, contains a reserve of undifferentiated progenitor cells, loosely termed stem cells, that participate in tissue regeneration and repair. Regeneration represents a coordinate process in which these stem cell populations are activated to maintain and preserve tissue structure and function upon injured stimuli. In this chapter we discuss the potential contribution of stem cell therapy in animal models of muscular dystrophy, in which constant degeneration and the exacerbating chronic inflammatory response cripples the regenerative capacity ability of the diseased muscle.
Muscular dystrophy and the pathogenetic role of inflammation The maintenance of a working skeletal musculature is conferred by its remarkable ability to regenerate after injury [1]. Most muscle pathologies are characterized by the progressive loss of muscle tissue due to chronic degeneration combined with the inability of regeneration machinery to replace damaged myofibers. Among pathologies leading to muscle wasting, muscular dystrophies severely compromise the func-
In Vivo Models of Inflammation, Vol. I, edited by Christopher S. Stevenson, Lisa A. Marshall and Douglas W. Morgan © 2006 Birkhäuser Verlag Basel/Switzerland
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Figure 1 The molecular organization of cytoskeleton protein. The dystrophin-glycoprotein complex (DGC) at the sarcolemma connects the cytoskeleton of a muscle fiber to its surrounding extracellular matrix (modified from [93]).
tional performance of skeletal muscle. Muscular dystrophies are degenerative disorders characterized by progressive weakness in specific muscle groups, persistent protein degradation and alteration in the regenerative capacity of muscle satellite cells [2]. Mutations in genes encoding proteins of the dystrophin-glycoprotein complex (DGC) (Fig. 1) lead to alteration in muscle structure and cause muscular dystrophy [2–4]. In this chapter we discuss advances in stem cell therapy in two experimental models of muscular dystrophy, Duchenne muscular dystrophy (DMD) and sarcoglycanopathies, where stem cell-mediated therapy has been extensively studied. Given the complexity of the field, this review should be regarded as a presentation
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of work in progress rather than a comprehensive description of dystrophic models and stem-cell-mediated-therapy. DMD is a X-linked genetic disease in which the dystrophin gene is mutated, resulting in dysfunctional or absent dystrophin protein. Without dystrophin, the DGC is unstable, leading to an increase in muscle damage. Sarcoglycanopathies are caused by the defective expression of components of the sarcoglycan (SG) complex, leading to a heterogeneous group of muscle diseases known as limb-girdle muscular dystrophy (LGMD) [4]. Different studies support the notion that loss of the link between the extracellular matrix and the cytoskeleton represents a critical parameter for the maintenance of the structural integrity of skeletal muscle [5, 6]. In addition to their structural role, the cytoskeletal proteins function as mediators of several signal transduction pathways, including those modulated by calcium. Calcium (Ca2+) is an important intracellular messenger in muscle, controlling numerous cellular process including proliferation, cell growth, differentiation, and apoptosis [7]. All muscle fibers use Ca2+ as their main regulatory and signal molecule [8]. However, in DMD and sarcoglycan-deficient muscle the Ca2+ homeostasis is altered, suggesting that the DGC contributes to muscle structural integrity and regulates intracellular Ca2+ levels [9–12]. In addition, the growth factor-regulated channel (GRC), which belongs to the transient receptor potential channel family, is elevated in the sarcolemma of skeletal and/or cardiac muscle in dystrophic human patients and animal models deficient in dystrophin or b-sarcoglycan [13]. Thus, in the absence of DGC, alterations in intracellular Ca2+ may contribute to an imbalance between muscle protein synthesis and protein degradation, culminating in necrosis, fibrosis, and shift in fiber content. A further complication that exacerbates muscular dystrophy is the persistence of inflammation. In normal skeletal muscle, damage is followed by an inflammatory response [1] involving multiple cell types that subsides after several days. This transient inflammatory response is a normal homeostatic reaction to myonecrosis and is necessary for efficient repair. However, in dystrophic muscle, activation of inflammatory pathways could be one of the critical mechanisms that render the damaged muscle incapable of sustaining and complete efficient muscle regeneration. A persistent inflammatory response is observed in dystrophic muscle, leading to an altered extracellular environment [14], including an increased presence of inflammatory cells (e.g., macrophages) and elevated levels of various inflammatory cytokines (e.g., TNF-_, TGF-`). From microarray-based expression profiling, it is clear that the inflammatory response dominates the molecular signature obtained from dystrophin-deficient muscles both in humans and mice [15, 16]. A vast body of evidence supports a pivotal role of inflammation in the progression of muscle loss. In DMD muscles, a chronic-type mononuclear cell infiltrate is present early in the disease, before the onset of muscle weakness. Macrophages and T lymphocytes are the predominant cell types, comprising 80% of the infiltrating cells [17]. T lymphocytes show an activated phenotype and V`2 T cell receptor rearrangement sug-
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gests antigen recognition. Although conclusive evidence is lacking, it is anticipated that inflammation associated to myonecrosis represents a critical parameter in the pathogenesis of sarcoglycanopathies [18, 19]. Inflammation has therefore emerged as a possible target of non-genetic intervention, leading to the development of therapeutic approaches aimed at inhibiting the inflammatory response in the muscles of patients with DMD and LGMD. Glucocorticoids (GC) have been demonstrated to stabilize muscle strength and function in DMD and in LGMD patients [19–21]. GC are the most powerful antiinflammatory and immunosuppressive agents available. Known effects of GC treatment in DMD patients include reducing in muscle inflammation and suppressing of cytotoxic T lymphocytes. Therefore, at least part of the benefit of GC treatment may stem from their effects on immune cells, further supporting the role of the inflammatory response in the progression of DMD. However, it is well known in that patients with chronic inflammatory diseases not specifically targeted to muscles, long-term GC treatment causes muscle atrophy secondary to protein catabolism and muscle proteolysis [22]. Therefore, the efficacy of GC treatment in DMD patients is the net benefit of positive effects (suppression of inflammation) and negative effects (muscle catabolism). Moreover, the use of long-term GC is hampered by the considerable side effects of this treatment that include excessive weight gain, retarded growth, bone loss, behavioral abnormalities and cushingoid appearance [23]. Therefore, GCs are not the ideal therapy to counteract inflammation in DMD and other, possibly more efficacious, therapies should be sought.
Animal models of muscular dystrophy Since muscular dystrophy has emerged as a complex muscle disease, reliable animal models of the human disease are necessary to predict drug, gene and cell therapy effectiveness in patients. Due to its different size and muscle physiology, the mouse is not the ideal preclinical animal for this disease. However, mice represent invaluable in vivo models to dissect the molecular and cellular mechanisms underlying the dystrophic phenotype. In the last decade transgenic and knockout mice have been become a powerful and exciting genetic research tools. Transgenesis and gene-targeting approaches have led to the definition of specific roles for tissue regulatory factors during embryogenesis, and have added new insights into the pathogenetic mechanisms of several diseases. This includes providing appropriate models in which to explore the contribution of stem cells to tissue homeostasis and remodeling. The mdx mouse strain, lacking a functional dystrophin gene, has served as the animal model for human DMD and Becker muscular dystrophy [24, 25]. Skeletal muscles of mdx mice undergo extensive necrosis early in neonatal life but, unlike the human pathology, the diseased muscle rapidly regenerates and regains structural
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and functional integrity, presumably due to differences in the proliferative capacity of murine satellite cells. The attenuated myopathy in mdx mice has therefore been attributed in part to an enhanced regenerative capacity of murine skeletal muscle. However, other factors such as small mass of the animal, quadrapedal gait, short life span, and differential expression of compensatory proteins such as utrophin may also be involved [26]. Another limitation using the mdx mouse as animal model for DMD is the relatively frequent occurrence of dystrophin-positive muscle cells called revertants. This natural phenomenon has hampered efforts to develop cellular and gene therapies by interfering with data interpretation. Notably, different mutations of the mouse dystrophin gene have been described in mdx mice. The mdx4cv and mdx5cv dystrophin mouse mutants have approximately tenfold fewer revertants than the mdx mutant at both 2 and 6 months [27, 28], and therefore represent more useful models in gene transfer studies and in development of cell therapy approaches. The mdx3cv dystrophin mouse mutant may be a useful model for some types of human dystrophin deficiencies in which the levels of dystrophin are low but not completely absent. To compensate for these shortcomings of the mdx mouse, other mouse models of muscular dystrophy have been generated, by crossing MyoD–/– mice with mdx mice. MyoD–/–:mdx animals display several phenotypic traits typical of the human DMD [29]. However, the value of this model has been called into question by the findings of Inanlou et al. [30], who reported that the failure of mdx:MyoD–/– diaphragm to develop normally is not caused by a reduced number of satellite cells, but from the inability of stem cells to progress through the myogenic program, which is not considered the case in humans. Thus, to date, the mdx mutants remain the only mouse models for human DMD and Becker muscular dystrophy. Sarcoglycanopathies are a group of autosomal recessive LGMD caused by mutation in the _, `, a, and b sarcoglycan gene, respectively [4]. Sarcoglycan-null mutant mice start to develop a progressive muscular dystrophy during the first week of age and, in contrast to mdx mice, showed ongoing muscle necrosis with increasing age (Tab. 1).
Inflammation in the dystrophic models The role of inflammation in the pathogenesis of dystrophy in the mdx mouse has been the objective of numerous studies. Depletion of CD4+ and CD8+ T lymphocytes causes significant reduction in muscle pathology, and the use of perforin-deficient mice shows direct cytotoxicity of myofibers by T cells [31, 32], suggesting that T cells have an important role in the pathology of DMD. Macrophages represent a quantitatively important component of the infiltrate [17]. Macrophages are a major source of oxygen radicals and inflammatory cytokines that have been shown to produce myofiber damage [33]. Indeed, macrophage depletion results in decreased
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Table 1 - Main characteristics between two animal models of muscular dystrophy mdx
Sarcoglycan (SG) null
Model of Duchenne and Becker muscular dystrophy Lacks a functional dystrophin gene Skeletal muscles undergo extensive necrosis only early in neonatal life Higher regenerative capacity of murine skeletal muscle compared to human disease Differential expression of compensatory proteins
Models of limb-girdle muscular dystrophy (LGMD) -2C, -2D, -2E, -2F mutation in either _, `, a, or b sarcoglycan gene Progressive muscle necrosis with increasing age
Short life span Relatively frequent occurrence of dystrophin-positive muscle cells called revertants Shift in myosin heavy chain expression to slower isoforms Chronic inflammation Altered calcium handling Elevated serum creatine kinase levels
Develop a progressive muscular dystrophy during the first week of age, present excess of collagen and fatty infiltration Primary deficiency of any single SG leads to partial or complete absence of all other sarcoglycans Short life span Soleus muscle of SG_-null mice contain a higher number of regenerating fibers compared to EDL muscle Fibers of SG_-null mice contain inclusion bodies within the contractile structure Inflammatory involvement cardiomyopathy characterized by focal degeneration Elevated serum creatine kinase levels
myonecrosis in mdx mice [34]. In addition, treatment with cyclosporine A (CsA), a drug with a wide immunosuppressant action, counteracted muscle decline, fully preventing the 60% drop of forelimb strength induced by exercise [35]. A significant amelioration was observed in histological profile of CsA-treated gastrocnemius muscle, with reductions in fibrosis, centronucleated fibers, and degenerating area compared to untreated exercised mdx mice [35]. While the evidence supporting a major role of the inflammatory infiltrate in the progression of muscle damage in DMD is rather convincing, the molecular mechanisms are still unclear. The definition of these mechanisms and of the pivotal mediators involved may provide the necessary background for anti-inflammatory treatments that are more efficacious, more specific and with less side effects. A potential target is tumor necrosis factor (TNF). However, mdx mice crossed with TNF-deficient mice do not show a marked amelioration in muscle pathology [36] and pharmacological blockade of TNF activity delayed the appearance of the
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inflammatory infiltrate, but did not affect significantly long-term necrosis in mdx mice [37]. A role for inflammation is also hypothesized in the exacerbation of sarcoglycanopathies [18, 19], although compared to the information available for the mdx mouse model of muscular dystrophy, definitive evidence of inflammatory involvement is not yet conclusive for sarcoglycan-null mouse models. Collectively, the current data indicate that immunosuppression in the dystrophic mouse models has beneficial effects on some indices of muscle dysfunction, indicating that targeted immunosuppression may offer some promise in delaying the pathological progression of this insidious muscular disease.
Muscular dystrophy, regeneration and stem cells The contribution of satellite cells to injured muscle In addition to the chronic inflammatory insult in muscular dystrophy, the reconstruction of damaged muscle tissue is hampered by the lack of functional substitution of the injured myofibers. Mammalian skeletal muscle includes a population of myogenic progenitors, known as satellite cells, which form the major source of myogenic precursors in mammalian muscle regeneration [1]. Satellite cells reside between the basal lamina and plasmalemma [38] and are rapidly activated in response to appropriate stimuli. Once activated, satellite cells express factors involved in the specification of the myogenic program such as Pax-7, desmin, MNF_, myf-5, and MyoD [39]. Activated satellite cells proliferate as indicated by the expression of factors involved in cell cycle progression such as proliferating cell nuclear antigen [40, 41] and by incorporation of BrdU [42]. Ultimately the committed satellite cells fuse together or to the existing fibers to form new muscle fibers during regeneration and muscle repair. Taken together, these data confirm that regeneration is a highly coordinated program in adult skeletal muscle that partially recapitulates the embryonic developmental program. This aspect of muscle regeneration is hampered in several muscle diseases, including aging and muscular dystrophies. It has been suggested that the decline in the regenerative potential of senescent muscle is mainly due to decline in satellite cell number [43]. In contrast, Conboy et al. [44] reported that the dramatic age-related decline in myoblast generation in response to injury is due to an impairment of activation rather than a decline in number of satellite cells [44], demonstrating that Notch signaling plays a pivotal role in satellite cell activation and cell fate determination. Indeed, to examine the influence of systemic factors on aged progenitor muscle cells, this group recently established parabiotic pairings (that is, a shared circulatory system) between young and old mice (heterochronic parabioses), exposing old mice to factors present in young serum [45]. Notably, heterochronic parabiosis
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restored the activation of Notch signaling as well as the proliferation and regenerative capacity of aged satellite cells [45]. Among other factors, insulin-like growth factor 1 (IGF-1) plays critical role in muscle regeneration [46]. IGF-1 expression decreases during post-natal life, raising the prospect that this decline contributes to the progress of muscle atrophy in senescence, and limits the ability of skeletal muscle tissue to effect repair or regeneration. This hypothesis was confirmed by our work demonstrating that localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent and diseases skeletal muscle [41, 47, 48].
Myoblast transplantation in muscular dystrophy Satellite cells represent an important cellular mechanism to counteract the decline of skeletal muscle, participating in both the formation of new muscle fibers and to rescue of damaged muscle. However, satellite cell number decreases substantially in muscular dystrophy [49, 50], leading to altered regenerative capacity of skeletal muscle. Myoblast cell therapy has therefore been extensively explored as a promising alternative to correct genetic diseases by contributing to tissue regeneration. Replacement of diseased muscles with healthy and functional muscle fibers has long been a major therapeutic strategy for muscular dystrophies [51–53]. However, the failure of injected committed cells to survive in the recipient animals and successfully engraft within their target organs has proven disappointing. Indeed, even in an optimized environment for myoblast transplantation, such as in an immunodeficient, irradiated mdx host, the majority of transplanted cells underwent rapid death [54, 55]. Therefore, the poor survival of injected cells (less than 1%), minimal migration from injection site (1 mm), and rapid senescence of the surviving population, has failed to produce satisfactory protocols of muscle regeneration that might be considered for therapeutic purposes [51, 53, 55]. Several lines of research have been employed to increase the survival of injected myoblasts. Myoblast death observed after transplantation was significantly reduced when the hosts were irradiated [56, 57], suggesting that modification of the host cell environment contributes to this phenomenon. In addition, efficient myoblast transplantation was observed when mice were immunosuppressed with monoclonal antibodies against CD8, CD4 and CTLA4 Ig [56, 58, 59]. The authors postulated that neutrophils mediate myoblast mortality by an LFA-1-dependent mechanism. In another study, Kinoshita et al. [60] reported that immunosuppression with FK 506 insured good success of myoblast transplantation in mdx mice. Modulation of the inflammatory reaction to foreign cells is emerging as a necessary prerequisite for effective clinical applications of myoblast transplantation. Thus, integrating cell therapy approaches with anti-inflammatory treatments may circumvent the major problem associated with the survival of transplanted cells,
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enhancing cell engraftment and improving muscle regeneration. In summary, these studies emphasize how the restorative potential of pathological muscle is dependent not only on the presence of satellite cells, but also on the support of optimal environmental cues.
The contribution of stem cells to muscle regeneration Given the disappointing performance of transplanted myoblasts in treatment of muscular dystrophies, a wide range of alternate stem or progenitor cell types have been proposed for muscle cell therapy [1, 53, 61–63], including embryonic stem cells, adult bone marrow (BM) stem cells and endogenous pools of muscle progenitor cells. Although stem cells offer a new tool for regeneration in muscle disease, the signaling and molecular pathways involved in recruitment and myogenic commitment of progenitors cells is an important question that remains to be satisfactorily addressed. In addition, the environment in which these stem cells operate represents another important determinant for cell survival and differentiation, which may be compromised in the dystrophic milieu.
Embryonic stem cell therapy A true stem cell must satisfy two major operational criteria. First, it must be clonogenic, capable of unlimited self renewal. Second, it must be able to divide asymmetrically; one daughter resembling its mother with self-renewing capabilities, the other daughter giving rise to multiple types of differentiated cells. Embryonic stem (ES) cells are a classic example of a multipotent stem cell that are derived from a fertilized egg that has divided for 5 days to form the blastocyst, giving rise to every adult cell type [64]. In theory, ES cells are the ideal starting point for any stem cell therapy, since they survive and proliferate indefinitely in tissue culture when removed from the embryo. However, aside from the ethical and socio-political impediments [65], many factors have slowed research into ES cell-based therapies. ES cells derived from humans differ significantly from ES cells derived from mice, which proliferate much more rapidly than their human counterparts [64]. Whereas many of the molecular mechanisms that underlie mouse ES cell growth are well characterized, it is not clear if these are shared by human ES cells. Not all human ES cell lines are the same, but rather reflect the genetic diversity of the embryos from which they were derived. The hurdle of graft rejection still plagues any potential use of these cell lines in the clinic [66]. Finally, the robust proliferative capacity of ES cells brings with it the threat of cancer. Such factors need to be resolved before ES cells can be successfully delivered in the context of ageing or diseased tissues.
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Adult stem cell therapy The discovery of stem cell lineages in many adult tissues has challenged the classic concept that stem cells in the adult are present in only a few locations, such as the skin or BM, and are committed to differentiate into the tissue in which they reside [63]. Nevertheless, the criteria for defining stem cells in the adult are still difficult to satisfy experimentally. There is no predictable location for stem cells in most adult tissues, and markers for identifying them are limited, based on information derived primarily from studies of the BM (Tab. 2). Mouse BM stem cells proliferate constantly to renew circulating blood yet are as rare as 1 in 10 000, and may be even less common in humans. BM-derived mesenchymal stem cells readily proliferate in culture, which makes them attractive candidates for therapy. Hematopoietic stem cells (HSC) display additional cell surface markers that allow them to be labeled and tracked in the bloodstream to target tissues, or to be isolated and cultured in vitro [67–69]. Accounts of the repopulation of adult organs by BM-derived stem cells suggest that under the right conditions, they can contribute to virtually any part of the body [70–72] (Fig. 2). Mobilization of HSC from the BM into the circulation is increasingly used for other clinical applications [73]; however, HSC participation in normal regeneration processes is not yet proven. Local commitment of these cells to myogenesis and replenishment of the satellite cell compartment has been proposed as a potential route to improved response to injury [74]; alternatively, they may fuse directly into regenerating muscle fibers [75]. However, in all animal studies to date, it has been necessary to replace host BM with marked progenitor cells to prove their provenance. This experimental manipulation inevitably involves lethal irradiation of the host animal, a process that is emerging as a necessary prerequisite for BM engraftment into injured muscle [76]. In any case, the total number of BM stem cells recruited to a muscle fate in these studies still appears to be insufficient to be of therapeutic benefit [77]. Additional concerns that adult HSC do not normally adopt the phenotype of other cells are supported by studies that failed to detect descendants of a single labeled stem cell in other tissues, and implicate other BM stem cell types in observed regenerative events. Of these alternate cell types, marrow stromal cells can differentiate into osteocytes, adipocytes, chondrocytes, skeletal myocytes and smooth muscle myocytes [70]. The extent to which adult stem cells fuse with other cell types is also variable: fusion is the most prevalent source of hepatocytes in the liver, and is commonplace in damaged skeletal muscle beds: fusion with a circulating stem cell may actually rescue a damaged myocyte. These findings offer an alternative explanation for the presumed transdifferentiation of adult stem cells in a new environment. It is likely that aside from the BM and skin, pluripotent stem cells also exist in specialized niches within many other adult tissues, and that these common progen-
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Table 2 - Type, location, molecular markers and potential plasticity of stem cells Stem cells isolated from different sources can potentially differentiate in several lineages. Stem cells express several molecular markers; however, it is the combination of two or more markers that identify a potential stem cell population. Type and location of stem cells
Molecular markers
Type of cells/ tissues produced
Hematopoietic stem cell: bone marrow
ckit+; Sca1+; CD11b+; CD43+; CD45+; CD34–; Lineage marker (B220, Mac-1, Gr-1; CD4, CD5, CD8–) D13+; CD29+; CD44+; CD73+; CD90+; CD105+; CD14–; CD28–; CD31–; CD34– CD45– CD34+; M-cadherin+; VCAM+; NCAM+; Pax3+; Pax7+; MNF+ Sca1+; Pax3+; CD45+; CD34+; ckit– ckit+; sca1+; MDR1+; CD34–; CD45–; CD20–; CD8–; TER119– Ckit+; CD34+; Ov6+; CK7+; CK19+; Thy1+ Bcl2+; CD133+; nestin+ GFAP+; CD24–; CD34–; CD45–
Blood cells, cardiomyocytes, skeletal muscle, neuronal cells, hepatocytes, thymus, kidney, pancreas Bone, cartilage, muscle, ligament, tendon, adipose tissue, stroma, neural cells
Mesenchymal stem cells: bone marrow, peripheral blood Satellite cells: skeletal muscle Muscle derived stem cells (MDSC): skeletal muscle Cardiac stem cells: myocardium Hepatic stem cells: canals of Hering Neural stem cells: brain (hippocampus, subventricular zone) Testis stem cells: testis
Tai1+; _6integrin+; CD9+; ckit–; sca1– Stem cells of the skin: skin Cytokeratin-8, 18, 19+; E-cadherin+; cytokeratin-1, 5, 6, 14, 20–; vimentin–; nestin–; ckit– Mesoangioblasts: embryonic Flk1+; Sca1+; CD34+; dorsal aorta; vessels VEcadherin+
Skeletal muscle, bone, fat, smooth muscle Skeletal muscle, neural cells Myocytes, endothelial cells Oval cells, hepatocytes, biliary epithelial cells Neuron, astrocytes, oligodendrocytes, skeletal muscle, blood Gonads Keratinocytes
Mesoderm-derived tissues
itor cells are capable of regenerating and repairing tissues throughout the body. One of these was identified as a side population (SP), on the basis of their Hoechst dye exclusion properties [78]. Originally isolated from hematopoietic tissues in the BM, these stem cell populations have been described in several tissue compartments. In
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Figure 2 Versatility of stem cells An understanding of the plasticity of adult stem cells initially grew from observations that donor cells were found in nonhematopoietic tissues in the recipients of BM transplants. An increasing number of reports also showed that stem cells from a variety of tissues contribute to repair of different organs (for detailed information see [70–72]). However, reports of in vivo and in vitro cell-cell fusion suggest caution in stem cell biology [94].
particular, the identification of multi-potent stem cells residing in extra-hematopoietic adult tissues, including skeletal muscle, has offered new perspectives in cellmediated therapy for genetic diseases. Muscle SP cells were greatly enriched for cells competent to form hematopoietic colonies. However, muscle SP cells did not differentiate into myocytes in vitro, but, importantly, muscle SP cells exhibited the potential to give rise to both myocytes and satellite cells after co-culture with myoblasts, or after intramuscular transplantation in response to Wnt molecules [79, 80].
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These studies raise the intriguing possibility that small numbers of HSC reside in the muscle beds of non-injured animals [81], and more migrate into sites of injury, providing a potential mechanism by which damaged tissues are repaired [82–84]. However, BM-SP engraftment is a rare event and presents limitations for an efficient tissue repair. Indeed, even in a chronically regenerating environment as that of mdx dystrophic muscle, marrow-derived stem cells are unable to give rise to dystrophinpositive muscle fibers in clinically relevant numbers [77]. It is tempting to speculate that the poor recruitment of HSC into the dystrophic muscle of the mdx mouse may be the major obstacle for muscle regeneration and, therefore, for the rescue of the genetic disease. In addition, it is also possible that the mdx background does not provide enough selective advantage to the recruited cells to rescue the genetic disease. In fact, as also previously reported, despite the defect in dystrophin gene expression, mdx mice do not develop a dystrophic clinical phenotype observed in human patients, where the regenerative potential of satellite cells is progressively lost and skeletal muscle undergo extensive and irreversible degeneration [85]. More recently, other animal models of muscular dystrophy, such as sarcoglycannull mice, have been used to address any efficiency of cell-mediated therapy. In one of these studies, BM-SP cells from normal male mice readily incorporated into skeletal and cardiac muscle of transplanted b-sarcoglycan null female mice, but failed to express sarcoglycan [86], indicating impaired differentiation and/or maturation of BM-derived stem cells. The inability of BM-SP cells to express this protein severely limits their utility for cardiac and skeletal muscle regeneration. BM-SP stem cells have become very popular in experimental stem cell therapy for primary myopathies [87]. However, if their differentiation toward a mature muscle phenotype cannot be achieved, their prospective use in stem cell therapy for the regeneration of both skeletal muscle becomes unrealistic [87]. A new class of vessel-associated fetal stem cells, termed mesoangioblasts, has been isolated [88]. These cells show profiles of gene expression similar to that reported for hematopoietic, neural, and embryonic stem cells [88]. Mesoangioblasts can differentiate into most mesoderm (but not other germ layer) cell types when exposed to certain cytokines or to differentiating cells [88]. Intra-arterial mesoangioblast delivery was effective in restoring expression of _-SG protein and of the other members of the dystrophin-glycoprotein complex in treated _-SG null mice [89]. Restoration of sarcoglycans expression was also associated to a marked reduction of the fibrosis and complete functional recovery of treated muscle. Interestingly, no immune reaction occurred against reconstituted fibers, even though low-titer serum antibodies to _-SG were detected in treated mice [89]. However, as the authors discussed [89], one of the limitations of using mesoangioblast in human diseases is that human mesoangioblasts have been isolated only from fetal vessels but not yet from the patient’s own vessels. In summary, clinical application of stem cells is still hampered by the small number of cells that can be isolated from any adult tissue. Once cells are in the culture
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dish, unknown extrinsic and intrinsic factors may control cell fate. Extensive culture of human adult stem cells may also subtly change their intrinsic properties and render them unfit for restoring injured or diseased tissues in patients. It is important to note that stem cells found in tissues such as skeletal muscle or fat may be nothing more than the descendants of circulating BM stem cells, which are far more prevalent and easier to harvest from patients.
Enhancing stem cell–mediated muscle regeneration Although in recent years considerable evidence has been accumulated regarding the pathophysiology of muscle disease, it is still an open question what molecular mechanisms regulate the phenotypic changes leading to the pathology of dystrophic skeletal muscle and to associated limitations in cell therapy. Despite problems in harvesting and maintaining adult stem cells in culture, small numbers of stem cells may act locally in a paracrine fashion, to induce the regeneration of injured or diseased organs. Increasing evidence suggests that stem cells, like metastatic tumor cells, use common chemo-attractive mechanisms that guide them to damaged zones, where they control inflammation and induce regeneration in surrounding tissues [73]. Circulating blood cells are also known to secrete survival molecules or other growth factors that promote local regeneration at sites of injury [1, 90]. Activated progenitor cells may also help repair damaged dystrophic tissue by dissolving scar tissue and reconstructing appropriate matrix scaffolds that provide niches for new cells to inhabit. This is well illustrated by the mechanism whereby expression of a transgene encoding a locally acting isoform of insulin-like growth factor 1 (mIGF-1), normally involved in muscle regeneration, enhances repair of skeletal muscle damage. Expression of the MLC/mIGF-1 cassette, delivered either as an inherited transgene [41] or somatically on an AAV vector [91], induces muscle hypertrophy and strength, and preserves regenerative capacity in senescent and dystrophic mice [41, 47, 91]. Increased recruitment of proliferating BM cells to injured mIGF-1 transgenic muscles was accompanied by elevated BM stem cell production in response to distal trauma [92]. These data implicate mIGF-1 as a powerful enhancer of the regeneration response, mediating the recruitment of BM cells to sites of tissue damage and augmenting local repair mechanisms. In addition, our results suggest that while stem cells represent an important determinant for tissue regeneration, a “qualified” environment is necessary to guarantee and achieve functional results.
Future prospects Although stem cell therapy has not yet solved the major problem related to cell transplantation, namely the capacity to survive and to improve muscle regeneration,
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recent studies are beginning to elucidate the signals and mechanisms whereby regenerating muscle recruits circulating cells to sites of injury or degeneration. These cells need not be stem cells as long as they maintain sufficient plasticity to participate in muscle repair, either by rebuilding the damaged tissue or by instructing resident precursors. Current advances in stem cell biology justify a cautious optimism, yet the presence of stem cells seems to be not sufficient to guarantee an efficient tissue regeneration and repair. Specific factors are required to trigger stem cells toward a specific lineage, to improve their survival, and to render them effective in contributing to tissue repair. Inflammation, resulting from tissue damage, is a functional necessity to activate the regenerative process. However, the persistence of inflammation alters the normal tissue metabolism, leading to exacerbation of disease. In this context the recruited stem cells entering a compromised environment are exposed to apoptotic/necrotic signals, which in turn can inhibit their potential to participate to tissue regeneration and repair. Research is now directed at identifying the optimal progenitor cell type for cell therapy, at enhancing the recruitment of the patient’s own reserves or transplanted cells to damaged muscle and to characterize factors that render the injured tissues qualitatively advantageous to improve stem cell-mediated regeneration and repair. Such cells could also be used to deliver genes that replace missing functions or counter degenerative processes, a promising avenue for designing future therapies of muscle wasting.
Acknowledgments Work in the authors’ laboratories has been supported by Italian Telethon, Ministero della Salute, ASI and MDA.
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expression after engraftment into cardiac and skeletal muscle. J Clin Invest 114: 1577–1585 Cossu G (2004) Fusion of bone marrow-derived stem cells with striated muscle may not be sufficient to activate muscle genes. J Clin Invest 114: 1540–1543 Cossu G, Bianco P (2003) Mesoangioblasts – vascular progenitors for extravascular mesodermal tissues. Curr Opin Genet Dev 13: 537–542 Sampaolesi M, Torrente Y, Innocenzi A, Tonlorenzi R, D’Antona G, Pellegrino MA, Barresi R, Bresolin N, De Angelis MG, Campbell KP et al (2003) Cell therapy of alphasarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts. Science 301: 487–492 Tidball JG (2005) Inflammatory processes in muscle injury and repair. Am J Physiol Regul Integr Comp Physiol 288: R345–353 Barton-Davis ER, Shoturma DI, Musaro A, Rosenthal N, Sweeney HL (1998) Viral mediated expression of insulin-like growth factor I blocks the aging-related loss of skeletal muscle function. Proc Natl Acad Sci USA 95: 15603–15607 Musaro A, Giacinti C, Borsellino G, Dobrowolny G, Pelosi L, Cairns L, Ottolenghi S, Cossu G, Bernardi G, Battistini L et al (2004) Stem cell-mediated muscle regeneration is enhanced by local isoform of insulin-like growth factor 1. Proc Natl Acad Sci USA 101: 1206–1210 Musarò A, Rosenthal N (2003) Attenuating muscle wasting: cell and gene therapy approaches. Current Genomics 4: 575–585 Wurmser AE, Gage FH (2002) Stem cells: cell fusion causes confusion. Nature 416: 485–487
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Gene transfer technology Karen F. Kozarsky Biopharmaceuticals CEDD, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA
Introduction The process of developing gene transfer technology as a therapeutic has led to the use of the tools of gene therapy for efficient gene transfer in animals. In vivo gene transfer can be used to generate models of disease, as well as to determine which gene targets may be therapeutic. In the latter case, overexpression of a gene or inhibition of its expression/activity can be used for target validation, i.e., to determine what targets may be suitable for development of a therapeutic. The therapeutic molecule may be a small molecular weight compound or a biopharmaceutical, such as protein therapeutics, monoclonal antibodies, or gene therapy. Why use in vivo gene transfer to study inflammation? Administration of purified proteins can be useful in animal models of disease; however, there are limitations due to the half-life of the protein relative to the frequency with which it can be administered, and the need for relatively large amounts of purified protein which retains its native activity. An alternative is to use gene transfer or to generate a transgenic model so there is a constant source of protein production in the body. The protein produced is likely to be in an active conformation, since it is being synthesized in vivo. Because gene transfer vectors, in contrast to transgenic or knockout technology, are typically delivered to somatic cells of the body and not to germ cells, in vivo gene transfer is often referred to as somatic gene transfer. Some of the advantages of in vivo gene transfer over transgenic models include a faster generation of animal models compared to a conventional transgenic or knockout mouse. For example, once a cDNA has been cloned, a recombinant virus can usually be generated and administered in vivo within 3 months. In contrast, generating a transgenic mouse involves microinjection of an embryo; generating a knockout mouse involves gene transfer into embryonic stem cells by site-specific integration, and disruption of the target gene. Establishment of a line of these mice typically takes a minimum of 6 months to 1 year. An additional benefit of somatic gene transfer is that the same vector used for gene transfer can usually be used in a wide variety of strains without the need for
In Vivo Models of Inflammation, Vol. I, edited by Christopher S. Stevenson, Lisa A. Marshall and Douglas W. Morgan © 2006 Birkhäuser Verlag Basel/Switzerland
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cross-breeding (as with transgenics) and in a wide variety of species without further modification. Recombinant adenovirus and adeno-associated virus (AAV), for example, typically work with similar efficiency in mice, rats, rabbits, dogs, and nonhuman primates. Some transgenic and knockout mouse strains have proved to be impossible to generate and maintain as a result of embryonic or neonatal lethality due to either overexpression or the absence of target gene expression. This problem can be avoided by the use of somatic gene transfer in newborn or adult animals. These animals may also be good models for acquired disease, as the disease state can be induced at any time with gene transfer. In addition, each animal can serve as its own control, such as when genes affecting cholesterol metabolism are introduced. In this case, the animal’s baseline cholesterol levels and can be measured immediately before and after introduction of the gene transfer vector, to determine how transgene expression affects lipid metabolism in that animal. In this chapter, the most common methods for somatic gene transfer are described, along with their advantages and disadvantages, and how they can be used to validate targets in inflammation pathways as well as to develop animal models of inflammatory disease. Technologies are evaluated with regard to in vivo models, rather than human therapeutics, with a focus on models of joint disease.
Principles of gene transfer Gene transfer involves the introduction of foreign DNA sequences into a host cell. Because DNA is a large, highly charged particle, introduction of DNA into a cell can be inefficient. The mechanisms for introducing DNA into a cell include the use of liposomes to facilitate the entry of DNA into the cell, and the use of viruses, which have naturally evolved to efficiently deliver their RNA or DNA-based genomes into cells. Viruses used for somatic gene transfer include retrovirus, lentivirus, adenovirus, AAV, and herpes simplex virus. The criteria for choosing a viral vector depend primarily on the tissue and cell type in which gene expression is desired, and the duration of transgene expression (i.e., transient vs. long-term). As described below, different viral vectors are needed to efficiently deliver their transgene to, or transduce, different cell types [1]. The payloads that are delivered are most typically an expression cassette containing a promoter, the cDNA of interest, and a polyadenylation site for the resulting mRNA. This self-contained expression cassette will drive expression of the desired protein, resulting in either overexpression of the protein in a tissue, replacement of a missing protein, or ectopic protein expression (i.e., in a tissue in which it is not normally expressed). For instance, expression of a secreted protein can be accomplished either by gene transfer to the tissue in which it is normally expressed, or may be accomplished even more effectively by ectopic gene transfer to a different tissue such as the liver [1]. Other payloads may modulate gene expression, such
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Table 1 - Vectors for gene transfer in animal models Advantages Plasmid DNA Simple to engineer Safe Accepts any size DNA Retrovirus Stable integration of DNA Few viral sequences
Disadvantages
Tissue tropism
Inefficient Expression is usually transient Transfects only dividing cells High expression only with ex vivo transduction Limited cloning capacity Can cause insertional mutagenesis Limited cloning capacity In some tissues, less efficient than other viral vectors Can cause insertional mutagenesis Expression usually transient Usually causes local inflammation, is immunogenic Limited cloning capacity
Depends on site of physical delivery of the DNA Dividing cells, including hematopoietic cells and cancer cells
Lentivirus
Transfects dividing and non-dividing cells Transfects hematopoietic stem cells
Adenovirus
Easy to grow to high titer Transfects dividing and non-dividing cells
Adenoassociated virus
Easy to grow to high titer Transfects dividing and non-dividing cells Transfects cells of Complex system neuronal origin efficiently Can cause inflammation Accepts large amounts of DNA
Herpes simplex virus
Depends on the envelope glycoprotein used (pseudotyping)
Most tissues except hematopoietic cells
Many tissues, including liver, lung, brain, muscle, heart Neurological tissues
as a dominant negative mutant of a protein to inhibit protein activity, delivery of antisense RNA, ribozymes, or inhibitory RNA (RNAi, usually in the form of short hairpin RNA encoded by double-stranded DNA), all of which inhibit protein expression by causing degradation of the target RNA or inhibition of translation of the RNA [2, 3]. For these RNA-targeting mechanisms, it is essential to transduce the cell types which are expressing the desired RNA/protein. The ability of gene transfer vectors to target different tissues is summarized in Table 1.
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Non-viral methods for gene transfer These methods use plasmid DNA only, rather than viral vectors, to deliver the gene of interest. Because of the low efficiency with which naked DNA can enter a cell and be delivered to the nucleus intact, both physical and chemical methods are used to enhance DNA penetrance into a cell. Chemical methods include complexing of DNA with lipids into liposomes, typically for localized delivery, such as to the lung or muscle [4]. Physical methods include electroporation, which has been used in vivo to enhance uptake into skin and into muscle, and a gene gun method in which a tissue site such as the skin is bombarded with tiny gold particles coated with DNA. In hydrodynamic injection, a mouse is injected systemically with DNA in a large volume of buffer rapidly; the transient increase in pressure enhances transfection of the liver. In all of these cases, gene expression is quite transient, so these techniques are most useful when up to a week of gene expression is adequate for the animal model [4]. Recently, a technique has been described whereby plasmid DNA can give relatively high-level and stable gene expression. This is based on the hypothesis that the bacterial DNA sequences in a plasmid lead to silencing of gene expression from the plasmid. DNA fragments containing only the mammalian expression cassette were isolated from the original plasmids, so that they contained no bacterially derived sequences. When these were injected into mice using the hydrodynamic injection method, gene expression of the soluble proteins Factor IX or alpha-1 antitrypsin persisted for at least 9 months [5–7]. So far, this method has been only shown to be useful for liverdirected gene transfer; however, it is likely to be useful in other somatic tissues.
In vivo gene transfer with plasmid DNA: Arthritis models For gene delivery to the joint using methods where in vivo gene transfer may not be efficient, chondrocytes can be transfected ex vivo and reintroduced into the damaged joint. In a study using plasmid DNA complexed with lipids, rabbit articular chondrocytes were transfected with liposomes, encapsulated in alginate spheres, and injected into rabbit knees into which a cartilage defect had been introduced. Gene expression peaked at approximately day 5, but was detectable up to day 32 following transfection [8]. A subsequent study using insulin-like growth factor 1 (IGF-1) cDNA demonstrated transgene expression up to day 36, and an accelerated rate of repair of the damaged cartilage [9].
Viral vectors for gene transfer Given the relatively inefficient rates of delivery of plasmid DNA to the nucleus of a cell, naturally occurring viruses have been harnessed as vehicles for efficient deliv-
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ery of their payload (DNA or RNA) to a cell. The life cycle of a virus typically includes entry into the cell, delivery of their genome to the nucleus, at which point the virus uses the host cell’s machinery to replicate its DNA and produce viral proteins to enable production of new virus particles. For in vivo gene transfer, however, only the first part of the virus life cycle is desired. Therefore, the recombinant viruses are rendered replication-defective, so that they can deliver their DNA or RNA to cells to enable expression of a transgene, but subsequently remain inactive rather than replicate. Three of the most common types of viruses used for in vivo gene transfer are described below.
Retrovirus/lentivirus Retroviruses (such as murine leukemia virus) and the retroviral subclass of lentiviruses (such as HIV) are enveloped viruses which carry an RNA genome. Retroviruses have simpler genomes, containing only three open reading frames, encoding gag (structural proteins), pol (reverse transcriptase) and env (envelope glycoprotein). Retroviruses bind to cellular receptors through the envelope glycoprotein; the virus particle enters the cell by receptor-mediated endocytosis. The envelope is removed, and the viral RNA genome is reverse transcribed into DNA by the pol enzyme. Following second-strand DNA synthesis, the viral-derived DNA can then integrate into the host cell genome. This is a critical step for retroviruses, since their genes will not be expressed unless the integration step occurs, and it can only occur in actively dividing cells. The viral DNA can then drive expression of gag, pol, and env, as well as expression of new viral genomes for packaging into new particles. These new particles bud off from the cell membrane. When a recombinant retrovirus is generated, it is deleted of the gag, pol, and env genes, and instead carries an expression cassette encoding the transgene of interest. Thus, after integration of DNA into the genome, no viral replication occurs, just transgene expression. It should be noted that an advantage of retroviruses in transducing dividing cells is that due to the integration process, the viral genome will be maintained in all daughter cells. Lentiviruses have genomes similar to those of retroviruses, but with several additional open reading frames encoding proteins which are involved in virus replication and persistence. A critical difference affecting the utility of these vectors in vivo is that lentiviruses do not require the host cell to be dividing for the lentiviral genome to integrate and drive gene expression. The tissue tropism of these vectors is defined by several criteria. First, and arguably, most important, is the distribution of the cellular receptors for the virus itself. Additional steps which can impact the ability of the virus to successfully transduce the target cell are the intracellular trafficking of the virus, which has to not only escape the endosome to enter the cytoplasm, but also enter the nucleus itself, and whether or not the target cell is dividing. For receptor entry, the tissue tropism
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can be manipulated with the retroviral and lentiviral systems. A producer cell line can be generated in which the viral env gene is replaced with a different envelope glycoprotein, generating a pseudotyped virus. Such glycoproteins can include those from other retroviruses, or viruses such as vesicular stomatitis virus (VSV), Ebola, or rabies. These pseudotyped viruses have been characterized to identify viruses that efficiently transducer pancreatic islet cells [10] and airway epithelia in vitro and in vivo [11, 12]. Because recombinant retroviruses bud off of the membranes of the host cell, they can be generated from stable cell lines. To make a recombinant retrovirus-producing cell, cells are stably transfected with DNA encoding the desired recombinant retroviral genome, along with helper plasmids encoding the needed genes (i.e., gag, env, and pol). These helper genes are expressed, so that the proteins can generate new virus particles. However, the only genes packaged into the virus particles are in the expression cassette, due to the genetic elements flanking it: the viral long-terminal repeats (LTRs) on each end, and a packaging signal inside the LTRs. When a stably transfected cell line is selected and grown, the recombinant retrovirus particles are secreted into the media. After the medium is harvested, it can be used directly for ex vivo transfection of cells in culture; alternatively, the virus particles can be purified by centrifugation or by column purification for in vivo administration [13].
In vivo gene transfer with recombinant retroviruses: Arthritis models Arthritis models in animals include both an immunization model (collagen-induced arthritis) and a physical model in which the cartilage of a weight-bearing joint such as the knee is surgically injured. Several studies have used recombinant retrovirus to deliver an anti-inflammatory gene such as interleukin-1 receptor antagonist (IL1Ra). In these protocols, chondrocytes or synovial cells are transduced ex vivo with a recombinant retrovirus encoding IL-1Ra, and then injected into the affected joint. Studies in rabbits [14], rats [15], and dogs [16, 17] have all demonstrated chondroprotective effects of the expression of IL-1Ra. To test the effects on human tissue, SCID mice were implanted with retrovirally transduced human synovial fibroblasts along with human cartilage; cartilage degradation was prevented when the fibroblasts expressed IL-1Ra as opposed to a marker gene [18].
Adenovirus Adenoviruses are non-enveloped viruses which contain a linear double-stranded DNA genome enclosed in a protein capsid. Adenoviruses have been isolated from many tissues, and the human adenoviruses include at least 50 serotypes. The most commonly used serotypes for in vivo gene transfer are serotypes that primarily cause
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respiratory infections. Adenovirus binds to cells usually through two receptors: for adenovirus serotype 5, the primary receptor is the coxsackie-adenovirus receptor (CAR), with an integrin serving as a co-receptor required for the virus to be internalized. One of the adenovirus proteins, designated penton, helps mediate endosomal lysis, allowing the adenovirus genome to enter the cytoplasm of the cell and eventually be transported into the nucleus. In the wild-type adenovirus life cycle, the adenovirus genes are expressed and the DNA replicated and packaged into new adenovirus particles that remain within the cell. Eventually, the host cell dies of attrition, and virus is released from the cell. For the recombinant adenovirus, the genome remains in the nucleus without replicating, and is primarily retained in an episomal (i.e., non-integrated) form. Similar to retrovirus, the tissue tropism of adenovirus is determined primarily by the expression of the appropriate receptors on the cell surface, and by the route of administration. For example, if injected intratracheally, the virus will primarily transduce the lung, while if injected systemically, the virus will primarily target the liver [19]. Recombinant adenoviruses can transduce most somatic cell types, whether dividing or non-dividing, except for many cells of hematopoietic origin, such as mature B and T cells [1]. Importantly, because recombinant adenovirus DNA usually does not integrate into the host cell genome, if cells that are actively dividing are transduced with recombinant adenovirus, the DNA will eventually be lost by dilution. The adenovirus genome is fairly complex, containing 36 kb of DNA, and contains many open reading frames, which are grouped into early genes (expressed before the onset of adenoviral DNA replication) and late genes (expressed after the onset of DNA replication). In general, the early genes control viral gene expression and replication, while the late genes encode primarily structural proteins of the virus. To render an adenovirus replication defective, the early 1 (E1) region DNA is deleted, since the E1 region encodes genes which turn on expression of other adenoviral transcription units. In first-generation adenoviral vectors, the expression cassette containing the transgene of interest is inserted into the deleted E1 region of the viral genome. This recombinant adenoviral DNA is then transfected into a cell line such as 293, which contains the E1 region genes, thus providing the proteins needed for the recombinant virus to replicate. Because adenovirus is not a lytic virus, the virus is usually isolated by harvesting the cells, rather than the medium. The cells are then lysed, and the virus can be propagated by serial infection of 293 cells. Once produced on a larger scale, recombinant virus is usually purified by cesium chloride gradient centrifugation or by column chromatography. The virus can usually be purified to a high titer, approximately 1 × 1013 particles/ml. As a result, it is possible to use fairly large amounts of virus in vivo and obtain high-level transgene expression [1]. The major disadvantage of first-generation adenoviral vectors is that they can cause local inflammation, and transgene expression is typically transient, although
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longer-lasting than with plasmid DNA. If the transgene product is immunogenic, gene expression may last in vivo up to only about 3 weeks, whereas if the transgene product is non-immunogenic, gene expression may last for months [20]. At least some of the inflammation is caused by low-level expression of adenoviral proteins in the host cell. To overcome this problem, second- and third-generation adenoviral vectors were generated that contained additional deletions in other early region genes. In addition, helper-dependent (or “gutted”) adenoviruses were generated that contain no adenoviral genes, but retain the inverted terminal repeats (ITRs) and packaging signal from the adenovirus genome. The problems with these viruses are primarily in generating them; titers can be lower, and with the helper-dependent adenoviruses, it is difficult to separate them from the wild-type virus needed to assist in replication. The advantage is that gene expression from these viruses is typically very stable [1].
In vivo gene transfer with recombinant adenovirus: Arthritis and asthma models Recombinant adenoviruses have been used extensively to determine whether specific proteins are pro-inflammatory or anti-inflammatory. Because recombinant adenoviruses themselves can cause local inflammation, it has been important to include virus controls, such as a recombinant adenovirus expressing an irrelevant transgene, or none at all. In arthritis models, first-generation adenoviruses have an adequate time course of gene expression to determine their effects on inflammation and joint destruction. For example, overexpression of IL-18 in an arthritis model increased inflammation and accelerated the rate of cartilage degradation, suggesting that inhibition of IL-18 may be beneficial in inflammatory arthritis. Using the same virus, it was shown that in an IL-1-deficient mouse, IL-18 did not promote cartilage destruction, although it still increased inflammation [21]. This indicated that IL-18 and IL1 can affect inflammation through separate pathways. Thus, a combination of in vivo gene transfer and different animal models can be used together to tease out pathways of inflammation. Recombinant adenovirus has also been used to generate new mouse models of inflammatory arthritis. Joints were injected with recombinant adenoviruses encoding oncostatin M along with IL-1 or tumor necrosis factor alpha (TNF-_) [22, 23]. In this model, the cytokines not only caused inflammation and loss of cartilage, but also bone destruction. In mouse models of asthma and airway hyperreactivity (AHR), recombinant adenoviruses have been used to identify proteins that can prevent or reverse inflammation. In vivo gene transfer of nuclear factor-kappa B (NF-gB) inhibitory protein ABIN-1 reduced eosinophil infiltration and reduced levels of inflammatory cytokines [24]. Interferon-gamma prevented AHR when delivered prophylactically
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via recombinant adenovirus, and even reversed established AHR when delivered after induction of AHR. These effects were shown to work through a pathway that includes IL-12 and signal transducer and activator of transcription 4 (STAT4) [25]. In a model in which AHR is exacerbated by adenovirus-mediated expression of GM-CSF, concomitant transduction with an adenovirus encoding IL-10 reduced inflammation and cytokine expression [26]. Interestingly, in contrast to the arthritis model, IL-18 inhibited disease, perhaps due to its ability to shift the immune response from a Th2- to a Th1-biased immune response [27].
Adeno-associated virus The parvovirus AAV, like adenovirus, is a non-enveloped virus, although smaller in size, and its genome consists of single-stranded DNA. AAVs were originally isolated in cultures associated with adenoviruses. Originally, only 6 human serotypes were identified, none of which have been associated with human disease. They are naturally replication defective, and require helper genes from another virus such as adenovirus or herpes simplex virus to replicate. As with the other viruses described here, AAV must bind to a cell-surface receptor to enter the cell. For the serotypes for which receptors are known, different receptors have been identified. AAV2 binds to heparan sulfate proteoglycan [28] and uses a co-receptor [29, 30], while AAV4 and AAV5 bind to sialic acid-containing proteins [31]. When the AAV genome reaches the nucleus, the single-stranded DNA must be converted to double-stranded DNA for transgene expression to occur. The inverted terminal repeats at each end of the genome form a hairpin structure that can act as a primer for DNA replication. Most of the AAV DNA remains episomal, although some may integrate into the genome [32]. Tissue tropism is determined by several factors. As with the other viruses, one important step is that of cell entry, i.e., where the appropriate receptor is expressed. The rate of onset of gene expression as well as the level is also determined by the rate of conversion of the single-stranded AAV genome to a double-stranded DNA genome. As a result, transgene expression from AAV usually takes several weeks to reach a maximal level, as compared to adenovirus which reaches peak levels of gene expression within several days. However, expression from recombinant AAV, unlike most recombinant adenoviruses, is usually very stable, and can last for the lifetime of the transduced cell. Most tissues can be transduced with recombinant AAV, depending on the serotype used. Both the cellular entry and the conversion to double-stranded DNA are affected by the serotype of AAV. Recently, additional serotypes of AAV have been isolated from human and non-human primate tissues; some of which have very high levels of transduction as compared to AAV2. These newer serotypes have dramatically increased the utility of AAV as a vector for in vivo gene transfer [33, 34].
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The AAV genome is relatively simple, with only two genes: rep, which encodes the proteins needed for replication, and cap, which encodes the proteins that make up the capsid of AAV. To generate recombinant AAV, three components need to be introduced into cells; usually a co-transfection system of three different plasmids is used. The components include the recombinant AAV genome, containing the ITRs and the expression cassette; the AAV “trans” plasmid which contains the rep and cap genes without the ITRs; and a “helper” plasmid containing several genes from adenovirus which provide the help for AAV replication. Using this helper plasmid, rather than providing the genes by adenovirus infection, only AAV virus particles are produced. As with adenovirus, AAV is purified from harvested cells by either cesium chloride centrifugation or by column chromatography [1, 35].
In vivo gene transfer with recombinant AAV: Arthritis and asthma models Recombinant AAV can confer an advantage over recombinant adenovirus in that AAV administration usually does not cause inflammation. In addition, the longer duration of transgene expression can be useful in many disease models. Initially, mouse joints were injected with recombinant AAVs to determine the duration of transgene expression as well as efficacy. Expression was observed for at least 7 months with no detectable local inflammation. In the collagen-induced arthritis model, AAV-mediated expression of IL-4 in the joint resulted in decreased cartilage destruction [36]. In a separate study, AAV encoding the anti-angiogenic protein angiopoietin was injected into the knee joints in a mouse model of collagen-induced arthritis. The local expression of angiopoietin resulted in diminished inflammation as well as a reduction in the number of blood vessels in the arthritic joint [37]. In an AHR model, AAV was used to express a mutant IL-4 protein, IL-4Ra. IL4Ra blocks the action of both IL-4 and IL-13, which play an important role in the pathogenesis of asthma. AAV was introduced into a mouse model of AHR either by systemic administration (for systemic production of IL-4Ra) or by intratracheal administration. In both cases, IL-4Ra expression resulted in reduced eosinophilia, and reduced cytokine levels and AHR [38].
Conclusion In vivo gene transfer is a useful tool for generating animal models of disease, and for validating pathways that are involved in disease. The understanding that is gained through this technology can provide the information needed to develop new therapeutics specifically targeted at the pathways of interest. The choice of gene transfer vector will depend on the tissue and cell types to be transduced, the levels of gene expression needed, and the duration of gene expression. Recent
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advances in generating recombinant viral vectors, and the identification of new serotypes with different tropism, allow for the generation of a wide range of animal models.
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dana M (1999) Interleukin-10 gene transfer to the airway regulates allergic mucosal sensitization in mice. Am J Respir Cell Mol Biol 21: 586–596 Walter DM, Wong CP, DeKruyff RH, Berry GJ, Levy S, Umetsu DT (2001) IL-18 gene transfer by adenovirus prevents the development of and reverses established allergeninduced airway hyperreactivity. J Immunol 166: 6392–6398 Summerford C, Samulski RJ (1998) Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions. J Virol 72: 1438–1445 Summerford C, Bartlett JS, Samulski RJ (1999) Alpha V beta 5 integrin: a co-receptor for adeno-associated virus type 2 infection. Nat Med 5: 78–82 Qing K, Mah C, Hansen J, Zhou SZ, Dwarki V, Srivastava A (1999) Human fibroblast growth factor receptor 1 is a co-receptor for infection by adeno-associated virus 2. Nat Med 5: 71–77 Kaludov N, Brown KE, Walters RW, Zabner J, Chiorini JA (2001) Adeno-associated virus serotype 4 (AAV4) and AAV5 both require sialic acid binding for hemagglutination and efficient transduction but differ in sialic acid linkage specificity. J Virol 75: 6884–6893 Nakai H, Wu XL, Fuess S, Storm TA, Munroe D, Montini E, Burgess SM, Grompe M, Kay MA (2005) Large-scale molecular characterization of adeno-associated virus vector integration in mouse liver. J Virol 79: 3606–3614 Choi VW, McCarty DM, Samulski RJ (2005) AAV hybrid serotypes: Improved vectors for gene delivery. Curr Gene Ther 5: 299–310 Gao GP, Vandenberghe LH, Wilson JM (2005) New recombinant serotypes of AAV vectors. Curr Gene Ther 5: 285–297 Monahan PE, Samulski RJ (2000) Adeno-associated virus vectors for gene therapy: more pros than cons? Mol Med Today 6: 433–440 Watanabe S, Imagawa T, Boivin GP, Gao GP, Wilson JM, Hirsch R (2000) Adeno-associated virus mediates long-term gene transfer and delivery of chondroprotective IL-4 to murine synovium. Mol Ther 2: 147 Takahashi H, Kato K, Miyake K, Hirai Y, Yoshino S, Shimada I (2005) Adeno-associated virus vector-mediated anti-angiogenic gene therapy for collagen-induced arthritis in mice. Clin Exp Rheumatol 23: 455–461 Zavorotinskaya T, Tomkinson A, Murphy JE (2003) Treatment of experimental asthma by long-term gene therapy directed against IL-4 and IL-13. Mol Ther 7: 155–162
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Transgenics Matthias Müller1 and Nicole Avitahl-Curtis2 1Novartis
Institute of Biomedical Research, 4002 Basel, Switzerland; 2Novartis Institute for Biomedical Research, 100 Technology Square, Cambridge, MA, USA
Introduction Over the past decade, biological tools have been developed that allow the manipulation of genes in whole organisms in a well-defined manner. The completion of the sequencing of both the human genome and the mouse genome will help to select candidate genes that are involved in multifactorial diseases. Following identification of such genes, decisions can be made on which models are appropriate for further study. Knockout and transgenic mouse or rat models have resulted in significant advances in our understanding of the function of many of these genes and their roles in the pathophysiology of disease. Transgenic and knockout technologies, developed during the past two decades, allow gene function studies in the mouse that are not possible in most other organisms. Null mutations, as well as subtle missense or gainof-function mutations, can be introduced into virtually any gene in the mouse germ line using homologous-recombination-based, gene-targeting technology. In addition, the conditional or even inducible inactivation of gene expression in vivo can be used to inactivate a gene in only a subset of cells and/or at well-defined stages of development. Phenotypes of these mice not only unmask the role of individual genes in the pathophysiology of disease, but also provide further insights into the critical nature of a given gene in normal homeostasis. Indeed, a retrospective analysis of knockout phenotypes for targets of best selling drugs exemplifies the predictive power of transgenic technology in validating therapeutic targets [1]. Dysregulated immune function, which leads to inflammatory disease, is one of the most-well-studied biological disciplines in mice. Decades of intense effort to replicate human inflammatory diseases in mice have led to standardized immune manipulation methodologies. Application of these techniques in genetically altered mice is providing important clues to the biology of inflammation. This chapter focuses on new transgenic mouse technologies developed during the past years. They will help to understand the roles of specific genes in the pathophysiology of major inflammatory diseases. Genetically engineered mice with tar-
In Vivo Models of Inflammation, Vol. I, edited by Christopher S. Stevenson, Lisa A. Marshall and Douglas W. Morgan © 2006 Birkhäuser Verlag Basel/Switzerland
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geted deletion or overexpression of specific genes are among the most important tools of target validation in the modern drug discovery process.
Background/literature summary Pronuclear microinjection The introduction of foreign DNA (transgenes) into the genome of laboratory animals allows for in vivo studies of gene expression and protein function [2]. These genes are usually inserted by microinjection of purified DNA into the pronucleus of the fertilized egg. When inserted in this way, the new genetic material becomes integrated into the chromosomal DNA of the host organism, and is transmitted as a heritable trait to succeeding generations of progeny. These “transgenes”, which may be derived from any species (e.g., human genes in transgenic mice) can be efficiently expressed by tissue-specific promoters and enhancers. Normally, the mouse is the species of choice for most investigators due to the relatively low cost and the extensive research that has been done in mice, resulting in extensive knowledge of the genetics and physiology of various strains. In some cases, however, investigators prefer to use rats, rabbits, chickens, goats, sheep or other animals, due to influencing factors such as larger body size or because of greater similarities to humans in terms of physiology and disease pathology. Pronuclear microinjection has the advantage of being fast (in mice models about 3 months to get the first founder animal), and normally the transgene can be introduced into a pure mouse background, eliminating the problems which arise with mixed genetic backgrounds [3]. Challenges of this technology are that the transgene integrates into the genome: (1) in an uncontrolled way and (2) in multiple copy numbers, resulting in an unexpected expression pattern and level of the transgene. Such transgenic lines have to be carefully analyzed and good experimental practices means working thereafter with two independent lines to exclude an integration site effects on neighboring genes. To overcome this limitation, transgenes can also be integrated by homologous recombination into a defined locus such as the Rosa 26, LC1 or HPRT locus [4]. Such a controlled integration should result in a more predictable expression level and pattern. On the other hand, you can not work with transgenic lines with different expression levels which you normally get with classical pronuclear injections. Different phenotypes correlating with the different expression levels of course can improve the interpretation of a transgenic experiment. There are many cases in which transgenic technology has allowed the creation of novel rodent models for studying human disease, which have substituted for models that previously existed only in larger animals. Perhaps the best example of an emerging transgenic model for a disease, which could previously only be studied in larger species, relates to Alzheimer's disease (AD). This disorder has no known
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counterpart in rodents. However, lesions reminiscent of AD occur in older primates and long-term maintenance of primates has been necessary for studies of AD. Now, several laboratories have shown that overexpression of amyloid precursor protein (APP) as a transgene produces a disorder highly similar to AD. These mice offer enormous promise for the study of the etiology and pathogenesis of AD, and could replace the primate model.
Homologous recombination The “loss-of-function” approach for studying gene function in mice has been very successful in recent years. The most commonly used “loss-of-function” approach is to study the consequence of eliminating a gene in the mouse. This technique, termed gene “knockout”, has been well established in the mouse. Analysis of the “knockout” mouse phenotype often leads to the discovery of the function of this gene in a biological process. In addition, it offers a genetic tool useful in the study of complex genetic pathways during development or disease processes. The first step in creating a knockout mouse is the preparation of the knockout embryonic stem (ES) cell line. This process utilizes homologous recombination to target and replace the endogenous gene with a modified nonfunctional copy. These targeted ES cells are injected into the blastocysts of donor mice. The injected blastocysts are then transferred to the uterus of a pseudo-pregnant foster mouse. The injected ES cells aggregate with the ES cells of the inner cell mass of the donor blastocyst, thus resulting in a chimeric pup, with both the injected and donor cells contributing to the structure of the mouse. Chimeric mice are easily identified by coat color, as the chimeric mouse will have bands of fur displaying the colors of both the background donor and ES cells. In addition, the development of phage-based homologous recombination systems has greatly simplified the generation of transgenic and knockout constructs, making it possible to engineer large segments of genomic DNA, such as bacterial artificial chromosomes (BACs) in Escherichia coli [5]. It offers exciting new opportunities for creating mouse models of human diseases [6]. Inactivation of a gene often does not reveal its complete spectrum of functions – especially if the deletion results in embryonic lethality. To circumvent this problem, many new tools and novel applications of classic techniques have been developed to place spatial and temporal restrictions on the genomic alterations.
Conditional gene targeting Conditional gene targeting is a variation of knockout technology very successfully used for gene validation in drug discovery and research applications [7]. The most
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popular concept of a conditional gene knockout is the strategy that combines homologous and site-specific recombination. Site-specific recombinases are enzymes that recognize specific DNA sites and recombine the DNA between them. Many have been identified; however, two of them, the Cre recombinase of the P1 bacteriophage [8] and the FLP recombinase of yeast [9], have been the choice for experiments in mammalian systems because they require only a specific, short (34-bp) consensus recognition site (loxP and FRT sites, respectively) to catalyze recombination. If two recombinase sites are placed in the same orientation in cis, recombinase excision will result in deletion of the recombinase-sites-flanked region of DNA [7]. Therefore, the general strategy for conditional knockouts is to place two recognition sites for a site-specific recombinase flanking an essential exon, in such a way that these small insertions of sequence do not alter the gene function. Thus, completely normal mice carrying this altered allele in homozygous form can be established. If a transgene expressing the recombinase under the control of a tissue/cell type-specific promoter is introduced into this homozygous animal, it will remove the essential part of the gene in the lineage of specificity, rendering it nonfunctional. There exists a well-developed and validated “Recombinase Zoo” (a good overview is presented on homepage http://www.mshri.on.ca/nagy/) to modify selected mouse genes in a temporally and spatially controlled way. This technology makes it possible to inactivate or overexpress a given gene in selected tissues of the adult mouse.
Inducible transgenic systems One of the most powerful tools in transgenesis is the ability to turn genes on and off at one’s discretion. In the mouse, this has been accomplished by using binary systems in which gene expression is dependent on the interaction of two components, resulting in either transcriptional transactivation or DNA recombination. During the last few years, these systems have been used to analyze complex and multistaged biological processes. Traditionally, tissue-specific promoters had been used to directly express transgenes in the target tissue. These studies, for example in lung, provided impressive insights into the chronic respiratory effector functions of inflammatory mediators and the pathogenesis of asthma and pulmonary fibrosis [10, 11]. In these systems, the transgene is activated in utero and expressed in a constitutive fashion thereafter. As a result, these modeling systems are unable to differentiate between effects on development versus effects on function of the fully differentiated tissue. Alternatively, genetic compensatory mechanisms may mask the effect of the transgene. Finally, this method cannot be used to study genes whose products are toxic in early life. To deal with the limitations inherent in standard overexpression modeling, a number of investigators have established transgenic systems in which the expression of the transgene can be externally regulated. Although a variety of approaches have
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Figure 1 The tissue-specific expression of a fusion gene that encodes Cre and a mutated ligand-binding domain (LBD) from the estrogen receptor (ER) results in a chimeric protein that sequestered in the cytoplasm by binding to heat-shock proteins 90 (HSP90). In the presence of the inducer 4-OH tamoxifen, the chimeric protein can translocate from the cytoplasm to the nucleus and results in recombination of a loxP flanked target gene.
been utilized, tetracycline-controlled expression systems have been employed most frequently [12, 13]. Recently there is a trend to work with inducible Cre fusion proteins, in which Cre recombinase is fused to a mutated ligand-binding domain of the estrogen receptor (Fig. 1). This fusion protein is insensitive to endogenous `-estradiol but responsive to the synthetic estrogen antagonist 4-OH tamoxifen [14]. Because a robust inducible system is still not clearly established, a lot of other inducible systems are being developed [15].
Recombinase-mediated cassette exchange (RMCE) To ensure a transgene is expressed in the desired fashion, a commonly utilized technique is to insert a transgene into an endogenously expressed mouse locus by homologous recombination. However, this process is quite labor intensive. Alternatively, a DNA construct can be integrated into a specific well-defined tagged locus via site-specific recombinases, such as Cre and/or Flp. For such a strategy, an endogenous locus has first to be manipulated by integration of the recognition sides for the recombinase. Fundamental to such a strategy is the finding that Cre and Flp
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Figure 2 RMCE exploits the fact that recombination is efficiently mediated only between pairs of homotypic, but not heterotypic target sites that vary in spacer sequence. (A) The sequences of the wild-type loxP and mutated lox511 sites are listed. The point mutation in the core region of the lox511 site is highlighted in bold type. (B) A selectable marker gene under the control of an endogenous promoter is flanked by wild-type loxP and the mutated lox511 sites. A plasmid containing the gene-of-interest flanked by similar lox sites is coinjected with either Cre protein or a plasmid encoding Cre. As the Cre protein mediates recombination between the loxP and lox511 sites flanking both the marker gene and gene of interest, the gene of interest replaces the marker gene. As a result, the gene of interest comes under transcriptional control of the endogenous promoter.
each tolerate certain variation in their target sequence, but effectively recombine only particular combinations of the alternative sides [16]. One strategy takes advantage of loxP and the heterospecific lox-mutant lox511 (Fig. 2). LoxP and lox511 recombine very inefficiently due to a point mutation at the cleavage site of lox 511,
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while two lox511 sites are fully recombinogenic. Therefore, an endogenous gene segment flanked by loxP and lox511 can be replaced in a directional manner with a targeting cassette equally flanked by loxP and lox511 (Fig. 2). Using this technique any cDNA can be placed into a specific gene locus in a fast and efficient way compared to conventional transgenics. This process works very efficiently in ES-cells, but also at a low frequency directly in oocytes [17]. Recombinase-mediated cassette exchange (RMCE) creates the possibility to produce transgenic mice with high efficiency and predictability. Moreover, since the transgene goes into the same locus, this method eliminates effects on expression level due to integration site. Furthermore, it offers the possibility to test several genes in the same genetic environment, for example to compare mutants in a fast and efficient way due to highly reproducible expression profiles through endogenous loci. RMCE can also be a strong system for screening unknown genes for their function. The time-consuming characterization of the transgenic line is no longer mandatory. The limitation of such screening projects might still be the relatively labor-intensive phenotypical analysis of the transgenic mice.
RNA interference RNA interference (RNAi) offers enormous promise for rapid analysis of gene function in mice. RNAi is a highly evolutionary conserved mechanism involved in posttranscriptional gene regulation. During the initial steps of RNAi, the RNase III-like enzyme Dicer processes long double-stranded RNAs and complex hairpin RNAs into small interfering RNAs (siRNAs). siRNAs are 21–23-bp RNA duplexes with characteristic dinucleotide overhangs. These duplexes are unwound by an RNA helicase, and single-stranded siRNAs are then incorporated into the multi-component RNA-induced silencing complex (RISC). RISC functions as an siRNA-induced endonuclease and mediates the cleavage of target RNA that is perfectly complementary to the siRNA [18]. Transgene-driven stable RNAi is generally achieved using RNA polymerase III-dependent promoters expressing short hairpin RNAs (shRNAs), which bear a fold-back stem-loop structure and can mediate silencing of target genes in mammalian cells. Several reports describe the stable, shRNA-mediated gene silencing in mice using random transgenesis [19]. Seibler et al. [20] demonstrate that a single copy shRNA transgene, under the control of either the U6 or the H1 promoter, mediates efficient and ubiquitous gene knockdown in mice when integrated at the rosa26 locus. By combining RMCE at the rosa26 locus and the tetraploid blastocyst complementation approach [21], they presented a real state of the art system. A great advantage of RNAi-mediated knockdown is that mice can be developed within 4–5 months, representing a significant shortening of the typical yearlong process by conventional knockout techniques. A further potential advantage
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for pharmaceutical research is that a knockdown of a gene should simulate the effect of a test compound better than a complete knockout. Thus, the method of shRNA in the mouse appears attractive for drug target validation and target identification.
Imaging technologies Optical imaging of live animals has also grown to be an important tool in biomedical research, as advances in photonic technology and reporter strategies have led to widespread exploration of biological processes in vivo [22]. In vivo optical imaging technology provides a “window” into the organism, and makes possible the tracking of biological activity in real-time, at the molecular or cellular level. The most popular in vivo imaging utilizes luciferase – the enzyme that makes some insects, jellyfish, and bacteria glow. When active, it leads to a reaction that emits light. A sensitive camera and software system captures this image and analyzes it. By measuring and analyzing the light emission, researchers can monitor cellular or genetic activity and use the results to track gene expression, the spread of a disease, or the effect of a new drug candidate in vivo. More sophisticated systems are already on the market, which can also follow the expression of the red fluorescent protein or even enhanced green fluorescent protein (EGFP) [23].
Experimental descriptions Transgenics and new disease models The use of transgenic technology has lead to the creation of new mouse models of autoimmune disorders. The “K/BxN” mouse which spontaneously develops arthritis was fortuitously discovered by crossing mice of C57BL/6 background transgenic for a T cell receptor specific for the bovine pancreatic ribonuclease with the nonobese diabetic (NOD) mouse strain. The resulting offspring spontaneously develop chronic and progressive joint inflammation beginning at 25–35 days of age. Joint histology reveals several features that are reminiscent of human rheumatoid arthritis, such as leukocyte infiltration, synoviocyte hyperplasia, pannus formation and cartilage destruction and bone remodeling [24, 25]. The fortuitous autoantigen in this model turned out to be glucose-6-phosphate isomerase [26]. While the spontaneous development of arthritis is dependent on the NOD background, serum from arthritic animals can be transferred to normal mice of several different strain backgrounds to induce arthritis, usually within 2–3 days of serum transfer [27]. This new model has thus turned out to be a useful complement or alternative to the widely
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used method of administering a cocktail of anti-collagen antibodies. Models for colitis have also resulted from either overexpression of proinflammtory cytokines or elimination of cytokines important for functions of regulatory T cells. An example of the former is the deletion of the AU-rich element in the gene encoding the proinflammatory cytokine TNF-_, which results in increased stability of the TNF-_ mRNA and increased levels of TNF-_ protein. The overexpression of TNF-_ in this manner produces a mucosal inflammation and pathology that resemble Crohn’s disease [28]. Examples of the elimination of regulatory cytokines are mice that are deficient either for IL-2 [29–31] or IL-10 [32, 33].
Using conditional knockouts to elucidate gene functions The role that a gene of interest plays in inflammation can be tested using mice deficient for that gene, i.e., knockout mice. For example, testing of mice deficient for the intercellular adhesion molecule ICAM in inflammation models (such as delayedtype hypersensitivity and thioglycollate-induced peritonitis) revealed that knockout animals had significantly reduced leukocyte infiltrates compared to wild-type animals, thus revealing a critical role for ICAM in leukocyte trafficking and inflammation [34]. Similarly, the complexity of the role of TNF-_ in autoimmune diseases was revealed by studying mice deficient for either TNF-_ or its receptor, TNFR [35–37]. In cases where knockout of the gene of interest results in embryonic lethality, the use of conditional knockout technology provides a powerful approach for gaining insight to the functions of such genes. An example of this is the sphingosine-1-phosphate receptor, S1P1. A constitutive knockout of this G-protein coupled receptor (GPCR) results in embryonic lethality due to deficient smooth muscle cell migration and endothelial cell maturation [38]. However, conditional knockout of S1P1 in T cells gives rise to viable mice and has allowed the function of S1P1 in T cells to be studied [39]. Analyses of conditional knockout mice together with bone marrow reconstitution studies [39, 40] have provided important insights into the mechanism of action of immunomodulatory compounds that interact with S1P1, such as FTY720 [41]. Specifically, S1P1 deficiency in T cells abrogated their capacity to migrate in response to S1P, and resulted in failure of the cells to exit from thymus and secondary lymphoid organs in a S1P-dependent manner [39, 40]. A similar phenotype was induced through down-modulation of S1P1 by the phosphorylated form of FTY720 [40]. Thus, the analysis of S1P1 conditional knockout mice facilitated the elucidation of S1P/S1P1-dependent lymphocyte egress. Moreover, these studies provided insights into the mechanism of action of FYY720, indicating that inhibition of S1P/S1P1-dependent lymphocyte egress could be an effective means to reduce recirculation of pathogenic effector T cells [42].
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Invasive and noninvasive methods to study inflammatory diseases An invasive method to detect the origin of cells Transplanted arteries develop concentric and generalized intimal thickening, socalled graft vessel disease (GVD). The mechanism leading to GVD is characterized by an early acute cellular rejection process, followed by a less intense chronic inflammation [43]. Concurrently, donor medial smooth muscle cells (SMCs) dedifferentiate and migrate to form the subintima [44]. Thus, it has been assumed that neointimal smooth muscle-like cells in GVD originate from graft tissue and therefore are donor derived. However, accumulating evidence indicates that host-derived cells play a key role in the neointima formation after transplantation [45]. To answer this question Matsumoto and colleagues used a transgenic BALB/c strain ubiquitously expressing EGFP as the recipient of wild-type C57BL/6 carotids [46]. By this approach they demonstrated directional infiltration of host leukocytes, which were EGFP positive, outwards from the graft lumen, demonstrating that indeed cells of the circulation are involved in the disease.
Observation of IL-4 cytokine production by EGFP expression Development or activation processes can be tracked by expressing a reporter construct from the endogenous promoter of a gene of interest. This is achieved by introducing, into the 3’ untranslated region of the gene of interest, a construct containing an internal ribosomal entry site (IRES) upstream of a reporter gene such as `geo or EGFP. Thus, the reporter is contained in the same transcript as the gene of interest, but it is translated from a separate initiation site, thus making it possible to have a reporter protein whose expression should mirror the gene of interest without being a fusion protein. Mohrs et al. [47] have used this approach to monitor IL-4 expression by introducing into the 3’ UTR of IL-4 an IRES EGFP construct. Reporter T cells primed under Th2 conditions showed that EGFP expression correlated well with IL-4 expression. After type 2 immune challenge (infecting transgenic mice with the intestinal helminth, Nippostrongylus brasiliensis), FACS sorting and analysis of EGFP-positive cells could be used to identify IL-4-producing cells, monitor the percentage of T cells undergoing Th2 differentiation, and assess the cell types producing IL-4 [47–49].
An indirect noninvasive method to detect endothelial cell activation One of the hallmarks of endothelial cell activation by inflammatory stimuli is the de novo expression of the adhesion molecule E-selectin. Since E-selectin is a trans-
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membrane protein, it can only be monitored in vivo in terminal experiments by immunohistochemistry. To overcome this limitation, Luyten et al. [50] developed a novel and noninvasive animal model in which the activity of a marker enzyme in plasma was used to measure endothelial cell activation. Secreted heat-stable human placental alkaline phosphatase (SeAP) was placed under the transcriptional control of the E-selectin promoter by targeted integration into the mouse Eselectin locus. To test the system animals were treated with lipopolysaccharide or tumor necrosis factor TNF-_ and indeed SeAP activity increased 7–11-fold in plasma. Enzyme histochemistry indicated an endothelial origin of the alkaline phosphatase activity. Administration of dexamethasone, an anti-inflammatory steroid, inhibited TNF-_-induced SeAP expression in transgenic mice. The model described here allows monitoring of the E-selectin expression in vivo, offering a noninvasive means to quantitate inflammatory endothelial cell activation and to evaluate therapeutic approaches designed to inhibit endothelial cell activation. This novel animal model has the potential for a first line in vivo screening system in research programs.
A direct noninvasive method for the detection of reporter constructs by in vivo bioluminescence imaging In addition to the ability to identify or track populations of cells by ex vivo analyses such as immunohistochemistry or FACS, reporter constructs can be used for in vivo imaging of cells or cell functions. This is achieved through the use of a CCD camera to visualize light emission from luciferase-expressing cells after systemic injection of the luciferin substrate. This method can be used to visualize in vivo the trafficking of inflammatory cells in disease models. For example, CD4+ T cells expressing a luciferase reporter and specific for the autoantigen collagen type II can be adoptively transferred into mice. Upon induction of experimental arthritis, the specific homing of these cells to inflamed joints could be visualized [51]. Similarly, adoptively transferred CD4+ T cells expressing a luciferase reporter and specific for the autoantigen myelin basic protein were visualized as localizing in the central nervous system upon induction of experimental autoimmune encephalomyelitis [52]. Through another strategy, sites of inflammation can be visualized using NF-gB activity as a readout. Transgenic reporter constructs containing an NF-gB-responsive element driving expression of luciferase are powerful for studying inflammation as well as pathways that converge on the activity of this important transcriptional regulator of proinflammatory genes [53–56]. The power of in vivo imaging lies in the ability to track disease processes in single animals over time. As this technology develops, various transgenic lines may be developed that should greatly enhance the ability to test specific mechanisms of action of therapeutic compounds in disease models.
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Conclusions Knockout and transgenic mouse technologies have become well-established target validation tools in drug discovery research. The use of these technologies to confirm genes of interest as key mediators of inflammatory diseases has provided important insights and greatly enabled the development of novel small molecule and antibody therapeutics. To date, a knockout exists for about 10% of all potential mouse genes described in the literature. Recently, the Knockout Mouse Project was launched by a scientist consortium with the ultimate goal of producing deletions in all genes of the mouse genome [57, 58]. The phenotypic analyses of these mouse lines will be performed by standardized protocols and should generate a vast database of information that can be used to develop further novel therapeutic strategies to gain insights into pathways. Crossing various lines of mutant mice to generate doubleand triple-knockout mice will aid in dissecting the functional overlap and complex interplay of genes in inflammation. It is also expected that as the genetics of disease becomes better-defined in humans, these discoveries will be applied to generating mouse models that may mirror human disease more closely, both in mechanism and pathology. Finally, technologies such as inducible/reversible gene expression and RNAi hold the promise of making it possible to fine-tune the timing and extent of reducing gene expression. With respect to drug development, significant (as opposed to complete) gene knockdown should more accurately model the effect that an inhibitory compound might have and provide clearer insights into efficacy and potential toxicities.
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Nanotechnology: Towards the detection and treatment of inflammatory diseases Sreekant Murthy1, Elisabeth Papazoglou2, Nandhakumar Kanagarajan1 and Narasim S. Murthy3 1Division
of Gastroenterology and Hepatology, Drexel University College of Medicine, Philadelphia, USA; 2School of Biomedical Engineering, Drexel University, Philadelphia, USA; 3Associated Radiologists, PA, 322 E. Antietam Street, Suite 106, Hagerstown, MD 21740, USA
Introduction Inflammatory diseases comprise of a whole list of conditions such as rheumatoid arthritis, multiple sclerosis, asthma and various other life-altering diseases including myocarditis. Inflammation begins as a defensive process in which our body is equipped to protect itself from harmful pathogens and chemicals. When this defense mechanism is uncontrolled, the integrity of tissue may be breached resulting in damage to the vital organs, nervous, musculoskeletal systems and blood vessels. This breach permits soluble mediators of inflammation to be produced at the site, which causes leukocytes, monocytes and lymphocytes to migrate towards the site and to activate the immune system in a dysregulated manner, effecting hyper-reactivity of lymphocytes eliciting Th1 and Th2 type immune responses. As result of this abnormal immune activation, the surrounding tissue expresses variety of enzymes, adhesion molecules, cytokines, proteins, lipid mediators, and growth factors that participate in tissue destruction and repair [1]. Sometimes this abnormal immune response attacks self, resulting in further tissue damage. Although inflammatory reactions on the skin and other open areas on the body can be superficially visualized, in many cases when vital organs are affected, clinicians await until overt signs and symptoms are presented, and by that time destruction of the tissue in those vital organs has already progressed. Thus, in many cases it remains difficult to clearly diagnose these diseases. New technologies are needed to speed the diagnostic processes and help the basic scientist and clinician in the initiation of targeted treatments and to follow up treatment responses. An important milestone in this process has been the advances made by researchers in biochemistry, immunology and drug discovery fields in the identification of molecular signatures of inflammation and cancer, using complicated and cumbersome wet laboratory techniques. The objective now is to exploit those initial
In Vivo Models of Inflammation, Vol. I, edited by Christopher S. Stevenson, Lisa A. Marshall and Douglas W. Morgan © 2006 Birkhäuser Verlag Basel/Switzerland
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accomplishments, combining them with available new technologies to identify the earliest signatures of inflammation and cancer. Such developments will allow us to provide immediate and specific intervention and monitor the progress before it cascades into chronic inflammation and malignancy. To fulfill this objective, it requires the development of technologies of 1–100 nm size, which display unique mechanical, electrical, chemical, and optical properties and assist in visualizing or interacting with receptors, cytoskeleton, specific organelles and nuclear components within the cells. It will be very rewarding when many of these technologies can migrate into monitoring the disease condition through non-invasive methods in vivo in a physically undisturbed state, thus minimizing the influence of artifacts induced by physical methods while securing biological samples. The integration of nanotechnology with biology and medicine has given birth to a new field of science called “Nanomedicine”. The ultimate goal of nanomedicine is to develop well-engineered nanotools for the prevention, diagnosis and treatment of many diseases. In the past decade, extraordinary growth in nanotechnology has brought us closer to being able to vividly visualize molecular and cellular structures. These technologies are beginning to assist us in our ability to differentiate between normal and abnormal cells and to detect and quantify minute amounts of signature molecules produced by these cells. Most of these represent real time measurements, relating to the dynamic relationship among structures in the damaged area and also to repair of damaged tissues. Novel pharmaceutical preparations have been developed to fabricate nanovehicles to deliver drugs, proteins and genes, contrast enhancement agents for imaging, and hyperthermia agents to kill cancer cells. Several of these inventions have already transitioned into basic medical research and clinical applications. Because of this, some social, ethical, legal and environmental issues have emerged. Thus, regulatory and educational strategy needs to be developed for the society to gain benefit from these discoveries. The focus of this chapter is to provide an overview of the state-of–the-art in nanotechnology with particular reference to detecting and treating inflammation and cancer at the earliest settings.
Nanotechnology for inflammation scientists Nanotechnology encompasses multiple scientific disciplines, which exploit materials and devices with functional co-assembled molecules or sensors that has been engineered at the nanometer scale typically ranging from 0.1 to 100 nm [2]. In medical fields, it offers a wide range of tools that can be used as drug delivery platforms [3], better contrast agents in imaging [4], chip-based bio-laboratories [5] and nanoscale probes [6] that are able to track cell movements and manipulate molecules. Multifunctional nanostructures can combine diagnostic and therapeutic modalities and target cellular events.
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The concept of molecular medicine to develop personalized treatments can be made possible with the information available with the developed nanotools. These devices, systems and functionalized structures contain unique properties as a result of their nanosize. For example, gold nanoparticles and carbon nanotubes possess different properties [7] at the micron scale. Semiconductor particles exemplified by quantum dots exhibit quantum confinement effects, hence they fluoresce at various wavelengths compared to the semiconductor particles at micron size which do not exhibit the same optical or magnetic properties [8]. Macromolecular structures such as dendrimers and liposomes at the nanoscale are also considered valid nanotools [9], while biological molecules of nanometer size in their native state such as DNA and monoclonal antibodies are not examples of “Nanotools”. Thus, nanometer size is of critical importance to the cell and the living organisms. Interestingly, fabricated nanoscale devices are of the same size as subcellular organelles. Some nanoscale structures are of the order of enzymes, receptors and key molecules within the cell membrane or cytoplasm. For example, the lipid bilayer surrounding cells is 6 nm and hemoglobin is 5 nm in size. By modifying the surface chemistry of these nanostructures, which permits covalent or ligand-receptor (lock-key-type) or electrostatic interaction with key molecules, we can identify biomolecules on the cell surface and in the cytoplasm. An advantage of this will be our ability to map the transport of those molecules from the cytoplasm across the cell membrane so we can understand the cellular behavior in health and disease. Nanoparticles smaller than 20 nm can pass through blood vessel walls [10], which opens opportunities of diagnostic imaging and targeted delivery of drugs when non-toxic nanoparticles are used. What is critical for scientists engaged in inflammation science and engineering is that these technologies should be applicable to detect or monitor: -
Host biochemical and immune responses Bacterial and viral pathogens interacting within the local immune system Effect of noxious chemicals and pharmaceutical agents Thrombosis Neurogenic inflammation Wound healing and remodeling Imaging Diagnosing and treating vulnerable plaques Drug delivery and therapeutics for local delivery and retention
Nanostructures and nanosystems Nanotechnology in the medical field offers a wide range of tools that can be used as drug delivery platforms, better contrast agents in imaging, chip-based biolabs and nanoscale probes able to track cell movements and manipulate molecules [10].
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Combination of these multifunctional nanostructures through cross-disciplinary interactions may further enhance our diagnostic and therapeutic capabilities and to monitor intra- and extracellular cellular events in inflammation and cancer. Existing and emerging technologies, which may impact on early detection of inflammation, prevention and early detection of cancer, include several diverse technological innovations. They are bio-mimicry self-assembling peptide systems, which serve as building blocks to produce nucleotides, peptides and phospholipids, which support cell proliferation and differentiation and give insights into protein-protein interactions [11]. Microchip drug release systems, micromachining hollow needles and two-dimensional needle arrays from single crystal silicon for painless drug infusion, intracellular injections, microsurgeries and needle-stick blood diagnosis form another group of tools [12, 13]. All of these inventions could one day lead to develop personalized treatments [14]. The creation, control and use of structures, devices and systems with a length scale of 1–100 nm is the domain of Nanotechnology. Macromolecular structures such as dendrimers and liposomes at the nanoscale are also considered valid nanotools [9, 15]. The application of various nanotools in various areas of medicine is depicted in Figure 1. This list in the figure is by no means exhaustive as nanotechnology is continuing to grow with new technologies emerging each day.
Nanopore technology Biomolecular nanopore detector technology was first developed to rapidly discriminate between nearly identical strands of DNA thereby replacing the tedious process of running billions of copies of DNA through sequencing machines and minimizing errors and saving time [16]. In this technology single molecules of DNA are drawn through pores that are 1–2 nm in size and serve as a sensitive detector. The detection system through its electronic signature process can sequence more than one base pair per millisecond. This technology has the potential to detect DNA polyploidy, and DNA mutations.
Nano self-assembling systems This field includes biomimetics encouraged to mimic nature and create biomolecular nanomachines to handle various biological problems. Many biological systems use self assembly to assemble various complex molecules and structures [17]. Numerous man-made self-assembling systems that mimic natural self assembly of molecules are created to snap together fundamental building blocks of complex polymer molecules structured easily, and inexpensively, on beads, tubes, wires, and flat supports, and in suspensions and liposomes. These assemblies can have geneti-
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Figure 1 The application of nanotools in various areas of medicine.
cally introduced bifunctionality so that nonspecific molecules are repelled from fusing with the cell membrane fusion layers. DNA, lipid bilayers, ATP synthase, peptides and protein foldings are target candidates for self assembly. Liposomes are an example of a human-made supramolecular structure.
Cantilevers Nanoscale cantilevers are about 50-mm-wide flexible diving board-like beams that can be coated with antibodies and DNA complementary to a specific protein or a gene. When molecules come in contact with these substrates coated on the surface of cantilevers, they bind to the substrate and make the cantilevers resonate or bend as a result of this binding event [18]. This bending deflection is proportional to the quantity of binding, thus making it a quantitative technique. Multiple cantilevers
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can be used simultaneously to differentiate between bound and unbound molecules. Likewise, multiple antibodies can be used in the same reaction set up to quantify several markers at a time. An important advantage of this technique is that there is no need to add fluorescent tags to detect and quantify the molecule. Any biological sample containing biomolecules of interest can be tested. Nanoscale cantilevers, constructed as part of a larger diagnostic device, can provide rapid and sensitive detection of inflammation and cancer-related molecules and to evaluate how various drugs bind to their targets at a concentration 20 times lower than clinical threshold.
Carbon nanotubes Carbon nanotubes, also known as “bucky tube” and “buckyballs” are a member of fullerene structural family potentially useful in a number of biological applications. They could be cylindrical (nanotubes), spherical (buckyballs) or branched (fullerenes). Nanotubes could be single-walled nanotubes (SWNT) or multi-walled nanotubes (MWNT). The usefulness of nanotubes in drug delivery and cancer therapy is accomplished through the transporting capabilities of carbon nanotubes via suitable functionalization chemistry and their intrinsic optical properties. Proper surface functionalization is necessary to make carbon nanotubes biocompatible. In their most recent applications, SWNTs have been used to transport DNA inside living cells [19]. Intracellular protein transport has also been accomplished [20], although they are suspected to cause severe immune responses. Most SWNTs have diameters close to 1 nm, with a tube length that can be many thousands of times larger. SWNTs with lengths up to the order of centimeters have been produced [21]. SWNTs are a very important class of carbon nanotubes because they exhibit important electrical properties not shared by the MWNT variants. On the other hand, MWNTs are fabricated as multiple concentric nanotubes precisely nested within one another for perfect linear or rotational bearing. The technology has now advanced into merging these MWNTs with magnetic nanomaterials like magnetite, which can be functionalized. Gadofullerenes offer the ability to concentrate more gadolinium at the site of disease, than traditional Gd-DTPA. This is the result of the shielding that the carbon structure provides and its ability to link more gadolinium per conjugate. Gadofullerenes also take advantage of the gadolinium-water interactions as the gadolinium is brought along the periphery of the structure and can maintain its interaction with water, which is the basis of traditional proton density magnetic resonance imagine (MRI). These properties lead to a greater signal, which can increase sensitivity to small lesions. Recently, the technology has been further improved by developing smart bionanotubes that could be manipulated to produce open or closed end nanotubes to
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encapsulate drugs or genes to deliver them in a particular location [22]. Thus, possibilities exist for using nanotubes to improve gene sensing, gene separation, drug delivery and detection of biomarkers to improve health care, protection against bioterrorism and other areas of molecular sensing.
Nanoparticles They are mostly spherical particles with specific properties that allow their detection, analysis and quantification. Fundamentally, they are nanoprobes where these particles are complexed with biomolecules, drugs and other reagents. They exhibit various physical and optical properties. For example, iron oxide nanoparticles exhibit super paramagnetic properties [23], and gold nanoparticles specific optical absorption properties depending on their size [24]. It is important to note that particles made from the same materials but of micron dimensions do not exhibit such unique optical or magnetic properties [8]. One can thus combine the immense surface to volume ratio of these nanostructures to deliver higher loads of compounds encapsulated or linked to their surface, while their presence can be measured due to their characteristic magnetic or optical properties.
Quantum dots Quantum dots (QDs) are tiny light-emitting particles on the nanometer scale. They are emerging as a new class of biological probes that could replace traditional organic dyes and fluorescent proteins. The fundamental benefit of using QDs is the high quantum yield and strong emission intensity. The emission spectrum of QDs is a function of the particle size, and hence by varying particle size QDs can emit from visible to infrared wavelengths [25]. They can be excited by UV light. Their broad excitation spectrum and narrow emission spectrum with little or no spectral overlap makes them attractive for imaging and resolving multiple species at the same time without complex optics and data acquisition systems. QDs offer higher signal to noise ratios compared to traditional fluorochromes. Their high sensitivity allows accurate detection even in the presence of strong autofluorescence signals encountered during in vivo imaging [26]. Their excellent resistance to photobleaching is particularly useful for long-term monitoring of biological phenomena, critical in live cell imaging and thick tissue specimens. Already QDs are finding increasing use in live cell imaging, by themselves or as fluorescence resonance energy transfer (FRET) donors combined with traditional fluorchromes, and in in vitro assays and live animal imaging for cancer and tumor diagnostics. Limitations of QDs can arise from the stability of the core shell structure. Most commercially available materials comprise a core of CdSe and a shell of ZnS. To
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render this inorganic structure hydrophilic, amphiphilic polymers are used to cap the shell layer and provide reactive sites for further linking to proteins. It is, therefore, the stability of this layer that controls the aggregation of QDs, as well as possible release of core materials (Cd ions) to their surroundings, which may result in toxicity [27]. This has limited the immediate clinical use of QDs, but has focused applications to animal testing and in vitro assay developments. Another reported problem is the blinking property of the QDs. QDs tends to blink at the single dot level and hence present some limitation in absolute fluorescence quantification. However, when used for imaging of biomarkers, this property does not have much of an effect as there are hundreds or thousands of QDs in a sample to allow proper averaging.
Paramagnetic iron oxide crystals Paramagnetic iron oxide nanoparticles are a new class of contrast agents that are finding increasing applications in the field of diagnostics and molecular imaging based on magnetic resonance (MR) [23]. Traditional MRI agents rely on the interaction of the proton density, i.e., water molecules and the magnetic properties of the tissue. These paramagnetic agents accelerate the rate of relaxation of protons in the longitudinal direction, resulting in bright images, and hence are highly dependent on water molecules. However, the super paramagnetic iron oxide nanoparticles, by the virtue of their nanoscale properties, disturb the magnetic field independently of their environment, and hence are not dependent on presence of water molecules. They are also called negative enhancers as they act as negative contrast agents and appear dark where they are sequestered. The traditional MR agents such as gadolinium–diethylenetriamine penta-acetic acid (DTPA) enhance the signal from the vascular compartments and are nonspecific, whereas the nanoparticle-based contrast agents impact the MR signal from tissues and cells. Iron oxide nanoparticles are classified into two types depending on their size: (1) superparamagnetic iron oxides (SPIOs) (50–500 nm), and (2) ultra-small super paramagnetic iron oxide (< 50 nm). The advantage of these contrast agents lies in their ability to get sequestered anywhere within a support matrix and still generate a contrast, whereas the traditional MR agents need water in their vicinity of generate contrast. These nanoparticles can be used for both passive and active targeting. Because of the small size of these particles, tissue macrophages readily take up these agents, and hence it is possible to image liver, spleen, lymph nodes, and lungs. In addition, it is also possible to functionalize these nanoparticles using a wide variety of ligands, antibodies, peptides, aptamers, drugs, etc., to achieve site-specific or biomarker-specific targeting. This is an added advantage since traditional paramagnetic formulations are difficult to conjugate to antibodies, and even when conjugated, owing to the small number of cellular receptors, the signal intensity is not sufficient for accu-
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rate imaging. Thus, as a result of their superparamagnetic properties, iron oxide nanoparticles have been used as contrast agent for imaging of cancer, brain inflammation, arthritis, and atherosclerotic plaques. Because of the small size, these iron oxide particles have been able to distinguish between the normal and tumor-bearing lymphatic nodes [28]. These nanoparticles may also distinguish very small metastases (less than 2 mm in diameter) within normal lymph nodes, a size well below the detection limit of the most sensitive imaging techniques such as positron-emission tomography (PET) available today. Using cells loaded with iron oxide nanoparticles, it has been shown that these particles are non toxic and are cleared from the cell after five to eight divisions. Lewin et al. [29] labeled stem cells with iron oxide particles using HIV TAT peptide and injected them systemically. The labeled stem cells homed on to the bone marrow, and the labeled stem cells did not cause any impairment. However, due to the small size of these particles, a long time is required (up to 24 h) to clear them from the organs and blood to reduce background signals. Thus, MRI using SPIOs may result in improved sensitivity and selectivity, and may assist diagnosis of tumors at the earliest stages of malignancy or metastasis.
Dendrimers Dendrimers are a new class of hyper-branched polymer macromolecules that radiate from a central core with structural symmetry. They can vary in shape, size, surface, flexibility and topography, enabling fabrication of functional nanoscale materials that would have unique properties [30, 31]. They may be useful for developing antiviral drugs, tissue repair scaffolds, and targeted carriers of chemotherapeutics. Certain dendrimers are now being used commercially as immuno-diagnostic agents and gene transfection vectors. Dendrimers complexed with gadolinium (III) ions (Gadomer–17) are being tested (Phase I clinical trial) for MRI angiography [32]. It is anticipated that many exciting developments will emerge from the use of dendrimers in the near future.
Nanosomes/polymersomes These are made up of phospholipid bilayers exhibiting multifunctioning characteristics. They facilitate encapsulating of various classes of drugs and diagnostic agents for their controlled delivery into cellular and therapeutic targets. Important drug delivery strategies utilizing these agents include polymersomes, hydrogel matrices, nanovesicles/nanofiber mats and biodegradables [33]. Both small and large molecules can be used. Biodegradeable polymersomes based on polyethylene oxide have been synthesized, and they may be used as a surface to anchor antibodies or other targeting molecules. Quite recently fluorescent materials have been embedded into
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these cell-like vesicles [34] to produce near-infrared emissive polymersomes that could be used to locate areas of inflammation and deliver a load of drug to inflammation sites. Interestingly, inflammation sites deeper than 1 cm could be imaged with this technique. Efforts are being made to target DNA complexes into hepatocytes and macrophages with the idea of enabling gene therapy and delivering genetically derived vaccines in a safe and efficacious manner. Polymeric micelles are useful as developing agents for a-scintigraphy, MRI, and computed tomography (CT) [35]. Liposomes can be injected intravenously and when they are modified with lipids that render their surface more hydrophilic, their circulation time in the bloodstream can be increased significantly. Another class of polymersomes, called polymer nanotubes, has been synthesized by directly pulling on the membrane of polymersomes using either optical tweezers or a micropipette [36]. These polymersomes are composed of amphiphilic diblock copolymers consisting of an aqueous core connected to the aqueous interior of the polymersome, which are less than 100 nm in diameter. They are unusually long (about 1 cm) and are stable enough to maintain their shape indefinitely. The pulled nanotubes are stabilized by subsequent chemical cross-linking. The aqueous core of the polymer nanotubes together with their robust character offer opportunities for nanofluidics and other applications in biotechnology, especially in the development of nanohyperdermic syringes [36].
In vitro diagnostics Development and use of analytical tools in diagnostic area possibly presents immediate benefits to the user. Many diagnostic tools have been developed to improve human health. The diagnostic detection methods involve measuring antibody or antigen-based complexes, enzyme-based reaction rates, and polymerase chain reactions using micro-electro-mechanical systems (MEMS) [37]. Other methods include whole-cell bacterial sensors and biosensors which utilize aptamers, which are biomimetic synthetic bioreceptors that can complex with proteins, nucleic acids and drugs. The signal processing in these systems may be optical, electrochemical, or mass-related, creating resonance and thermal detection. Some diagnostic methods utilize nanoparticles as nanoprobes where nanoparticles are interfaced with biological molecules such as antigens, antibodies or chemicals. The nanoparticles used in diagnostics include QDs, nanobarcodes, metallic nanobeads, silica, magnetic beads, carbon nanotubes, optical fibers and nanopores [37]. In antibody/antigen-based detection methods, for example, 1–2-nm-wide, boron-doped silicon wires laid down on a silicon grid can be coated with antigens to provide real time detection of antibodies. Antibody binding to immobilized antigen gives a measurable conductance change at antibody concentrations less than 10
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nM. Detection of single copies of multiple viruses has been accomplished via antibody-conjugated nanowire field effect transistors [38]. The dream of optical biopsy is closer to reality with antibody-functionalized semiconductor nanoparticles (QDs) detected by fluorescence microscopy. Multiplexed assays can be developed since the fluorescence emission of QDs is tunable by changing their size. Outstanding detection sensitivity of antibodies in whole blood (picogram per ml) has been obtained using gold nanoparticle conjugates [39]. For detection purposes classical tools can be used as well as nanobased methods, such as atomic force microscopy (AFM) and near-field scanning optical microscopy (NSOM), methods where quantum tunneling plays a key role in amplifying detection capability. Again, such phenomena are related to the nanometer distance between the instrument probe and the surface/specimen under examination. AFM is used to elucidate structures of biomolecules under physiological conditions [40], to determine antibody/antigen binding properties [41], to image the topology of viruses [42], and to image pathologies at the molecular scale [43].
Nanoarrays Researchers in academia and the pharmaceutical industries traditionally use bioassays, which are often cumbersome and riddled with errors. The recent explosive development in the field of microfluidics, biotechnology and functional genomics has resulted in the miniaturization of these bioanalytical assays to micron scales for routine and throughput screening [44]. These assays have been used for genomic and proteomics analysis, though their application to proteomics still requires refinement since replication of proteins as opposed to DNA is yet to be fully realized. Efforts are being made to improve miniature microarrays, which are still used for analyzing proteins. These include fabrication of AFM-based Dip-pen nanolithography (DPN), which can probe complex mixtures of proteins, reactions involving the protein features and antigens in complex solutions, and can aid the study of cellular adhesion at the submicrometer scale. Protein nanoarrays generated by Dip-pen nanolithography are emerging [45]. With further advances in miniaturization techniques like DPN, it will be possible to design nanoarrays that can detect biological entities on a single particle level in a time- and cost-efficient manner and also profiling of new diagnostic biomarkers at a detection level beyond our imagination.
Application of nanosystems and nanoparticles in inflammation and cancer The pharmaceutical industry, physicians and patients have long desired better pharmaceutical formulations to improve and extend the economic life of proprietary
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drugs, to reduce the costs of preparation and treatment, and reduce toxicity or even death. Nanotechnology has already made significant inroads into the problems of improving delivery of injectibles, oral formulations, drug device implants, and topical and transdermal delivery of drugs. More is expected from nanotechnology in improving the delivery of drugs to the brain, as many of the formulations aimed at treating diseases of the brain fail to cross the blood-brain barrier. Various methods have been tested for drug delivery. For example, carbon-based materials, nanostructures, silicone-based materials, polymers and liposomes, which are capable of delivering drug molecules directly into cells, tumors and sites of inflammation either actively or passively. There is no question that there are many limitations such as opsonization, problems with encapsulation and leakiness of drug that needs to be tackled. Some of these obstacles have been overcome by the development of agents like “stealth liposomes”, which escape attack by the immune system. Thus, nanotechnology is expanding our capabilities through promising approaches for delivery of therapeutic agents. Nanosystems and nanoparticles have opened up hitherto unforeseen avenues in diagnostics and therapeutics in medicine, especially in the fields of inflammation and cancer. The previous treatment strategies in the fields of autoimmune diseases and cancer involved non-targeted treatment options with extensive “collateral damage”. Nanodelivery of drugs is envisioned to reduce this collateral damage, extend a drug’s availability and effectiveness at the site, and reduce toxicity, cost and storage. The focus of this section is to highlight several nanomedicine applications that have made an immediate major impact in these fields. Biological nanostructures used in drug delivery systems include lipid-, silica-, polymer-, fullerene (carbonbased buckyballs, bucky tubes)-based nanostructures such as liposomes, micelles and nanoparticle systems. Liposomes have been widely used as drug delivery systems, but current knowledge extends the use of any nanoparticle as an efficient carrier with necessary modifications.
Liposomal formulations for drug delivery Liposomes are vesicles with phospholipid membranes that contain hydrophilic substances in their core. The properties vary widely based on the size, lipid composition, surface charge and method of preparation [46]. Liposomal formulations have been used as anti-cancer and anti- fungal drugs, and have helped reduce the adverse effects of these drugs, while improving the efficacy and pharmacokinetics. Conventional liposomes are short-lived in vivo, and are rapidly cleared by the reticuloendothelial system (RES). A novel liposomal formulation with a polyethylene glycol (PEG) coating avoids RES-mediated clearing, and is called a stealth liposome. These
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stealth liposomes have favorable properties like long circulation half-life and targeted accumulation in tumor tissues [47]. Liposomes have been extensively used in cancer therapy. Some of the major classes of anti-cancer drugs in liposomal formulations that are currently available or in late stages of development include anthracyclines, camptothecins, platinum derivatives, anti-metabolites and cell-cycle-specific drugs like vincristine and doxorubicin. Liposomal formulations have been shown by clinical trials to decrease cardiotoxicity as compared to conventional formulations [48]. Current liposomal formulations include pegylated liposomal doxorubicin (Doxil® Orthobiotech, Caelyx® Schering-Plough) non-pegylated doxorubicin (Myocet® Elan Pharma) and liposomal daunorubicin (DaunoXome®, Gilead Sciences). This protective strategy to limit toxicity has aided in limiting the cumulative dose of anthracyclines and administration of dexrazoxane, a highly effective cardioprotective agent, prior to anthracycline administration [49]. Liposomal platinum derivatives like cisplatin and carboplatin are used in the treatment of head and neck cancers, testicular cancer, lung cancer and many other malignancies. They have shown significantly reduced toxicity and better pharmacokinetic profiles compared to conventional formulations [50]. Some formulations have not yielded the best results. For example, SPI-077, a stealth liposomal cisplatin showed low clinical efficacy in Phase I/II clinical trials, possibly secondary to inadequate release of drug from liposomes [51, 52]. Lipoplatin, which has shown lipid bilayer fusing properties [53], has shown significant nephrotoxicity, but further clinical research is awaited to see if this translates into improved clinical efficacy. The use of liposomes has also been extended for enhancing immunotherapeutic effects. It is now known that liposomal targeting can be achieved by passive targeting or active targeting. Passive targeting is achieved in both inflammatory and cancerous conditions taking advantage of the leakiness caused by many vascular factors that enhance permeability. This opens up a window of opportunity to increase the drug delivery, with accumulation of drug at higher concentration at the targeted site by extravasation, thereby reducing toxicity and collateral damage. On the other hand, active targeting depends on certain unique properties and molecular strategies involving monoclonal antibody-liposomal conjugates (immunoliposomes), which enables specific tumor cell targeting by antigen identification and drug delivery by internalization of the liposome by tumor cells [54]. Promising examples are the enhanced anti-tumor activity of anti-HER2 immunoliposomes containing doxorubicin [55], and the use of anti-epidermal growth factor receptor (anti-EGFR) immunoliposome, which showed increased cytotoxic effect in vitro against tumor cells overexpressing EGFR and enhanced efficacy in vivo in xenograft models [56]. The recent advent of the widespread use of monoclonal antibodies in cancer therapy promises more such targeted therapeutic agents. Liposomal preparations have also been studied in various autoimmune and chronic inflammatory diseases. Targeted delivery of anti-inflammatory agents to
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inflamed tissue is a promising approach limiting the adverse impact of these agents on healthy tissues. In animal colitis models, liposomal formulations of 5-ASA achieved significantly higher local concentrations of 5-ASA in inflamed colonic tissues compared to current treatment methods. On the other hand, liposomal preparation of Mercaptopurine (6-MP) failed to improve local delivery [57] because the drug is metabolized before it reaches the inflammation site. When PEG-liposomes containing glucocorticoids were injected in mouse collagen arthritis models, longlasting reduction in joint inflammation was achieved with a single dose, while regular steroids needed multiple injections [58]. Weekly inhaled liposomal budesonide was as effective as daily inhaled budesonide in a mouse model of asthma [59]. If this result can be replicated in clinical trials, it can greatly enhance patient compliance. This is particularly important since treatment of chronic inflammatory diseases is hampered by patient non-compliance. Similarly, it is reported that liposomal preparations of anti-oxidants can also be used in diseases like adult respiratory distress syndrome (ARDS), sepsis, radiation lung injury and emphysema [60]. Liposomes have also been shown to be effective in diverse clinical applications such as enhanced drug delivery systems for analgesics [61–63].
Application of other nanoparticles in medicine The carbon nanostructures that have gained most attention have been fullerene nanotubes and the geodesic dome-shaped C60 fullerenes. They have wide ranging applications as drug carriers and can also be used as vaccine delivery tools enhancing the immune response [64]. They have demonstrated neuroprotective properties in cortical cell cultures and have potential therapeutic applications in neuronal-inflammation and neurodegenerative disorders like Parkinson’s disease, amyotropic lateral sclerosis (ALS) and cerebral ischemia [65]. Carbon nanotubes can cross the cell membrane without causing damage and they can act as “nanoneedles” [66]. Tectodendrimers are multicomponent dendrimers capable of multiple functions like identifying defective cells, delivering imaging and therapeutic agents to the cell and reporting the response to therapy. They can be individualized for each specific disease state and can be mass produced. Baker and colleagues [67] designed dendrimers with folic acid, fluorescein and methotrexate, and showed a 100-fold increase in the cytotoxic response of cells to methotrexate. In some cases, nanoparticles also aided in avoiding harmful adverse effects of drug vehicles, as in the case of Abraxane® (American Bioscience), nanospheres of albumin-bound paclitaxel thus avoiding the need for toxic solvents like cremophor [68]. Recently, Kriz et al. [69] described a new sensing technology platform integrating a magnetic permeability detection and a two-site heterogeneous immunoassay using monoclonal anti-CRP-conjugated superparamagnetic nanoparticles and solid-
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phase polyclonal anti-CRP-conjugated silica microparticles to assay CRP in blood samples. The results were comparable to assays performed in a clinical laboratory. The methodology seems applicable for rapid screening of biomarkers and drugs in a rapid and cost-effective manner using whole blood samples.
Nanoparticles in molecular imaging and targeted radiation therapy The field of molecular imaging has exploded in recent times. Significant advances have been made in real-time cellular imaging and for detecting cellular pathophysiology. Over the last few years, varieties of nanostructures containing novel contrast agents and nanomaterials such as QDs, gold nanoparticles or nanoshells, supramagnetic nanoparticles complexed with biological agents that can specifically bind molecular signatures of inflammation and cancer have been described [70, 71]. QDs have enabled in vivo live imaging, down to the level of a single QD inside a cell. QDs provide several advantages over organic fluorochromes since they are photostable permitting imaging over extended periods of time, avoid interference with cellular autofluorescence, permit tracking of multiple processes simultaneously in the cells and are less toxic than organic dyes [72]. Although there is a possibility of cellular toxicity from the metallic components of QDs, no cellular toxicity was seen, even under selection pressure, when QDs were used to track metastatic tumor cell extravasations in an animal model [73]. However, toxicity to humans is still being debated. QDs have multimodal applications as contrast agents in bioimaging, microarrays, and FACS analysis, in monitoring pharmacokinetics of therapeutic agents, and in multicolor optical coding for high throughput screening [74]. QDs have been successfully been used for sentinel lymph node sampling in gastrointestinal tract in pig models. QDs can have immediate applications in oncological surgery if the safety profile can be established for humans [75]. There are several potential pitfalls, including lack of convincing evidence for absence of cytotoxicity. Further research is needed before we move forward towards widespread use of QDs in biological systems [76]. QDs can also potentially replace conventional fluorochromes in complex fluoroimaging techniques like FRET and fluorescence lifetime imaging microscopy, but intensive research is needed before that can happen. SPIO crystal core nanoparticles have magnetic properties that can be used to enhance current MRI techniques due to their selective activity during T2 relaxation times. They can also act as ‘negative enhancers’ [77]. Utilizing the lymphotropic properties of these nanoparticles, Weissleder and colleagues [78] showed that superparamagnetic nanoparticle enhanced high-resolution MRI. It was far superior to conventional high-resolution MRI in detecting clinically occult prostatic cancer metastasis to lymph nodes. The combined use of QDs with superoxide paramag-
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netic crystals may provide additional information by targeting specific molecular targets for imaging [79]. A particularly interesting application is the use of SPIO particles for the study of nucleic acid sequences and surface topography of subcellular organelles. This is achieved by a modified AFM with a nanoneedle mounted on a cantilever beam that deflects when it comes in contact with a paramagnetic nanoparticle. This response can be quantified and mapped [80]. Metal nanoshells are nanoparticles that can serve as strong near infrared absorbers. This property has been exploited to provide targeted thermal therapy selective to tumor cells without damaging normal tissue using gold nanoshells [81]. Gadolinium neutron capture therapy has several advantages, including more efficient tumor killing effects and the potential for simultaneous MRI to assess response. Fukumori and colleagues [82] utilized cationic polymer chitosan nanoparticles that incorporated gadolinium for efficient cellular uptake, and demonstrated significant in vitro tumoricidal effect at relatively low concentrations. Recently, Bankiewicz and colleagues [83] described an integrated strategy to delivery drugs to the brain. The combined technology involved conventionenhanced delivery (CED) to deliver liposomes containing Gadoteridol, with DIL-DS and MRI to obtain detailed images of drugs moving through a living primate brain following CED for imaging, and to induce better clinical efficacy. Molecular imaging has now crossed-over into medical imaging through the use of smart imaging agents for in vivo molecular imaging and imaging of animal models [84–86]. A recent study showed that magnetic nanoparticle conjugated with antiVCAM-1 antibodies can detect VCAM-1 expression through fluorescence and magnetic resonance on endothelial cells in vivo and in vitro [87]. This is an important step, opening up opportunities to use many specific markers specific for inflammation and cancer to diagnose and monitor many inflammatory diseases and cancers.
Summary and conclusions Biological systems operate at the nanoscale. Nanomedicine is the application of nanotechnology to monitor and treat biological systems in health and disease. This is accomplished by real time monitoring of molecular signaling at the cellular and tissue level. During the past decade, there has been an explosion in this field, resulting in revolutionary advances in determining the microstructure and function of living systems. These discoveries have led to the development of powerful tools for fundamental biological and medical research. Nanotechnology has been applied to targeted drug delivery to minimize side effects, creating implantable materials as scaffolds for tissue engineering, creating implantable devices, surgical aids and nanorobotics, as well as throughput drug screening and medical diagnostic imaging. The nanoinitiatives are funded by governments and private sources throughout the world to develop or further refine the technology to provide the beyond-imag-
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inable, most sophisticated tools to a physician and scientists to inflammatory diseases. No doubt, there will be many technical, regulatory and legal challenges in the deployment of these technologies. Unquestionably, there is enough desire and commitment to meet these challenges for the good of society and betterment of the quality of life.
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UK legislation of in vivo aspects in inflammation research Susan Brain King’s College London, Cardiovascular Division, Guy’s Campus, London SE1 1UL, UK
Introduction The inflammatory response plays an essential role in protecting the body against insults, including environmental and immunological activators that relate to chemical, thermal and mechanical stimuli. The body is designed to do this with an elaborate network of mechanisms that comprise the defensive aspects of the inflammatory response. These include mechanisms involved in acute inflammation and wound healing that are essential for the normal pathophysiological function of human as well as animal species. However, often the tissues of the body act in concert with a range of chemical mediators and inflammatory cells to mediate pain as well as inflammation that becomes damaging, chronic in nature and associated with poor wound healing. The precise role of a substantial number of inflammatory mediators, and their relative importance in inflammatory disease remain unclear in many situations. Some of the mediators are released early in the acute inflammatory process (e.g. histamine) and are established as primary mediators in specific conditions (e.g. histamine in acute allergy, with well-established drugs available). Others, especially those released later (e.g. novel cytokines and chemokines), may play either primary or secondary roles depending on situation and act in either a proinflammatory or anti-inflammatory manner. An increased understanding of such molecules is critical to determining common and selective pathways that can direct the development of novel targets for the treatment of human and animal inflammatory disease and in turn the discovery of new medicines. This requires research programmes that involves a range of fundamental and applied biomedical research approaches. The use of animals in inflammation research has been essential for learning more about basic mechanisms and about specific mechanisms in disease models relevant to the discovery of new therapeutic agents [1]. Without doubt, there is a large body of evidence in the literature to show that studies involving animals have identified novel therapeutic anti-inflammatory targets. In addition, the correct
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animal model, with biomarkers that also correlate to the human condition, plays an essential role in the first screening of promising novel treatments [2, 3]. Inflammation research encompasses a wide range of molecular, chemical and biological techniques. While the majority of research can be performed using complimentary non-animal experimental approaches, the therapeutic nature of drug administration requires researchers to integrate knowledge of in vitro and in vivo systems. It is, therefore, essential to evaluate the effects of drugs on complex whole animal systems. Figure 1 describes the major components of inflammation drug discovery programmes, with detailing of the various research components. This chapter details the legislation that is required to carry out inflammation research experiments involving animal species, examining the process from a UK perspective, and noting some of the problems that are faced.
UK legislation Research protocols involving animals in the UK are carried out under the Animals (Scientific Procedures) Act 1986 (ASPA) [4, 5]. Research carried out under the ASPA is monitored through an Inspectorate that resides in the UK Government ‘Home Office’. The large majority of the procedures (> 90%) are conducted on common laboratory species. Indeed, the vast majority of experiments for inflammation research, carried out on animal tissue in the UK, involve rodent tissues, utilised in a combination of in vitro and in vivo assays. A range of animal models of inflammation exist, each with specific attributes, as detailed elsewhere in this book. The UK system involves three major components. The first is that the Research Establishment holds a Certificate of Designation, the second is that the Principal Research Investigator holds a Project Licence(s) to cover the in vivo research experiments and the third is that the research scientist holds a Personal Licence. In addition, there are a range of compulsory Home Office Licence courses for Personnel, currently called Modules 1–5. Modules 1–4 are for prospective personal licence holders and Module 5 is for prospective project licence holders or their deputies. The system is often criticised by animal activists for not being stringent enough and by scientists for being too bureaucratic. In reality, the laws relating to the use of animals in biomedical research in the UK are probably the toughest of those found in any country in the world. Furthermore, the Home Office Inspectorate is continually working to refine the system through a continual appraisal of their systems and the paperwork that is required. Each research establishment will come under the jurisdiction of a specific Home Office Inspector who is either medically or veterinary qualified, and who will regularly visit the Establishment. The visits will comprise both regular appointments, especially when he/she has specific business to carry out (such as discussions with the Certificate Holder about a new animal house) or, more likely, unannounced visits (to observe the level of care of the animal
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Figure 1 An inflammatory situation, with the pivotal interactions between cells and mediators depicted. Some of the primary cell and tissue components are present, together with an indication of the range of chemical mediators that may be involved. The three main areas of research are (i) effects on cellular systems; (ii) in vivo/ex vivo studies of animal systems and (iii) in vivo/ex vivo studies of human systems.
facility and the work of the scientists). There is also an advisory committee, the Animal Procedures Committee. Their role is to advise the Government on any matter relating to the Act. The members of the committee reflect a range of people, including people who oppose use of animals in medical research. More details of the UK system can be found below and in further reading [5–8].
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The Certificate of Designation A Certificate of Designation is required by each research establishment that performs in vivo research in the UK. The Certificate is held by an individual, who will be either a senior administrator within the organisation or a professional person (often a vet or senior scientist). The vast majority of UK establishments use a senior administrator or academic who is not directly involved in managing the animal facilities. The Certificate will include a plan that shows rooms that have been passed. These rooms must be in an excellent state of repair and possess appropriate environmental regulation for the species and purpose that they will be used. The precise requirements are detailed elsewhere [6]. The environmental controls for animals are considerably more stringent that those required for hospital wards for humans. Thus, the process of building or refurbishing an animal facility is very expensive. In addition, there is a security requirement that further increases costs. The Certificate will state the type of procedure that can be carried out in each room. For example, many rooms will be designated as animal holding areas, others will be for minor procedures only and others, with the highest specifications, will be operating rooms for recovery surgery. Moreover, the Certificate will only be given if appropriate levels of animal care and local Ethical Review Procedures (ERP) are in place. The ERP Committee comprises of named persons under the ASPA who are the Named Veterinary Surgeon (NVS), the Named Animal Care and Welfare Officer (NACWO, normally an animal technologist), lay member (not normally directly involved in animal research) and a number of people who are licence holders or who have experience in medical research/experimental design.
The Project Licence Traditionally many scientists who hold a project licence approach the writing or amending of this project licence with more hesitancy than they approach writing the major research application that will fund the research. This is partly because the bureaucratic procedure in the UK requires substantial time, involving discussion and revision steps, in addition to a very specialised writing manner. Indeed project licence holders have to attend a Home Office Module 5 training course to learn of the responsibilities and are also encouraged to attend other training courses available at the time. It is also partly because the computer interfaces available for the forms have, until very recently, been poor. However, the project licence application form was revised in 2004 and this section refers to the revised form, which is more user friendly [7]. It should be noted that in many pharmaceutical companies there are key officers employed to facilitate the process. However, in UK universities, where traditionally there will be a much wider research base, covering a range of
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disciplines (that may include education per se), it is rarely considered cost-efficient to employ specific individuals to deal with this aspect. However, some argue that the actual writing of the project licence allows the scientist to refine their experimental design and objectives. The project licence can be held for up to 5 years and will permit procedures where it has been determined, through ERP consideration and cost/benefit analysis, by both the host institute and the Home Office, to be an essential procedure for the required research. The granting of a licence is dependent on (i) the cost/benefit analysis establishing that the potential results are so important that they justify the use of animals, (ii) that non-animal methods cannot be used for this component of the research, (iii) that the minimum number of animals will be used (in reality a statistical evaluation is essential as too few conversely may invalidate the findings), (iv) ‘higher’ species such as cats, dogs and primates require special justification for their use, (v) that appropriate use of anaesthetics and analgesics is always made, and (vi) all those involved have the appropriate training, skills and experience. The official Project Licence application form is only considered for approval by the Inspectorate once the work has been passed by the local ERP. However, it is usually advisable for the applicant to contact the Home Office Inspector at an early point in order that the Home Office legal requirements are fully understood by the applicant for the specific research that they wish to carry out. The new form for project licence applications was developed in conjunction with all stake holders (project licence holders, scientific bodies, animal welfare agencies etc) and is divided into several sections. The first section is for personal details and is straight forward. The second section is for the Programme of Work. Here the objectives and background are explained, and the expected outcomes. References and supporting documents can be provided. The application is helped if it is clear from the wording that the project has been peer-reviewed (perhaps to secure funding from a Government Research Agency) and that funds and equipment exist to enable the research at the highest level. This is a major section, drafts of which can sometimes be very difficult to understand due to the scientists normal desire to use a range of technical terms that can only be understood by other scientists in a similar field. It is anticipated that the new application form, which has an objective-based approach, will enable the scientist to explain the science in a clearer and more coherent manner. The third section is for a Plan of Work and is again objective driven and demands a clear statement on why the particular animal studies have to be performed. In particular, it is important to clearly justify the chosen species and procedures. The fourth section includes an index, with predicted numbers of animals to be used. There is also a description of the protocols and possible adverse effects, with statements of how they will be alleviated. The final section is a lay summary that will be published on the Home Office Web site.
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Applicants, in keeping with their scientific education, have been too precise in terms of their wording in the past. For example, a typical mistake is for the applicant to name a specific anti-inflammatory agent (e.g. indomethacin) in the application that will then become the written word of the legally binding licence. Then later, after the protocol has been performed and the study submitted for publication, the licence holder may be asked by a referee for a scientific journal to examine the effects of a related drug (e.g. aspirin) before publication. This has meant that the holder then has to send the licence back to the ERP and Home Office for amendment, thus creating unnecessary bureaucracy and government work that is time consuming (usually takes 1–2 months) and is unrelated to any improvement in animal welfare. By comparison, if the licence holder had used the drug class description and said he/she would have used a ‘non-steroidal anti-inflammatory drug’, they would have been covered. It is hoped that applicants today are more aware of some of these problems than in the past. Certainly, the revised licence application form and the attached notes are more directed to ensuring that the applicant only writes about the details required by the government for their evaluation of the application. The objective should be to have what is known as a ‘low maintenance’ project licence, in that it requires few future amendments.
The Personal Licence The personal licence is essential to ensure that the research scientist or technician can carry out experimental and surgical procedure on the laboratory animal. The applicant has to prove that they have passed the appropriate module training course, through production of the relevant certificate that is sent to the Home Office with the personal licence application. The application is relatively straight forward in that it requires personal information, information of the project licence that it will come under, supervisor details and a list of procedures that the applicant will be able to do [8]. The Home Office will take into account the applicant’s experience and the licence will be granted with an appropriate supervision clause depending on experience. The Home Office requires regular review of this licence and charges an annual fee. It is also normal for most research establishments to have their own internal review systems for personal licence holders. There has been an ongoing dialogue between the Home Office and Licence Holders over the ability of visiting scientists from overseas to obtain personal licences in a timely manner to correlate with timings of visiting fellowships. The Home Office Inspector will look at these cases on an individual basis. Experienced in vivo scientists with appropriate experience may be able to apply for a Personal Licence with some modules of the statutory training course waived in some cases. This speeds up and simplifies the application process in situations where wavers are accepted.
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Other legislative systems in the United States and Europe While the UK operates a double system comprising of the government Home Office scheme twinned with a local ERP, other countries run just a single review system. In most other countries, the regulation that applies to the use of animals in medical research operates either through central government (or in the USA specific state-led statutory controls) or by the local ethical committees. This may involve obtaining a single level of clearance for the laboratory and everyone who works in it, or it may involve a project approval system, where each project has to receive clearance. The level of ethical discussion is often high, with the same clear need to justify protocols as is observed in the UK. There are an increasing range of basic and advanced training programmes for individuals, some of which are compulsory depending upon which country one is in. There is ongoing discussion concerning legislation within Europe, to which academics, industrialists and animal welfare groups are contributing [9]. The European Council of Ministers agreed Directive 86/609/EEC entitled “The protection of animals used for experimental and other scientific purposes” in 1986, but the controls within this directive were not that specific, for example in most cases the UK law superseded these requirements. There is now a need to update and improve the Directive. The outcome will not be known for some years, but it is generally assumed that European systems will follow a level of requirements that will be closer to those that exist already in the UK. More specifically, it is suggested that the following criteria should be established: (i) that the current standard of animal welfare should be improved, (ii) that existing, successful schemes/practices should be respected, (iii) that feasibility studies should take into account available resources, and that implementation should be feasible throughout the EU. Full details can be obtained from the EU website [9].
Opportunities for reduction, replacement and refinement in inflammation research The specific use of animals in research has changed radically in recent years. The choice of species has been driven by various factors. The use of larger animals is more emotive than the use of rodent species. In addition, the maintenance costs of large animals and the specialised facilities that they require have all acted to reduce their use. However, when the legal and scientific reasons are compulsive, larger animals are still used. Without doubt the most dramatic change in recent years has been the impact of the generation of genetically modified mice on research [1]. The provision of technology that allows the breeding of mice lacking specific receptors or mediators (knockouts), or those with increased levels (transgenics), or technology that enable tissue-specific genetic modification has revolutionised research
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approaches, especially when supported by traditional pharmacological approaches. The use of mice demands a technical excellence due to challenges related to their body size, and over the years this has been associated with many examples of refinement of techniques. However, the use of mice, when compared with larger species has not led to the expected reduction in the number of experiments carried out on animals per year. This is partly due to the required size of healthy genetically altered mouse breeding colonies. Furthermore, just the breeding of a genetically altered mouse is classed as a procedure. Certainly, in the UK, a small increase has been observed in the numbers used each year and this has been associated with an outcry from animal welfare groups. On the other hand emerging technologies that enable in vivo imaging are often accompanied by minimal invasive techniques [10]. While these techniques are at present expensive and dependent on a high level of computer knowledge, their potential in the inflammation field is clear. The use of appropriate equipment designed for mice will enable the study of appropriately tagged mediators and cells in the in vivo environment in murine models of inflammatory disease. The longitudional nature of potential studies should act to enable reductions in numbers used. The models have been substantially refined in recent years, for example in the arthritis field (see elsewhere in this book). There are a range of chemical and surgical techniques for inducing arthritis that can induce either mono-, poly-, or systemic arthritis. Today a mono-arthritis involving only one joint is now routine, compared with the previously used antigen-induced models involving multiple joints. Indeed, in the UK, the law requires that the most refined model is used for the required purpose. This does not mean that the most mild model will fit all purposes relevant to one disease. For example, the chosen model may depend on the mediator or cell pathway under investigation, or the component of the disease that is under study by the particular group. In addition, the study of genetically altered mice strains that spontaneously develop arthritis has been important for moving the field forward [11]. Thus, the cost/benefit analysis has to be applied carefully in every situation. The models may involve side effects, causing acute and/or chronic discomfort alongside the main inflammatory symptoms. From the view point of animal welfare, there is an obligatory need for humane end points of protocols to be carefully defined for every experimental procedures and determined through effective monitoring of the procedure. In addition, analgesics should be used when possible, but this may not be available when it would invalidate the experiment. This is a major area that involves detailed discussion among all stakeholders, when protocols are under review.
Skills shortages in integrative inflammation research in the 21st century It has become apparent that an understanding of integrative research approaches is required by biomedical graduates today to equip them for careers in inflammation
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research. There is a need to ensure a balanced education that covers molecular and cellular research approaches in addition to traditional and emerging approaches to in vitro and in vivo systems research. However, in recent years there has been a growing realisation that a decline of in vivo education in UK universities has occurred and that this has a negative impact on the UK academic and industrial biomedical research base. Indeed, a recent survey has suggested that this situation is now becoming critical as a number of university lecturers with in vivo skills reach retirement age [12]. Several pharmaceutical companies have, in association with learned societies, established a fund to help reverse this decline. The objective of the fund is to enhance the academic research and training base for in vivo pharmacology, physiology and toxicology, so there is a pool of well-trained scientists from whom academia and industry can recruit and a vibrant research base for collaboration activities. The strategy is to use the fund in partnership with other research funders to enhance the benefit. The major funding initiative to date has been to work with the UK Research Councils to fund eight New Blood lectureships in Integrative Pharmacology in the UK. These schemes provide funds for 5-year fellowships to include research and teaching with a commitment from the University to provide employment at the end of the Fellowship. They are an ideal way to rebuild the academic skills base, following the retirement of many academic staff. This will allow an appropriate integrative education that will hopefully help to reverse the skills shortage that is reported to be world wide. Indeed the pharmaceutical companies find themselves in a challenged position. An increased number of new drug targets have been delivered following the detailing of the human genome. These need substantial understanding before they are turned into new drug candidates. The increasing number of issues that surround efficacy and safety concerns have prompted the companies to ensure that their research systems are beyond reproach as assessed by the public end users. This can only achieved by sophisticated integrative studies as described in this book, that are in turn performed by staff educated and trained to a new level of excellence [3].
References 1 2
3
van den Berg WB (2005) Animal models of arthritis. What have we learned? J Rheumatol (Suppl) 72: 7–9 Jobe PC, Burks TF, Fuller RW, Peck CC, Ruffolo RR, Snead OC III, Woosley RL (1998) The essential role of integrative biomedical sciences in protecting and contributing to the wealth and well being of our nation. Pharmacologist 40: 32–37 U.S. Department of Health and Human Services, Food and Drug Administration (2004) Innovation or stagnation: challenge and opportunity on the critical path to new medical products. U.S. Department of Health and Human Services, Food and Drug Administration
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4 5 6 7 8 9 10 11
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Guidance on the Operation of the Animals (Scientific Procedures) Act 1986 (HMSO 1990:HC182, ISBN 0102182906) Wolfensohn S, Lloyd M (2002) Handbook of laboratory animal management and welfare, 2nd edn. Blackwell Science, Oxford The Home Office code of practice for the housing and care of animals used in scientific procedures (Her Majesty’s Statonary Office, London HC107 ISBN 0102107890) The Home Office Project licence form http://www.homeoffice.gov.uk/docs4/PPL_ Form.doc The Home Office Personal Licence form http://www.homeoffice.gov.uk/docs/personal_lic_form.doc The European Commission http://europa.eu.int/comm/environment/chemicals/lab_animals/revision_en.htm Dustin ML (2003) In vivo imaging approaches in animal models of rheumatoid arthritis. Arthritis Res Ther 5: 165–171 Keffer J, Probert L, Cazlaris H, Georgopoulos S, Kaslaris E, Kioussis D, Kollias G (1991) Transgenic mice expressing human tumour necrosis factor: a predictive genetic model of arthritis. EMBO J 10: 4025–4031 In Vivo Pharmacology Training Group (2002) The fall and rise of in vivo pharmacology. Trends Pharmacol Sci 23: 13–18
Japanese guidelines and regulations for scientific and ethical animal experimentation Naoko Kagiyama1, Takuya Ikeda2 and Tatsuji Nomura1 1Central
Institute for Experimental Animals, 1430 Nogawa, Miyamae, Kawasaki 216-0001, Japan; 2GlaxoSmithKline Tsukuba Research Laboratories, 43 Wadai, Tsukuba 300-4243, Japan
What risk did animal experimentation in Japan present in terms of laboratory animal welfare? First, the 3R (Replacement, Reduction, Refinement) principle was not legally specified [1]. This resulted in criticism from Western countries ([2] and personal communication). Second, no national guidelines for animal experimentation were established. This caused disparities in appropriateness of animal experimentation among diverse institutions.
Legal standing of the 3R principle With the amendment of the Law for the Humane Treatment and Management of Animals, the specifications for the use of animals for scientific purposes have been revised and the 3R principle has been given legal standing (Tab. 1). The amendment came into force on June 1, 2006. The Ministry of the Environment regulates humane treatment of animals under the Law, and “Refinement” is specified in the Standards Relating to the Care and Management of Laboratory Animals and Relief of Pain as shown on the left in Figure 1. “Replacement” and “Reduction” are considered as the items to be discussed when the animal experimental protocol is prepared and are not direct methods to implement well-being of animals. There was a critical debate on whether it is wise to regulate scientific procedures by legislative measures, and if yes, whether the Law is the most preferable one or not. Eventually, members of the Japanese Diet concluded that bioscience should not be regulated only from the aspect of humane treatment of animals. They thought that appropriate animal experimentation could be better accomplished based on guidelines and not by stringent legislation.
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Table 1 - Amended Law for the Humane Treatment and Management of Animals Article 41. Where an animal is used for the purposes of education, testing, research, manufacturing of biologic products or other scientific purposes, consideration shall be given to proper use of animals such as use of alternative methods not using animals and using as few animals as possible within the limits to fulfill the purposes. 2. When an animal is used for scientific purposes, methods that do not cause pain or distress to the animals should be used within the limits imposed by this use. 3. When an animal is beyond recovery after use for scientific purposes, the person who used the animal for such scientific purposes must immediately dispose of the animal by a method that causes the animal as little pain and distress as possible. 4. The Minister of the Environment may, after consultation with the heads of the administrative agencies concerned, prescribe applicable standards with regard to the methods in Paragraph 2 and the measures in the preceding Paragraph.
Basic guidelines established by regulatory agencies Animal experimentation in Japan had been conducted based on the local regulation system at each institution following the administrative guidance of the Ministry of Education since 1987. (The Ministry of Education was reorganized in 2001 as the Ministry of Education, Sports, Science and Technology, MEXT.) The guidance also had some influence on animal experiments conducted by pharmaceutical companies as a reference. However, the guidance, just recommending the formulation of local regulations, was too soft to rectify disparities among academic institutions. Therefore, in 2004, the Science Council of Japan (SCJ) proposed the establishment of national guidelines on animal experimentation with the objective of scientific rationalization of animal experimentation in balance with laboratory animal welfare [3]. In accordance with the proposal, MEXT as well as the Ministry of Health, Labor and Welfare (MHLW) established animal experimentation guidelines “Basic policies on animal experimentation” as quasi-regulations on June 1, 2006, based on the 3R principle as shown on the right in Figure 1. These guidelines focus on scientific rationalization of animal experimentation rather than humane treatment of laboratory animals and simply describe basic policies and institutional responsibilities for conducting animal experiments. The institutional animal care and use committee (IACUC), appointed by the director of the institution, should review the animal experimental protocol prepared by a principal investigator and determine if the protocol complies with the local regulations and the national guidelines of the compe-
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Figure 1 Framework for implementing appropriate animal experiments
tent authority. The IACUC reports the results of the review to the director and the director decides whether or not to approve the protocol. The director is also responsible for training and informing investigators and animal technicians about related regulations and policies. The guidelines established by the MHLW give more consideration to information protection than those of MEXT, so that the policies can be applied by profit-making organizations. The Ministry of Agriculture, Forestry and Fisheries also established basic guidelines on animal experimentation and notified the research institutions under its control on June 1, 2006 .
Detailed guidelines formulated by the SCJ The two regulatory authorities, MEXT and MHLW, requested the SCJ to formulate more detailed guidelines so that they can serve as a vital reference when institutions establish their local regulations in accordance with respective basic guidelines. Thus, the detailed guidelines “Guidelines for the proper conduct of animal experimenta-
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tion” were published by SCJ on June 1, 2006 in parallel with the two basic guidelines. The detailed guidelines cover both aspects of scientific rationality of in vivo research and humane treatment of animals involved, referring to not only MEXT and MHLW basic guidelines, but also the Standards Relating to the Care and Management of Laboratory Animals and Relief of Pain of the Ministry of the Environment. In this respect, the detailed guidelines resemble the ILAR Guide for the Care and Use of Laboratory Animals in the United States. However, the detailed guidelines do not contain any numerical specifications, based on the concept of “performance approach” that the required conditions should be investigated from the scientific standpoint and set voluntarily. When drafting an experimental protocol, the items to be considered are listed in the detailed guidelines. They include animal rooms, procedure rooms, necessary equipment, restraint of animals, restrictions on supply of food and drinking water, surgical procedures, anesthesia and postoperative care, humane endpoints, euthanasia, occupational health and safety, and record keeping. The principal investigator should estimate the degree of pain in the animals used, and include in the protocol the methods of alleviating the pain, to the extent that they do not restrict the objective of the research.
Deliberation on local self-regulation of animal experiments One of the major elements to ensure enforced self-regulation is clearly the education and training of personnel. The detailed guidelines contain items such as related laws, regional ordinances, policies and regulations; experimental procedures and handling of animals; health, care and management of animals; hygienic procedures and safety assurance; and utilization of facilities, as examples of a syllabus. Wet-hand education directly connected with the experimental procedures used is also essential for scientific and humane animal experimentation, and should be given by senior investigators with sufficient knowledge, experience and skills. As proposed by SCJ, the final step in adequate self-regulation should be validation by an outside organization. It also improves the social transparency of animal experimentation, a weak point in self-regulation. The verification method should be a peer-review system by specialists with scientific knowledge. In Japan, a cross check will start among academic institutions, whereas pharmaceutical companies seem to prefer internationally recognized system(s) with strict confidentiality. Finally, we would like to refer to penalties. The penalties prescribed in the Law can be applied to persons who kill, injure or mistreat laboratory animals without any scientific rationale and thought concerning the animals.
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References 1
2 3
Kagiyama N, Nomura T (2004) Japanese regulations on animal experiments. In: ILAR, National Research Council (ed): Development of science-based guidelines for laboratory animal care. The November 2003 International Workshop. National Academy Press, Washington D.C., 50–56 Research Defense Society: International comparison of animal experimentation regulations. RDS News. July 2001 The 7th Division, Science Council of Japan. Report concerning the promotion of public understanding of animal experimentation (Proposal), July 15, 2004
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United States guidelines and regulations in animal experimentation Joanne B. Morris, Jeffrey Everitt and Margaret S. Landi GlaxoSmithKline Pharmaceuticals, LAS, 709 Swedeland Rd, King of Prussia, PA 19406, USA
Introduction Animals used in research, testing and teaching in the United States are protected and regulated under both state and federal laws, several of which have overlapping mandates. This chapter focuses on the rules and regulations that govern animal research in the United States (U.S.). Research with laboratory animals is a privilege granted by society and, as such, comes with the responsibility for conduct in a humane way. In many arenas, this is formalized by reference to the three Rs of Reduction, Replacement and Refinement [1]. For animal studies, reduction of numbers is often accomplished through the utilization of optimized experimental design and the employment of proper statistical science. Replacement is driven by understanding of the science and knowing where and when lower phylogenic models and methods can be used, or potentially the use of non-animal models. In many experiments, refined study design is the first tool for improving the science and animal welfare. As societal interactions and science becomes more global, there are international efforts to standardize care for laboratory animals [2, 3]. It can be expected that U.S. rules and regulations will be impacted by an increasing number of international guidelines from agencies and committees such as the European Union and the Canadian Council of Animal Care. The constant increase in our knowledge base concerning species-specific requirements for animal care will result in continuing changes in regulations and guidance documents. Research investigators must stay abreast of these changes. This chapter focuses on current rules and regulations that govern animal research in the U.S. and reviews the major guidelines used by U.S. animal care and use programs for research, teaching and testing. Emphasis is placed on topics relevant for specific animal models of inflammation, as well as commonly used endpoints in inflammation-related research. It is not the intent of this chapter to provide all information required for the conduct of animal research, testing or teaching.
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Federal laws Animal Welfare Act In 1966, Congress passed the Animal Welfare Act (AWA) in response to growing public concern for the issues of pet theft [3]. It was revised and strengthened through modifications in 1970, 1976, 1985 and 1990 [3]. The AWA is enforced by United States Department of Agriculture (USDA) to protect regulated species from inhumane treatment and neglect. The Animal Plant Health Inspection Service (APHIS) of the USDA protects and promotes agricultural health by administering the standards and regulations of the Animal Welfare Act [4]. The species of animals that are covered or regulated by the AWA include any live or dead, warm-blooded animal that is being used in research, teaching, testing, experimentation, exhibition or as a pet. Birds, rats of the genus Rattus and mice of the genus Mus bred for use in research teaching or testing, horses not used for research purposes and other farm animals intended for use as food or fiber are not covered by the AWA. The requirements of the AWA are implemented and overseen by an Institutional Animal Care and Use Committee (IACUC). The IACUC represents the institutional commitment to the program of animal care and welfare. In this chapter, the AWA and IACUC function are described separately [4]. The AWA requires training documentation for all scientists, animal technicians and other personnel involved with animal care and treatment at research facilities. The research facility is responsible for training personnel in the subjects of humane practice of animal husbandry and experimentation, limiting the numbers of animals used to those necessary to achieve experimental objectives, minimizing pain and distress and the utilization of resources, such as the national agriculture library to seek alternatives to animal experimentation. In addition, the institution is responsible under the AWA for establishing a process whereby deficiencies can be reported. The USDA inspects each registered facility at least once per year and sets penalties for noncompliance. The facility is inspected more frequently, with follow-up inspections if deficiencies are found. The AWA mandates a role for an Attending Veterinarian and outlines a program of adequate veterinary care [4]. In 1985, the AWA was amended (Pub. L. 99-198 – Improved Standards for Laboratory Animal Act) to add stipulations, specifically outlining an exercise requirement for dogs, environmental enhancement to promote psychological well-being for nonhuman primates, limitations on multiple survival surgeries and a requirement for veterinary consultation and consideration of alternatives to animal use during protocol development. These amendments, more than any others before or since, enforce the need for qualified professional judgment when working with regulated species [5].
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IACUC function required by AWA Each institution must identify a senior person to be accountable for all aspects of the AWA. This person must have the authority and fiscal responsibility to enact change when needed. The Institutional Official (IO), often a Chief Executive Officer, Chairman, Dean or Provost, appoints all members to the IACUC. The USDA requires at least three members, including an IACUC chairperson, a Doctor of Veterinary Medicine (DVM) with training or experience in laboratory animal science and medicine and an individual who is not affiliated with the institution. The IACUC veterinarian is to have direct or delegated responsibilities for the institutional animal care and use program. The non-affiliated committee member’s function is to serve as a voice for the general public [4]. The IACUC is responsible for reviewing the animal care and use program at least once every 6 months, and inspecting and evaluating the research facilities on a semiannual basis. This includes all animal holding areas, as well as any area or laboratory in which research is performed on a covered species. Reports of the program evaluation and facility inspection are submitted to the IO of the research facility, and must include any major or minor deficiencies with a plan for correction. These reports need to be signed by a majority of the committee and updated every 6 months to reflect the evaluations. The research facility is required to maintain these reports and to make them available to the APHIS inspection and officials of federal funding upon request for copying and inspection. The AWA requires specific record keeping including minutes of IACUC meetings, animal record keeping requirements and recording of minority IACUC views [4] The committee is responsible for reviewing and, if necessary, investigating concerns raised by staff or the public involving the care and use of animals at the research facility. They are also responsible for reviewing and reporting acts of noncompliance received from laboratory or research personnel or employees. The IACUC makes recommendations to the IO regarding any aspect of the research facility’s animal program, facilities or personnel training [3]. An important aspect of IACUC function includes the review of all proposed activities involving the care and use of animals. The IACUC is charged with the responsibility to approve, require modifications to or withhold approval of these activities. It is also authorized to suspend activities involving animals if it determines that the activity is not being conducted in accordance with the description of that activity provided by the principle investigator and previously approved by the IACUC. If an activity is suspended, the IO with the assistance of the committee will review the reason for suspension, take appropriate corrective actions and report the actions and corrective measures to APHIS and any other federal agency funding that activity; such as the National Institutes of Health (NIH) [4].
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Public Health Service Policy on Humane Care and Use of Laboratory Animals The Health Research Extension Act of 1985, Public Law 99-158, or the Public Health Service (PHS) Policy, also governs animal research. The secretary, acting through the Director of the NIH, establishes these guidelines for PHS-sponsored research. Adherence to these guidelines is monitored by the PHS Office of Laboratory Animal Welfare (OLAW), formerly the Office for the Protection of Research Risks (OPRR). The PHS Policy ensures proper care and treatment of animals, requiring the appropriate use of analgesics, anesthetics and appropriate pre- and post-surgical and nursing care. The PHS Policy requires an institutional animal care committee at each entity that conducts biomedical and behavioral research with funds provided under this act, including NIH and the National Research Institute. These regulations apply to all PHS-conducted or -supported activities involving the use of live animals, including institutions in foreign countries receiving PHS funding. Animals are defined under the PHS Policy as any live, vertebrate animal used or intended for use in research, research training, experimentation or biological testing or for related purposes. This broadens the number of species used in research covered by federal regulation in comparison to the AWA, which excludes mice, rats and livestock. The PHS Policy does, however, require compliance with the AWA and with the ILAR 'Guide for Care and Use of Laboratory Animals (Guide)' [6, 7]. Prior to any activity involving animals being conducted at an institution receiving PHS funding, the institution must have a written animal welfare assurance accepted by the PHS and submitted to the office of the Director of the NIH OLAW. The assurance is signed by the IO, and is generally approved for no longer then 5 years. Each written assurance must contain a complete description of the animal care and use program. The assurance identifies the program veterinarian and the members of the institutional animal care and use committee. It also describes the program of training in humane care and use of animals. Under PHS Policy, an institution is placed into one of two categories for obtaining an Assurance. Category I states that the institution is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International and that the IACUC will evaluate the program and facility every 6 months. Category II states that the institution will undergo a self evaluation with IACUC program review every 6 months. The two most recent IACUC program reviews must be submitted to OLAW with the assurance [6].
IACUC for PHS The composition of the IACUC required by the PHS is very similar to the AWA, with a few minor differences. The committee consists of no less than five members as opposed to the AWA, described earlier, which required no less than three members
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[4]. PHS Policy requires an IACUC constituted with a chairperson, a DVM with laboratory animal medicine training with direct or delegated program authority, one practicing scientist with experience in research involving animals, one member whose primary concern is in a nonscientific area (for example, a lawyer, an ethicist, a member of the clergy) and one individual who is not affiliated with the institution. One individual may fill more than one of these categories, but the committee must not contain fewer than five members. The IACUC function is very similar to that stipulated in AWA requirements, with some minor differences. The PHS Policy requires animal care program review every 6 months, but requires the use of the 'Guide' as a basis for evaluation. The IACUC has the responsibility to investigate concerns regarding the care and use of animals at the institution; a requirement to make recommendations to the IO concerning the animal care and use program; and the responsibility to review research protocols with a charge to approve, recommend modification or withhold approval for the proposed activity. In addition, both the PHS Policy and the AWA give the IACUC authorization to suspend an activity where deemed appropriate [6].
Guide for the Care and Use of Laboratory Animals First published in 1963 as the Guide for Laboratory Animal Facilities and Care, later revised to the Guide for the Care and Use of Laboratory Animals, the Guide has become the primary reference on animal care and use in the U.S. [7]. It was modified and revised in 1965, 1968, 1972, 1978, 1985 and 1996. The most recent edition of the Guide covers all vertebrate species used in research, teaching or testing. It does not specifically address livestock used in agriculture, wildlife or aquatic animals but the general principles in the Guide can be applied to all species. The written language of the Guide is based on performance standards, which, in addition to being more flexible than engineering standards, encourages the use of professional judgment. Furthermore, the Guide requires that investigators take responsibility for the following principles: 1. Research should be based on human or animal health, the advancement of knowledge, or for the good of society. 2. Appropriate species, quality and number of animals should be used. 3. Pain and distress should be minimized. 4. Appropriate sedation, analgesics and anesthetics should be used. 5. Defined end points should be established. 6. Appropriate animal husbandry directed by qualified personal should be provided. 7. Experiments on live animals should be conducted by or under the supervision of experienced personal.
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The Guide is divided into four major chapters: Institutional Policies and Responsibilities; Animal Environment, Housing and Management; Veterinary Medical Care; and Physical Plant. The chapter titled “Institutional Policies and Responsibility” provides guidelines for the care and use of animals in research, teaching and testing. This includes guidelines for topics such as IACUC requirements including the evaluation and approval of protocols. It also discusses topics such as physical restraint, major multiple survival surgeries, food and fluid restriction and veterinary care. Also included in this chapter are personnel qualifications and training, and guidelines concerning an occupational health and safety program for personnel [7]. The chapter on Animal Environment, Housing and Management covers guidelines for the physical environment and reviews housing, space recommendations, temperature and humidity, ventilation, illumination and noise requirements. The husbandry requirements for food, water, bedding, sanitation, waste disposal, pest control and emergency weekend and holiday care are discussed. Husbandry topics, including species specific social needs and behavior, are also addressed in this section. This section of the Guide outlines requirements for population management including identification, record keeping, genetics and nomenclature [7]. The Guide contains a chapter devoted to veterinary medical care with discussion of animal procurement, transportation, preventive medicine, quarantine, disease control, surgery, pain management and euthanasia issues. Generalized recommendations are made throughout the chapter. Due to differing disease prevalence and susceptibility, the Guide recommends that there is a physical separation between species. Again the recommendations of the Guide are based on performance standards as opposed to engineering standards [7]. Physical plant requirements for animal care and use programs are discussed in the fourth chapter of the Guide and are based on professional judgment. They include recommendations for functional areas that take into account animal housing, sanitation, receipt and quarantine, separation of species and storage areas. Construction guidelines are presented in this chapter and include discussion of corridor space, animal-room doors, exterior window, floors, drainage, walls and ceilings. Heating, ventilation and air-conditioning (HVAC) recommendations are listed and depend on species requirements. Other physical plant topics include power and lighting, storage, noise control, areas for cage sanitation as well as specific guidelines for aseptic surgery [7].
Food and Drug Administration, Good Laboratory Practices Good Laboratory Practices (GLPs) were enacted in 1979 under the auspices of the Food and Drug Cosmetic Act (FDCA) and were amended in 1987. The GLPs are standards for conducting and reporting non-clinical safety testing for human and animal drugs, biologics and medical devices. The primary purpose of the GLPs is to
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provide authority for imposing standards on the conduct of safety testing [8]. The result of GLP compliance has been the assurance of quality and integrity of data, and allows for an accurate reconstruction of the study data. These regulations apply to non-clinical studies, i.e. in vivo or in vitro experiments in which test articles are studied prospectively in test systems under laboratory conditions to determine their safety. This does not include studies with human subjects, clinical studies or field trials in humans or animals. Nor does it include basic exploratory studies carried out to determine physical or chemical characteristics of a test article. Any study that is performed under grants or contract for the sponsor must adhere to GLP standards [9]. Personnel and organization are an important part of the GLP standards. Each person involved in the study must have properly documented training that allows them to perform their assigned function, and training records must be maintained by each facility. The testing facility management must appoint a study director who has the overall responsibility for the technical conduct of the study. The testing facility must assure that there is a Quality Assurance Unit that will monitor each study to assure management that facility, equipment, personnel, methods, practices, records and controls are in conformance with the regulations. The Quality Assurance Unit conducts periodic inspections to ensure the integrity of the study. GLPs define required test facilities and ensure standard operating procedures (SOPs) for many aspects of facility management. This includes, but is not limited to, animal room preparation, animal care, handling of animals found moribund or dead during a study, necropsy or post-mortem examination of animals, collection and identification of specimens, maintenance and calibration of equipment and the transfer, proper placement and identification of animals. GLPs define several aspects of animal care including SOPs for housing, feeding, handling and care of animals. The regulations also state that all newly received animals from external sources be isolated and their health status be evaluated in accordance with acceptable veterinary practices. Animals must be free of pathogens and disease that could interfere with the study. Feed and water used for animals must be analyzed periodically for contaminants that could influence experimental results, animal cages and racks must be sanitized periodically and bedding material should not interfere with the conduct of study [9]. In conducting studies under GLP compliance, all raw data, documentation, protocols, final reports and specimens generated as a result of a nonclinical laboratory study must be retained through specified archiving procedures [9].
Specific considerations for animal models of inflammation Animal models of inflammation represent a challenge for the investigator under the U.S. regulations that govern animal research. The AWA states that procedures caus-
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ing more than momentary or slight pain or distress to the animal will be performed with appropriate sedatives, analgesics or anesthetics, unless withholding such agents is justified for scientific reasons [4]. The principle investigator is required to consult with the attending veterinarian or designee when designing a study to discuss measurements of pain and distress, and methods for their amelioration. If an animal experiences severe or chronic pain or distress that cannot be relieved then euthanasia (humane death) should be used to terminate the experiment or procedure [4]. The PHS Policy and the Guide use similar language concerning minimizing pain and distress in laboratory animals [6, 7]. Inflammation research can be a source of discomfort or pain to animals; however, there is a possibility that the use of analgesics and anesthetics may interfere with research objectives [10]. Because the use of chemical methods of pain relief may not be appropriate for certain studies, it is imperative that alternative pain-relieving methods and appropriate endpoints are incorporated into the study design [10]. Alternative pain-relieving methods include adaptive measures such as the use of soft flooring or bedding, provision of easy access to food and water so that animals do not have to undergo reaching and locomotion and other modifications, as appropriate, to make the animal’s environment as comfortable as possible. Defining endpoints in the study design is useful for a variety of reasons, such as the minimization of pain and distress in animals, decreasing unwanted variables and providing optimal tissue and biosamples for pathological and clinical evaluation [11]. Effective, defined endpoints can result in diminished pain and distress to the animal while, at the same time, satisfying the experimental goals and objectives. They must be compatible with the experimental design and incorporate both subjective and objective assessments of animals [12]. Subjective assessments include overall appearance, behavior, mobility and activity and clinical symptoms. Objective criteria include quantitative assessment of temperature, pulse, respiration, locomotion, body weight, feed and water consumption, quantity and quality of feces and urine output and measurable physiological parameters [12]. Many governmental agencies have published guidelines for humane endpoints, such as NIH, ILAR, the Canadian Council on Animal Care and the Organization Economic Co-Operation and Development (OECD) Test Guidelines Programme [13–15]. All of these guidelines should be evaluated for the particular species being worked with and modified to incorporate species-specific behaviors. In species covered by the USDA, if the pain or distress can not be relieved, the animal must be reported in Category E of the USDA Annual Report. Animals in Category E need to have specific explanations as to why pain or distress cannot be relieved. All USDA inspections and annual reports are available to the public under the Freedom of Information Act [16].
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Table 1 - Internet resources for the rules and regulations of laboratory animal research Resource
Internet address
AAALAC International American Association of Laboratory Animal Practitioners American Association of Laboratory Animal Science American College of Laboratory Animal Medicine Animal Plant Health Inspection Service Animal Welfare Information Center Canadian Council on Animal Care Foundation for Biomedical Research Institute of Laboratory Animal Research
www.aaalac.org www.aslap.org www.aalas.org www.aclam.org http://www.aphis.usda.gov/ www.nal.usda.gov/awic/ http://www.ccac.ca/ http://www.fbresearch.org/ http://dels.nas.edu/ilar_n/ ilarhome/index.shtml www.iacuc.org http://www.nabr.org/ http://www.aphis.usda.gov/ac/
Institutional Animal Care and Use Committee National Association for Biomedical Research USDA Animal Care Page
Summary The use of animals in biomedical research is a privilege granted by society, and the rules and regulations that govern this usage are in place to ensure the welfare of the laboratory animal subjects. The IACUC is the institutional commitment to this protection, and IACUC members continually challenge themselves and others to find a balance between achieving both scientific and animal welfare objectives. This is accomplished by incorporating the three Rs (reduction replacement and refinement) into animal usage in research, teaching and testing. IACUC members can be advocates for both the animals and the scientist. A good working relationship between the committee and the attending and/or laboratory animal veterinarian will promote identification, interpretation and clarification of the major rules and regulations. It is ultimately the responsibility of each investigator to comply with regulations for the welfare and safety of the animals and people involved in any research project. Animal models used in inflammation research often have specific considerations that need to be addressed to minimize issues of pain and distress. This includes the use of well-defined endpoints, the proper use of pharmacological interventions and the use of other pain-alleviating methods.
Acknowledgement Thank you to Tiffany Yarnall for her assistance with the manuscript.
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References 1 2 3
4 5 6
7 8 9 10 11
12 13 14 15 16
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Russell WMS, Burch RL (1959) The Principles of Humane Experimental Technique. Methuen, London Institute for Laboratory Animal Research Journal (2002) Regulatory Testing and Animal Welfare. ILAR Online 43 Supplement Anderson LC (2002) Laws, regulations, and policies affecting the use of laboratory animals. In: J Fox, L Anderson, F Loew, F Quimby (eds): Laboratory Animal Medicine, 2nd edn. Academic Press, London, 19–32 Animal Welfare Act (Title 7 U.S.C. 2131–2159), as amended by P.L. 107–171 May 13, 2002. Kulpa-Eddy JA, Taylor S, Adams KM (2005) USDA perspective on environmental enrichment for animals. ILAR 46(2): 83–94 US Department of Health and Human Services, National Institute of Health, Office of Laboratory Animal Welfare. Public Health Service Policy of Humane Care and Use of Laboratory Animals, revised August 2002, pursuant to Health Research Extension Act of 1985 (P.L. 99-158, November 20, 1985.) National Research Council, Institute of Laboratory Animal Resources (1996) Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington D.C. Owiertniewicz J (2005) Introductions to the Good Laboratory Practice Regulations. Lab Animal 34(3): 29–32 US Department of Health and Human Services, Food and Drug Administration (2005) Good Laboratory Practice for Non Clinical Laboratory Studies (21 CFR Part 58) Olfert ED, Godson DL (2000) Humane Endpoints for Infectious Disease Animal Models. ILAR 41(2): 99–104 Morton DB (1998) Humane endpoints in animal experimentation for biomedical research: ethical, legal, and practical aspects. In: CFM Hendriksen, DB Morton (eds): Proceedings of the International Conference: Humane endpoints in animal experimentation for biomedical research, 22–25 Nov 1998, Zeist, The Netherlands Stasiak KL, Maul D, French E, Hellyer PW, Vandewoude S (2003) Species-specific assesment of pain in laboratory animals. Contemp Top 42(4): 13–20 National Institute of Health Office of Animal Care and Use (2005) ARAC Guidelines. Guidelines for Endpoints in Animal Study Proposals. Revised 1/12/05 Canadian Council on Animal Care (1998) Guidelines on: choosing an appropriate endpoint in experiments using animals for research, teaching and testing. Ottawa, Canada Institute for Laboratory Animal Research Journal (2000) Humane Endpoints for Animals Used in Biomedical Research and Testing. ILAR 41: 2 Freedom of Information Act (Title 5 of the United States Code, Section 552) as amended by P.L. No. 104-231, 110 Stat. 3048 March 31, 1997
Index
ABIN-1 132
B cell B29 11
acute-phase proteins 10
B cells 6
adalimumab 4
bacterial artificial chromosomes (BACs) 141
adeno-associated virus (AAV) 126, 127, 133,
basic policies on animal experimentation as
134 adenovirus 126, 127, 130, 131
quasi-regulations, animal experimentation guidelines 188
adjuvant-induced arthritis (AA) 1
body weight loss 18
alkaline phosphatase, secreted heat-stable
bone destruction 35, 36
(Se AP) 149
bone formation 7
alpha-1-acid glycoprotein 10
bone pathology 15
angiogenesis 86
bone roughness 15
angiogenic response 90
bone volume 15
angiopoietin 134
bone-associated markers 11
animal experimentation guidelines 188 Animal Welfare Act 194
cancer, nanoparticles in 165, 166
Animals (Scientific Procedures) Act 1986
cantilevers 159, 160
(ASPA) 178
cartilage 2, 128, 130
ankle swelling 9
cartilage and bone markers 2
anti-CCP antibodies 6
cartilage destruction 35, 36, 55, 57, 128, 132
anti-collagen type II IgG antibodies 6
cartilage pathology 13
antisense RNA 127
cartilage-associated oligomeric matrix protein
arginase I 93, 94
(COMP) 13
arthritis, chronic, pathogenesis of 35
cathepsin K 11
arthritis models 1, 35, 58, 128, 130, 132, 134
CC-chemokine receptor (CCR) 86
asthma and airway hyperreactivity (AHR)
CCL2 91
132–134
CD4 11
asthma models 132, 134
CD11b 11
autoantibodies 6
cell therapy approaches 110
autoimmune cartilage and bone destruction
cell transplantation 116
36 autoimmune disorders 146
cell-cell fusion 114 Certificate of Designation 178
203
Index
chemo-attractive mechanisms 116
E-selectin, reporter mouse 148
chemokines 84, 88, 91
etanercept 4
chitin 69 chondrocytes 69, 128, 130
fibrin 7
chondroitin 69, 70
flare, arthritic 9
chorioallantoic membrane (CAM) 87
FTY720 147
chromodacryorrhea 18 chronic inflammation and cancer 83, 84, 86
gadofullerenes 160
collagen emulsion, preparation of 20
gene expression 2, 11, 12
collagen type I fragments (Rat-LapsTM) 15
gene targeting,
collagen type II fragments
(CartilapsTM)
13
BAC 141
collagen-induced arthritis (CIA) 1, 36, 130, 134
conditional 141
complete Freund’s adjuvant (CFA) 1
homologous recombination 141
compound testing 17 cornea 88, 89, 92 corticosteroids 2
inducible 142 gene transfer 125, 126 gene transfer, in vivo 125
steroids 1
gene transfer, somatic 125, 126
prednisone 2
gene-targeted mice 85, 92
Crohn’s disease 147
genetic predisposition 1
CRP 11
GM-CSF 133
CTLA4-Ig 5
Good Laboratory Practices 198, 199
CXCL8 91
graft vessel disease (GVD) 148
cyclooxygenase-2 (COX-2) 1, 11, 87, 93
granulocytes 90, 93, 94
cyclosporine A 1, 4
granuloma 89
cytokines 7, 12, 47, 84, 85, 88, 90, 93
growth factor 70, 84, 116 Guide for the Care and Use of Laboratory
dendrimers 157, 163
Animals 197, 198
detailed guidelines as a vital reference, formulated by the SCJ 189 disease-modifying antirheumatic drug (DMARD) 3 DNA damage, initiation by inflammation 84 drug delivery, liposomal formulations 166 Duchenne muscular dystrophy (DMD) 104
haptoglobin 11 heat shock protein 6 herpes simplex virus 126, 127 human placental alkaline phosphatase, secreted heat-stable (SeAP) 149 humane treatment of animals 190
dystrophy and inflammation 107 ibuprofen 2, 3 endothelial cell activation 148
ICAM 147
enforced self-regulation 190
IFN-a 11, 132
enhanced green fluorescent protein (EGFP) 146,
IL-1 1, 71, 75, 132
148
IL-1` 11
eosinophils 92–94
IL-1R antagonist 7, 130
erythrocyte sedimentation rate (ESR) 10
IL-4 134
204
Index
IL-4, reporter mouse 148
liposomes 157, 166
IL-4Ra 134
liposomal formulations for drug delivery
IL-5 11
166–168
IL-6 11
loxP 142, 144, 145
IL-8 (CXCL8) 91
luciferase 149
IL-10 11, 133
lymph node 11
IL-12 133 IL-13 134
macrophages 7, 90, 92, 94
IL-18 132, 133
mast cells 92
imaging 149, 157, 169
Matrigel 88, 89
immune suppression 5, 84, 92
matrix metalloproteases (MMPs) 85
in vivo imaging 149
mdx mouse strain 106
indoleamine 2,3-dioxygenase (IDO) 93, 94
mesoangioblasts 115
inducible transgenic systems 142
methotrexate 1, 4
inflammation in the pathogenesis of dystrophy
MHC class II alleles 5
107
micro-computed tomography (µCT) 15
inflammation, nanoparticles in 165
model validation 68
infliximab 4
modulation of the inflammatory reaction 110
innate immunity 83, 84, 90
molecular imaging 169
iNOS 11
molecular targets 68
Institutional Animal Care and Use Committee
monoarticular 100P model 20
(IACUC) 194, 195 institutional responsibilities for conducting animal experiments 188
monocyte chemoattractant protein 1 (MCP-1) 91 mRNA expression 11
insulin-like growth factor (IGF-1) 110, 128
muscle damage 105
insulin-like growth factor 1 (mIGF-1), locally
muscle regeneration 109
acting isoform 116 interferon-gamma 11, 132
muscular dystrophies 103 Duchenne muscular dystrophy (DMD) 104
interleukin-1 receptor antagonist (IL-1Ra) 7, 130
myeloid lineage cells 7
invertebrate 90
myoblast cell therapy 110
iron oxide nanoparticles, paramagnetic 162
myonecrosis 106
joint destruction 35
nano self-assembling systems 158 nanoarrays 165
knockout mouse 125, 126
nanomedicine 156 nanoparticles 157, 162, 165, 166
laboratory animals, guide for the care and use 197, 198
nanopore 158 nanoscale probes 156
leflunomide 3
nanoscale structures 157
lentivirus 126, 127, 129
nanosomes 163
Lewis rats 9
nanostructures 157, 158
lipopolysaccharide (LPS) 85, 88
nanosystems 157, 158, 165
205
Index
nanotechnology 155
3R principle, legal standing of 187
nanotools 157
progenitor cell 117
nanotubes 160
Project Licence 178, 180-182
naproxen 2, 3
pronuclear microinjection 140
neutrophils 7, 91, 92
prophylactic treatments 2
neutrophil counts 9
proteases 7
nonsteroidal anti-inflammatory drugs (NSAIDs)
Public Health Service Policy 196
1, 2 nuclear factor-kappa B (NF-gB) 132
quantum dots 161
NF-gB-induced inflammatory cytokines 11 3R principle 187 oncostatin M 132
RANKL 11
osteoarthritis, therapeutic agents (table) 69–75
recombinase Cre/loxP 142
osteoarthritis animal models,
recombinase FLP/FRT 142
anterior cruciate ligament (ACL) 66
recombinase-mediated cassette exchange 143
dog 66, 67, 70-75
recruitment of BM cells 116
guinea pig 66, 67, 69, 70, 73, 75
regulatory T cells (T regs) 93, 94
hamster 66, 69, 73
retrovirus 126, 127, 129
induced 66, 67
rheumatoid arthritis 1, 35
meniscectomy 66
rheumatoid factor 6
mouse 67, 70, 74, 75
ribozymes 127
rabbit 66, 67, 69–75
RNA interference 145
rat 66, 67, 70, 72–74
RNAi, inhibitory RNA 127
spontaneous 66, 67 transgenic 66
S1P1 147
osteoclasts 7
sarcoglycan-null mutant mice 107
oxidative stress 93
sarcoglycanopathies 104
oxygen metabolites 7
satellite cells 109 scientific rationality of in vivo research 190
pannus tissue 7 paramagnetic iron oxide nanoparticles 162
secreted heat-stable alkaline phosphatase (SeAP) 149
peptidoglycan and polysaccharide (PG-PS) 2
self regulation, enforced 190
PG-PS 100P 5, 20
self-assembling systems 158
periosteal bone formation 7
serum acute-phase proteins 2
persistence of inflammation 105
serum fibrinogen levels 9
Personal Licence 178, 182
short hairpin RNAs (shRNAs) 145
pharmacokinetic (PK) analysis 17
side population (SP) 113
plasma/serum and tissue cytokine levels 12
signal transducer and activator of
plasmid DNA 127, 128
transcription 4 (STAT4) 133
plethysmometer 15
specific immunity 83
polymersomes 163
stem cells 112
prednisone 2
206
embryonic stem cells (ES) 111
Index
hematopoietic stem cells (HSC) 111
glucosamine 69, 70
plasticity of 113
growth factors 70
steroids 1
hyaluronan (HA) 69
streptococcal cell wall-induced arthritis (SCW)
interleukin-1 related 71, 75
1
manganese 70
synovial cells 130
matrixmetalloproteinase inhibitors 73, 74
synovial fluid 7, 67
nitric oxide inhibitors 74
synoviocyte 7
nonsteroidal antiinflammatories 72, 73
synovium 7 systemic 10S model 20
selenium 70 therapeutic dosing regimes 2 tissue regeneration/repair 103
T cells 4, 93, 94
TNF-_ 1, 11, 132, 147
T cell response 5
transdifferentiation of stem cells 112
regulatory T cells (T regs) 93, 94
transforming growth factor-beta 1 (TGF-`1)
TGF-` 11, 85 therapeutic agents, osteoarthritis (table) 69–75
11, 85 transgenic mouse 85, 125, 126
ascorbic acid 70
TRAP 11
bisphosphonates 72
tumor xenograft 90
calcitonin 71
type II collagen 18
chitin 69 chondroitin 69, 70 collagen 70
vascular endothelial growth factor (VEGF) 87, 90
estrogen related 70 exercise 69
wound healing 90
207
The PIR-Series Progress in Inflammation Research Homepage: http://www.birkhauser.ch
Up-to-date information on the latest developments in the pathology, mechanisms and therapy of inflammatory disease are provided in this monograph series. Areas covered include vascular responses, skin inflammation, pain, neuroinflammation, arthritis cartilage and bone, airways inflammation and asthma, allergy, cytokines and inflammatory mediators, cell signalling, and recent advances in drug therapy. Each volume is edited by acknowledged experts providing succinct overviews on specific topics intended to inform and explain. The series is of interest to academic and industrial biomedical researchers, drug development personnel and rheumatologists, allergists, pathologists, dermatologists and other clinicians requiring regular scientific updates.
Available volumes: T Cells in Arthritis, P. Miossec, W. van den Berg, G. Firestein (Editors), 1998 Chemokines and Skin, E. Kownatzki, J. Norgauer (Editors), 1998 Medicinal Fatty Acids, J. Kremer (Editor), 1998 Inducible Enzymes in the Inflammatory Response, D.A. Willoughby, A. Tomlinson (Editors), 1999 Cytokines in Severe Sepsis and Septic Shock, H. Redl, G. Schlag (Editors), 1999 Fatty Acids and Inflammatory Skin Diseases, J.-M. Schröder (Editor), 1999 Immunomodulatory Agents from Plants, H. Wagner (Editor), 1999 Cytokines and Pain, L. Watkins, S. Maier (Editors), 1999 In Vivo Models of Inflammation, D. Morgan, L. Marshall (Editors), 1999 Pain and Neurogenic Inflammation, S.D. Brain, P. Moore (Editors), 1999 Anti-Inflammatory Drugs in Asthma, A.P. Sampson, M.K. Church (Editors), 1999 Novel Inhibitors of Leukotrienes, G. Folco, B. Samuelsson, R.C. Murphy (Editors), 1999 Vascular Adhesion Molecules and Inflammation, J.D. Pearson (Editor), 1999 Metalloproteinases as Targets for Anti-Inflammatory Drugs, K.M.K. Bottomley, D. Bradshaw, J.S. Nixon (Editors), 1999 Free Radicals and Inflammation, P.G. Winyard, D.R. Blake, C.H. Evans (Editors), 1999 Gene Therapy in Inflammatory Diseases, C.H. Evans, P. Robbins (Editors), 2000 New Cytokines as Potential Drugs, S. K. Narula, R. Coffmann (Editors), 2000 High Throughput Screening for Novel Anti-inflammatories, M. Kahn (Editor), 2000 Immunology and Drug Therapy of Atopic Skin Diseases, C.A.F. Bruijnzeel-Komen, E.F. Knol (Editors), 2000 Novel Cytokine Inhibitors, G.A. Higgs, B. Henderson (Editors), 2000 Inflammatory Processes. Molecular Mechanisms and Therapeutic Opportunities, L.G. Letts, D.W. Morgan (Editors), 2000
Backlist
Cellular Mechanisms in Airways Inflammation, C. Page, K. Banner, D. Spina (Editors), 2000 Inflammatory and Infectious Basis of Atherosclerosis, J.L. Mehta (Editor), 2001 Muscarinic Receptors in Airways Diseases, J. Zaagsma, H. Meurs, A.F. Roffel (Editors), 2001 TGF-` and Related Cytokines in Inflammation, S.N. Breit, S. Wahl (Editors), 2001 Nitric Oxide and Inflammation, D. Salvemini, T.R. Billiar, Y. Vodovotz (Editors), 2001 Neuroinflammatory Mechanisms in Alzheimer’s Disease. Basic and Clinical Research, J. Rogers (Editor), 2001 Disease-modifying Therapy in Vasculitides, C.G.M. Kallenberg, J.W. Cohen Tervaert (Editors), 2001 Inflammation and Stroke, G.Z. Feuerstein (Editor), 2001 NMDA Antagonists as Potential Analgesic Drugs, D.J.S. Sirinathsinghji, R.G. Hill (Editors), 2002 Migraine: A Neuroinflammatory Disease? E.L.H. Spierings, M. Sanchez del Rio (Editors), 2002 Mechanisms and Mediators of Neuropathic pain, A.B. Malmberg, S.R. Chaplan (Editors), 2002 Bone Morphogenetic Proteins. From Laboratory to Clinical Practice, S. Vukicevic, K.T. Sampath (Editors), 2002 The Hereditary Basis of Allergic Diseases, J. Holloway, S. Holgate (Editors), 2002 Inflammation and Cardiac Diseases, G.Z. Feuerstein, P. Libby, D.L. Mann (Editors), 2003 Mind over Matter – Regulation of Peripheral Inflammation by the CNS, M. Schäfer, C. Stein (Editors), 2003 Heat Shock Proteins and Inflammation, W. van Eden (Editor), 2003 Pharmacotherapy of Gastrointestinal Inflammation, A. Guglietta (Editor), 2004 Arachidonate Remodeling and Inflammation, A.N. Fonteh, R.L. Wykle (Editors), 2004 Recent Advances in Pathophysiology of COPD, P.J. Barnes, T.T. Hansel (Editors), 2004 Cytokines and Joint Injury, W.B. van den Berg, P. Miossec (Editors), 2004 Cancer and Inflammation, D.W. Morgan, U. Forssmann, M.T. Nakada (Editors), 2004 Bone Morphogenetic Proteins: Bone Regeneration and Beyond, S. Vukicevic, K.T. Sampath (Editors), 2004 Antibiotics as Anti-Inflammatory and Immunomodulatory Agents, B.K. Rubin, J. Tamaoki (Editors), 2005 Antirheumatic Therapy: Actions and Outcomes, R.O. Day, D.E. Furst, P.L.C.M. van Riel, B. Bresnihan (Editors), 2005 Regulatory T-Cells in Inflammation, L. Taams, A.N. Akbar, M.H.M Wauben (Editors), 2005 Sodium Channels, Pain, and Analgesia, K. Coward, M. Baker (Editors), 2005 Turning up the Heat on Pain: TRPV1 Receptors in Pain and Inflammation, A.B Malmberg, K.R. Bley (Editors), 2005 The NPY Family of Peptides in Immune Disorders, Inflammation, Angiogenesis and Cancer, Z. Zukowska, G. Z. Feuerstein (Editors), 2005 Toll-like Receptors in Inflammation, L.A.J. O’Neill, E. Brint (Editors), 2005 Complement and Kidney Disease, P. F. Zipfel (Editor), 2006 Chemokine Biology – Basic Research and Clinical Application, Volume 1: Immunobiology of Chemokines, B. Moser, G. L. Letts, K. Neote (Editors), 2006 The Hereditary Basis of Rheumatic Diseases, R. Holmdahl (Editor), 2006 Lymphocyte Trafficking in Health and Disease, R. Badolato, S. Sozzani (Editors), 2006 In Vivo Models of Inflammation, 2nd Edition, Volume II, C.S. Stevenson, L.A. Marshall, D.W. Morgan (Editors), 2006